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Chronic exposure to methamphetamine causes adaptive changes in brain, which underlie dependence symptoms. We have found that the transmembrane protein 168 (TMEM168) is overexpressed in the nucleus accumbens of mice upon repeated methamphetamine administration. Here, we firstly demonstrate the inhibitory effect of TMEM168 on methamphetamine-induced behavioral changes in mice, and attempt to elucidate the mechanism of this inhibition. We overexpressed TMEM168 in the nucleus accumbens of mice by using an adeno-associated virus vector (NAc-TMEM mice). Methamphetamine-induced hyperlocomotion and conditioned place preference were attenuated in NAc-TMEM mice. Additionally, methamphetamine-induced extracellular dopamine elevation was suppressed in the nucleus accumbens of NAc-TMEM mice. Next, we identified extracellular matrix protein osteopontin as an interacting partner of TMEM168, by conducting immunoprecipitation in cultured COS-7 cells. TMEM168 overexpression in COS-7 cells induced the enhancement of extracellular and intracellular osteopontin. Similarly, osteopontin enhancement was also observed in the nucleus accumbens of NAc-TMEM mice, in in vivo studies. Furthermore, the infusion of osteopontin proteins into the nucleus accumbens of mice was found to inhibit methamphetamine-induced hyperlocomotion and conditioned place preference. Our studies suggest that the TMEM168-regulated osteopontin system is a novel target pathway for the therapy of methamphetamine dependence, via regulating the dopaminergic function in the nucleus accumbens.

A Lewis acid-catalyzed [3+1+1] cycloaddition reaction between aliphatic isocyanides and azomethine ylides generated in situ from aziridines, leading to pyrrolidine derivatives, has been developed. This reaction proceeds smoothly under mild conditions and can also be modified by employing aromatic isocyanides to generate four-membered heterocycles, azetidines, through a [3+1] cycloaddition reaction.

The degree of gradient wind balance was investigated in a number of tropical cyclones (TCs) simulated under realistic environments. The results of global-scale numerical simulations without cumulus parameterization were used, with a horizontal mesh size of 7 km. On average, azimuthally averaged maximum tangential velocities at 850 (925) hPa in the simulated TCs were 0.72% (1.95%) faster than gradient wind–balanced tangential velocity (GWV) during quasi-steady periods. Of the simulated TCs, 75% satisfied the gradient wind balance at the radius of maximum wind speed (RMW) at 850 and at 925 hPa to within about 4.0%. These results were qualitatively similar to those obtained during the intensification phase. In contrast, averages of the maximum and minimum deviations from the GWV, in all the azimuths at the RMW, achieved up to 40% of the maximum tangential velocity. Azimuthally averaged tangential velocities exceeded the GWV (i.e., supergradient) inside the RMW in the lower troposphere, whereas the velocities were close to or slightly slower than GWV (i.e., subgradient) in the other regions. The tangential velocities at 925 hPa were faster (slower) in the right-hand (left hand) side of the TC motion. When the tangential velocities at the RMW were supergradient, the primary circulation tended to decay rapidly in the vertical direction and slowly in the radial direction, and the eyewall updraft and the RMW were at larger radii. Statistical analyses revealed that the TC with supergradient wind at the RMW at 850 hPa was characterized by stronger intensity, larger RMW, more axisymmetric structure, and an intensity stronger than potential intensity.

A novel N-acetyltransferase, Shati/Nat8l, was identified in the nucleus accumbens (NAc) of mice with methamphetamine (METH) treatment. Previously we reported that suppression of Shati/Nat8l enhanced METH-induced behavioral alterations via dopaminergic neuronal regulation. However, the physiological mechanisms of Shati/Nat8l on the dopaminergic system in the brain are unclear. In this study, we injected adeno-associated virus (AAV) vector containing Shati/Nat8l into the NAc or dorsal striatum (dS) of mice, to increase Shati/Nat8l expression. Overexpression of Shati/Nat8l in the NAc, but not in the dS, attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference in mice. Moreover, the Shati/Nat8l overexpression in the NAc attenuated the elevation of extracellular dopamine levels induced by METH in in vivo microdialysis experiments. These behavioral and neurochemical alterations due to Shati/Nat8l overexpression in the NAc were inhibited by treatment with selective group II metabotropic glutamate receptor type 2 and 3 (mGluR2/3) antagonist LY341495. In the AAV vector-injected NAc, the tissue contents of both N-acetylaspartate and N-acetylaspartylglutamate (NAAG), endogenous mGluR3 agonist, were elevated. The injection of peptidase inhibitor of NAAG or the perfusion of NAAG itself reduced the basal levels of extracellular dopamine in the NAc of naive mice. These results indicate that Shati/Nat8l in the NAc, but not in the dS, plays an important suppressive role in the behavioral responses to METH by controlling the dopaminergic system via activation of group II mGluRs.

[1] Deep moist atmospheric convection is a key element of the weather and climate system for transporting mass, momentum, and thermal energy. It has been challenging to simulate convection realistically in global atmospheric models because of the large gap in spatial scales between convection (100 km) and global motions (104 km). We conducted the first ever subkilometer global simulation and described the features of convection. Through a series of grid-refinement resolution testing, we found that an essential change for convection statistics occurred around 2 km grid spacing. The convection structure, number of convective cells, and distance to the nearest convective cell dramatically changed at this resolution. The convection core was resolved using multiple grids in simulations with grid spacings less than 2.0 km.

Relapse of drug abuse after abstinence is a major challenge to the treatment of addicts. In our well-established mouse models of methamphetamine (Meth) self-administration and reinstatement, bilateral microinjection of adeno-associated virus vectors expressing GDNF (AAV-Gdnf) into the striatum significantly reduced Meth self-administration, without affecting locomotor activity. Moreover, the intrastriatal AAV-Gdnf attenuated cue-induced reinstatement of Meth-seeking behaviour in a sustainable manner. In addition, this manipulation showed that Meth-primed reinstatement of Meth-seeking behaviour was reduced. These findings suggest that the AAV vector-mediated Gdnf gene transfer into the striatum is an effective and sustainable approach to attenuate Meth self-administration and Meth-associated cue-induced relapsing behaviour and that the AAV-mediated Gdnf gene transfer in the brain may be a valuable gene therapy against drug dependence and protracted relapse in clinical settings.

The stereoselective direct transformation of N-(propargylic)hydroxylamines into cis-2-acylaziridines was achieved by the combined use of AgBF4 and CuCl. Copper salts were found to promote the transformation of the intermediary 4-isoxazolines into 2-acylaziridines and both 3-aryl- and 3-alkyl-substituted 2-acylaziridines could be prepared by using this method. Furthermore, subsequent 1,3-dipolar cycloaddition of azomethine ylides that were generated in situ from the intermediary 2-acylaziridines with maleimides was achieved in a stereoselective one-pot procedure to afford the corresponding 2-acylpyrrolidines, which consisted of an octahydropyrrolo[3,4-c]pyrrole skeleton. Love the way ylide: The transformation of N-(propargylic) hydroxylamines into cis-2-acylaziridines and subsequent 1,3-dipolar cycloaddition of the in situ generated azomethine ylides with maleimides stereoselectively afforded 2-acylpyrrolidines. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A mechanism for the transition of tropical cyclones (TCs) to the spontaneous rapid intensification (RI) phase is proposed based on numerical results of a three-dimensional full-physics model. The intensification phase of the simulated TC is divided into three subphases according to the rate of intensification: 1) a slowly intensifying phase, 2) an RI phase, and 3) an adjustment phase toward the quasi-steady state. The evolution of a TC vortex is diagnosed by the energy budget analysis and the degree of axisymmetric structure of the TC vortex, and the simulated TC is determined to be axisymmetrized 12 h before the onset of RI. It is found that equivalent potential temperature θe in the lowest layer suddenly increases inside the radius of maximum azimuthally averaged horizontal wind rma after the TC becomes nearly axisymmetric. Forward trajectory analyses revealed that the enhanced convective instability in the TC core region where the eyewall subsequently forms results from the increased inertial stability of the TC core after the axisymmetrization. Since fluid parcels remain longer inside rma, owing to the increased inertial stability, the parcels obtain more enthalpy from the underlying ocean. As a result, low-level θe and hence convective available potential energy (CAPE) increase. Under the condition with increased CAPE, the eyewall is intensified and the secondary circulation is enhanced, leading to the increased convergence of low-level inflow; this process is considered to be the trigger of RI. Once the eyewall forms, the simulated TC starts its RI.

The number of patients with schizophrenia has increased over the past decade. Previously, many studies have been performed to establish its diagnostic criteria, prophylactic methods, and effective therapies. In this study, we analyzed whether the ratios of DNA methylation in CpG islands of the Shati/Nat8l is decreased in model mice of schizophrenia-like phenotype using genomic DNA collected from brain regions and peripheral blood, since the mouse model of schizophrenia-like phenotype, mice treated repeatedly with methamphetamine showed increase of Shati/Nat8l mRNA expression in our previous experiment. The ratios of Shati/Nat8l CpG island methylation were significantly decreased in both the nucleus accumbens and the peripheral blood of model mice compared with those of control mice. We also investigated Shati/Nat8l methylation in the blood of patients with schizophrenia. We found that Shati/Nat8l CpG island methylation ratios were lower in the patients with schizophrenia than in the healthy controls, which is consistent with our findings in the mice model. To our knowledge, this is the first study to show similar alterations in methylation status of a particular genomic DNA site in both the brain and peripheral blood of mice. Furthermore, the same phenomenon was observed in corresponding human genomic sequences of the DNA extracted from the peripheral blood of patients with schizophrenia. Based on our findings, DNA methylation profiles of the CpG island of Shati/Nat8l might be a diagnostic biomarker of schizophrenia.

This study found that there is a radius within which the sea surface flux for moist enthalpy plays a vital role in determining the intensity of a tropical cyclone. From the results of the numerical experiments using an axisymmetric nonhydrostatic model, it was shown that when the sea surface fluxes are modified within the radius, the intensity suddenly changes on a short timescale. As long as the surface enthalpy flux diminishes outside this radius, the tropical cyclone intensity does not decrease. In the simulated tropical cyclone, this radius locates 7–8 times the radius of the maximum wind speed. Copyright © 2010 Royal Meteorological Society

We show that Ca(2+)/calmodulin(CaM)-dependent protein kinase I (CaMKI) is directly inhibited by its S-glutathionylation at the Cys(179). In vitro studies demonstrated that treatment of CaMKI with diamide and glutathione results in inactivation of the enzyme, with a concomitant S-glutathionylation of CaMKI at Cys(179) detected by mass spectrometry. Mutagenesis studies confirmed that S-glutathionylation of Cys(179) is both necessary and sufficient for the inhibition of CaMKI by diamide and glutathione. In transfected cells expressing CaMKI, treatment with diamide caused a reversible decrease in CaMKI activity. Cells expressing mutant CaMKI (179CV) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKI via its modification at Cys(179) is an important mechanism in processing calcium signal transduction in cells.

The mechanisms of NO inhibition of CaMK [Ca2+/CaM (calmodulin)-dependent protein kinase] II activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser847 was sensitive to an inhibitor of CaMKs, KN-93, and was enhanced by inhibition of nNOS with 7NI (7-nitroindazole). Enzyme activity of CaMKII following in situ treatment with 7NI was also increased. The in vitro activity of CaMKII was inhibited by co-incubation either with nNOS and L-arginine or with NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) and DEA-NONOate [diethylamine-NONOate (diazeniumdiolate)]. Once inhibited by these treatments, CaMKII was observed to undergo full reactivation on the addition of a reducing reagent, DTT (dithiothreitol). In transfected cells expressing CaMKII and nNOS, treatment with the calcium ionophore A23187 further revealed nNOS phosphorylation at Ser847, which was enhanced by 7NI and CaMKII S-nitrosylation. Mutated CaMKII (C6A), in which Cys6 was substituted with an alanine residue, was refractory to 7NI-induced enhancement of nNOS phosphorylation or to CaMKII S-nitrosylation. Furthermore, we could identify Cys6 as a direct target for S-nitrosylation of CaMKII using MS. In addition, treatment with glutamate caused an increase in CaMKII S-nitrosylation in rat hippocampal slices. This glutamate-induced S-nitrosylation was blocked by 7NI. These results suggest that inactivation of CaMKII mediated by S-nitrosylation at Cys6 may contribute to NO-induced neurotoxicity in the brain.

It is known that the adhesion molecule ALCAM (CD166) mediates metastasis of malignant cells and organogenesis in embryos. We show here that embryonic day 8.5 (E8.5) murine yolk sac cells express ALCAM protein and that ALCAM expression can be used to define endothelial and cardiac precursors from hematopoietic precursors in E8.5 yolk sacs. ALCAM high+ cells exclusively give rise to endothelial and cardiac cells in matrigel assays but generate no hematopoietic colonies in methylcellulose assays. ALCAM low+ and ALCAM− populations predominantly give rise to hematopoietic cells in methylcellulose, but do not generate any cell clusters in matrigel. The ALCAM high+ population contains both Flk-1+ and Flk-1− cells. The former population exclusively contains endothelial cells whereas the latter give rise to cardiac cells when cultured on OP9 stromal cells. We also show that cardiac lineage marker genes such as Nkx-2.5, and the endothelial marker VE-cadherin are expressed in the ALCAM high+ fraction, whereas the hematopoietic marker GATA1 and Runx1 are expressed in the ALCAM low+/− fraction. However, we did not detect expression of the cardiac structural protein cTn-T in cells from yolk sac cells until these had had been differentiated on OP9 for 5 days. Altogether, these results indicate that cells retaining a potential to differentiate to the cardiac lineage are present in E8.5 yolk sacs and can be isolated as ALCAM high+, Flk-1− cells. Our report provides novel insights into the origin and differentiation process of cardiac cells in the formation of the circulatory system.

An acute application of neurosteroid pregnenolone sulfate (PREGS) to hippocampal slices from adult rats induced a long-lasting potentiation (LLPPREGS) at the perforant path-granule cell synapse. As a partial mechanism of the LLPPREGS, we previously revealed that PREGS transiently increases the probability of presynaptic glutamate release via a sensitization of α7-nicotinic acetylcholine receptor (α7nAChR). We herein demonstrate that the LLPPREGS could be separated into two independent processes: the above-mentioned early presynaptic-origin short-term potentiation (STPPREGS) and a delayed postsynaptic N-methyl-d-aspartate receptor (NMDAr)-dependent long-term potentiation termed LTPPREGS. This study focused on the analysis of the signaling mechanism underlying the LTPPREGS. PREGS increased the tyrosine phosphorylation of NR2B, a subunit of NMDAr, and the NMDAr-mediated Ca2+ influx in the granule cells. The enhanced Ca2+ influx was largely attenuated by the NR2B subunit inhibitor ifenprodil and the Src kinase family inhibitor PP2. PREGS also triggered a persistent phosphorylation of extracellular signal-regulated kinase 2 (ERK2) followed by an ERK-dependent phosphorylation of cAMP-response element-binding protein (CREB), which was crucial for the LTPPREGS induction and was sensitive to ifenprodil. These results suggest that PREGS induces an acute increase in the NR2B tyrosine phosphorylation which enhances the Ca2+ influx through NMDAr, followed by an activation of the ERK/CREB signaling cascade that leads to LTPPREGS.

Nascent mesodermal cells derived from EB5 embryonic stem (ES) cells were sorted in terms of cardiogenic potential on the basis of their expression levels of platelet-derived growth factor receptor alpha (PDGFRalpha) and fetal liver kinase 1 (Flk-1). The sorted cells were cocultured with OP9 stromal cells to induce terminal differentiation into contractile cardiac colonies. A significant number of cardiac colonies were found in the Flk-1+/PDGFRalpha+ fraction. The enrichment double-positive fraction produced approximately fivefold more cardiac colonies than the Flk-1+/PDGFRalpha- fraction and 10-fold more than the Flk-1-/PDGFRalpha+ fraction. To investigate the involvement of these markers in embryonic cardiogenesis, the cells that disseminated from the E7.5-7.75 embryos were fractionated and seeded on OP9 cells. The cardiogenic potential was markedly enhanced in the Flk-1+/PDGFRalpha+ fraction. These results suggest that some of the precursor cells coexpressing these markers are selectively involved in cardiogenic events, and that the identification of ES-cell-derived precursors with these markers will contribute to the effective production of cardiomyocytes for cell therapies.

This study investigated the impact of several cloud microphysical schemes on the trade wind cumulus in the large eddy simulation model. To highlight the differences due to the cloud microphysical component, we developed a fully compressible large eddy simulation model, which excluded the implicit scheme and approximations as much as possible. The three microphysical schemes, the one-moment bulk, two-moment bulk, and spectral bin schemes were used for sensitivity experiments in which the other components were fixed. Our new large eddy simulation model using a spectral bin scheme successfully reproduced trade wind cumuli, and reliable model performance was confirmed. Results of the sensitivity experiments indicated that precipitation simulated by the one-moment bulk scheme started earlier, and its total amount was larger than that of the other models. By contrast, precipitation simulated by the two-moment scheme started late, and its total amount was small. These results support those of a previous study. The analyses revealed that the expression of two processes, (1) the generation of cloud particles and (2) the conversion from small droplets to raindrops, were crucial to the results. The fast conversion from cloud to rain and the large amount of newly generated cloud particles at the cloud base led to evaporative cooling and subsequent stabilization in the sub-cloud layer. The latent heat released at higher layers by the condensation of cloud particles resulted in the development of the boundary layer top height.

A Lewis acid-catalyzed [3+1+1] cycloaddition reaction between aliphatic isocyanides and azomethine ylides generated in situ from aziridines, leading to pyrrolidine derivatives, has been developed. This reaction proceeds smoothly under mild conditions and can also be modified by employing aromatic isocyanides to generate four-membered heterocycles, azetidines, through a [3+1] cycloaddition reaction.

Background: Addictive drugs lead to reinforcing properties by increasing dopamine in the nucleus accumbens, which is composed of a core and shell regions. Neurons in the nucleus accumbens are divided into 2 subtypes based on the differential gene expression of the dopamine D1 receptors and D2 receptors. Methods: In the present study, we investigated the role of D2 receptors in the nucleus accumbens core in behaviors and signal transduction induced by psychostimulant methamphetamine in mice that were microinjected with adeno-associated virus vectors containing a microRNA (miRNA) sequence for D2 receptor (adeno-associated virus-miD2r vectors) in the nucleus accumbens core. The adeno-associated virus vectors containing a miRNA sequence for D2 receptor-treated mice (miD2r mice) were assessed at a reduction in D2 receptor, but at no change in dopamine D1 receptor, in the nucleus accumbens core compared with the adeno-associated virus-Mock vectors-treated mice (Mock mice). Results: miD2r mice exhibited a reduction in hyperlocomotion that was induced by a single treatment with methamphetamine. The development of locomotor sensitization induced by repeated treatment with methamphetamine exhibited less extension in miD2r mice. In a place conditioning paradigm, the preferred effects of methamphetamine were significantly weaker in miD2r mice than in Mock mice. Furthermore, the single treatment with methamphetamine-induced phosphorylation of extracellular signal regulated kinase and cyclic adenosine monophosphate response element-binding protein in the nucleus accumbens core of miD2r mice was decreased compared with that in Mock mice. Repeated treatment with methamphetamine-induced delta FBJ murine osteosarcoma viral oncogene homolog B accumulation in the nucleus accumbens core of miD2r mice was also attenuated. Conclusions: These findings suggest that a D2 receptor-mediated neuronal pathway from the nucleus accumbens core plays an inhibitory role in the development of reinforcing properties.

ALCAM (activated leukocyte cell adhesion molecule, CD166) belongs to the immunoglobulin superfamily and is involved in axon guidance, hematopoiesis, immune response and tumor metastasis. During embryogenesis, mRNA encoding ALCAM was expressed in the cardiac crescent and the neural groove at embryonic day (E) 7.75 and predominately in the tubular heart at E8.5. A newly generated monoclonal antibody against the ALCAM molecule (ALC-48) exclusively stained cardiomyocytes at E8.25–10.5. However, ALCAM expression was lost by cardiomyocytes by E12.5 and its expression shifts to a variety of organs during later stages. ALCAM was found to be a prominent surface marker for cardiomyocytes in early embryonic hearts. The transient expression of ALCAM during early developmental stages marks specific developmental stages in cardiomyocyte differentiation.

数種のモモ台木品種と岐阜県飛騨地域在来のハナモモに‘白鳳’，‘昭和白桃’を接ぎ木して凍害による主幹部障害，枯死樹発生に及ぼす影響を検討した．‘おはつもも’台は3年生時から穂木部を中心に障害が発生し，4年生時にはすべて枯死した．これに対し‘モモ台木筑波1号’台，‘ハローブラッド’台および‘国府HM-1’台は3～4年生時に穂木部，台木・接ぎ木部ともに障害が発生したが，枯死には至らなかった．中でも‘国府HM-1’台は，障害程度が最も軽症であり，障害発生抑制に有効な台木であると考えられた．一方，台木品種による穂木品種の発芽期，開花始期，展葉期に差は認められなかった．また，樹体生育量には差は認められたが，障害発生との関係は明らかでなかった．

Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is now emerging as an important modulator of the function of the CNS. Methamphetamine (METH) is a widely abused psychostimulant that causes euphoria, hyperactivity, and drug dependence. High doses of METH cause long-term neurotoxicity in dopaminergic neurons. In this study, we investigated a role of TNF-α in METH-induced dependence and neurotoxicity. Repeated treatment with METH (2 mg/kg for 5 d) in rats induced a significant increase in TNF-α mRNA and protein expression in the brain. Exogenous TNF-α (1-4 μg) blocked locomotor-stimulating and rewarding effects of METH, as well as METH (4 mg/kg; four times at 2 hr intervals)-induced dopaminergic neurotoxicity in mice. To examine a role of endogenous TNF-α in behavioral and neurochemical effects of METH, we used mice with targeted deletions of the TNF-α gene. TNF-α-(-/-) mice showed enhanced responses to the locomotor-sensitizing, rewarding, and neurotoxic effects of METH compared with wild-type mice. We also examined the role of TNF-α in METH-induced dopamine (DA) release and uptakein vitroandin vivoin C57BL/6 mice. Exogenous TNF-α (4 μg) attenuated the METH-induced increase in extracellular striatal DAin vivoand potentiated striatal DA uptake into synaptosomesin vitroandin vivo. Furthermore, TNF-α activated vesicular DA uptake by itself and diminished the METH-induced decrease in vesicular DA uptake. Our findings suggest that TNF-α plays a neuroprotective role in METH-induced drug dependence and neurotoxicity by activating plasmalemmal and vesicular DA transporter as well as inhibiting METH-induced increase in extracellular DA levels.

Tissue plasminogen activator (tPA) is a serine protease that catalyzes the conversion of plasminogen (plg) to plasmin, which in turn functions to degrade extracellular matrix proteins in the central nervous system. The tPA-plasmin system plays a role in synaptic plasticity and remodeling. Here we show that this protease system participates in the rewarding effects of morphine by acutely regulating morphine-induced dopamine release in the nucleus accumbens (NAcc). A single morphine treatment induced tPA mRNA and protein expression in a naloxone-sensitive manner, which was associated with an increase in the enzyme activity in the NAcc. The acute effect of morphine in inducing tPA expression was diminished after repeated administration. Morphine-induced conditioned place preference and hyperlocomotion were significantly reduced in tPA -/- and plg -/- mice, being accompanied by a loss of morphine-induced dopamine release in the NAcc. The defect of morphine-induced dopamine release and hyperlocomotion in tPA -/- mice was reversed by microinjections of either exogenous tPA or plasmin into the NAcc. Our findings demonstrate a previously undescribed function of the tPA-plasmin system in regulating dopamine release, which is involved in the rewarding effects of morphine.

The NMDA subtype of glutamate receptor (GluR) plays an important role in excitatory neurotransmission, synaptic plasticity, brain development, and neurodegeneration. NMDA receptors are inherent high Ca(2+)-permeable channels, which are formed by heteromeric assembly of the GluR zeta 1 subunit (NR1) and any one of four GluR epsilon subunits (GluR epsilon 1-4; NR2A-D). Mice lacking the GluR epsilon 1 subunit exhibited a malfunction of NMDA receptors, as evidenced by reduction of NMDA receptor channel current, hippocampal long-term potentiation, [3H]MK-801 binding, and NMDA-stimulated 45Ca2+ uptake. A biochemical analysis revealed a hyperfunction of dopaminergic and serotonergic neuronal systems in the frontal cortex and striatum of GluR epsilon 1 mutant mice. The enhancement of dopaminergic neuronal activity in the striatum, at least, due to the disinhibition of inhibitory GABAergic neuronal input. GluR epsilon 1 mutant mice showed an increase of locomotor activity in a novel environment attributed to the hyperfunction of the dopaminergic neuronal system, and an impairment of spatial, contextual, and latent learning. These findings provide evidence that NMDA receptors regulate behavior through the modulation of not only glutamatergic but also GABAergic and dopaminergic neuronal systems. Moreover, it is suggested that GluR epsilon 1 mutant mice are useful as an animal model, which is associated with the malfunction of NMDA receptors and hyperfunction of the dopaminergic neuronal system.

Shati/Nat8L significantly increased in the nucleus accumbens (NAc) of mice after repeated methamphetamine (METH) treatment. We reported that Shati/Nat8L overexpression in mouse NAc attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference. We recently found that Shati/Nat8L overexpression in NAc regulates the dopaminergic neuronal system via the activation of group II mGluRs by elevated N-acetylaspartylglutamate following N-acetylaspartate increase due to the overexpression. These findings suggest that Shati/Nat8L suppresses METH-induced responses. However, the mechanism by which METH increases the Shati/Nat8L mRNA expression in NAc is unclear. To investigate the regulatory mechanism of Shati/Nat8L mRNA expression, we performed a mouse Shati/Nat8L luciferase assay using PC12 cells. Next, we investigated the response of METH to Shati/Nat8L expression and CREB activity using mouse brain slices of NAc, METH administration to mice, and western blotting for CREB activity of specific dopamine receptor signals in vivo and ex vivo. We found that METH activates CREB binding to the Shati/Nat8L promoter to induce the Shati/Nat8L mRNA expression. Furthermore, the dopamine D1 receptor antagonist SCH23390, but not the dopamine D2 receptor antagonist sulpiride, inhibited the upregulation of Shati/Nat8L and CREB activities in the mouse NAc slices. Thus, the administration of the dopamine D1 receptor agonist SKF38393 increased the Shati/Nat8L mRNA expression in mouse NAc. These results showed that the Shati/Nat8L mRNA was increased by METH-induced CREB pathway via dopamine D1 receptor signaling in mouse NAc. These findings may contribute to development of a clinical tool for METH addiction.

Various molecules are involved in drug addiction induced by drugs of abuse. Therefore, the mechanism of drug addiction is still not clear, and it has been a difficulty in the development of preventive and curative drugs for drug dependence. We tried to identify the molecules associated with drug dependence, and found three molecules including shati/nat81. Recently, it has been demonstrated that the substrate for shati/nat81 is aspaltate and shati/nat8l biosynthesizes N-acetylaspartate, which exists abundantly in the mammalian brain. In this study, we investigated the physiological function of shati/nat81 and the role of shati/nat81 in drug dependence. The overexpression of shati/nat81 in the dorsal striatum of mice led to social abnormality and depression-like behavior, and worsened a part of the motor dysfunction induced by Ca2+ channel agonist BAY-K 8644. The overexpression of shati/nat81 in the nucleus accumbens of mice inhibited methamphetamine-induced behavioral and biochemical abnormalities. These findings suggest that the shati/nat81-associated system could play a role in the regulation of mental activity and motor action, and be a new target in the development of therapeutic drugs for drug dependence.

NMDA receptors, an ionotropic subtype of glutamate receptors (GluRs) forming high Ca2+-permeable cation channels, are composed by assembly of the GluRζ subunit (NR1) with any one of four GluRε subunits (GluRε1–4; NR2A-D). In the present study, we investigated neuronal functions in mice lacking the GluRε1 subunit. GluRε1 mutant mice exhibited a malfunction of NMDA receptors, as evidenced by alterations of [3H]MK-801 binding as well as45Ca2+uptake through the NMDA receptors. A postmortem brain analysis revealed that both dopamine and serotonin metabolism were increased in the frontal cortex and striatum of GluRε1 mutant mice. The NMDA-stimulated [3H]dopamine release from the striatum was increased, whereas [3H]GABA release was markedly diminished in GluRε1 mutant mice. When (+)bicuculline, a GABAAreceptor antagonist, was added to the superfusion buffer, NMDA-stimulated [3H]dopamine release was significantly increased in wild-type, but not in the mutant mice. GluRε1 mutant mice exhibited an increased spontaneous locomotor activity in a novel environment and an impairment of latent learning in a water-finding task. Hyperlocomotion in GluRε1 mutant mice was attenuated by treatment with haloperidol and risperidone, both of which are clinically used antipsychotic drugs, at doses that had no effect in wild-type mice. These findings provide evidence that NMDA receptors are involved in the regulation of behavior through the modulation of dopaminergic and serotonergic neuronal systems. In addition, our findings suggest that GluRε1 mutant mice are useful as an animal model of psychosis that is associated with NMDA receptor malfunction and hyperfunction of dopaminergic and serotonergic neuronal systems.

Esmolol, an ultra-short-acting beta-adrenergic blocker, is a racemate. In this study, to compare the stereoselective pharmacokinetics of enantiomers, we have investigated the blood level profile after a single intravenous administration (10 mg/kg) of 14C-esmolol hydrochloride (14C-esmolol) to dogs. The distribution into the heart, a target organ, was tested after a single intravenous administration (20 mg/kg) of 14C-esmolol to rats. Each enantiomer was tested for metabolic conversion by blood esterase (in vitro), and the protein binding. In addition, the chiral inversion was investigated using human (in vitro) and dog blood (in vitro and in vivo). 1. The blood concentration and the AUC of d-esmolol were 1.6-fold higher than those of l-esmolol, and the half-life of d-esmolol was 1.7-fold longer than that of l-esmolol after administration of 14C-esmolol to dogs. 2. The half-life of d-esmolol was 1.4-fold longer than that of l-esmolol after incubation with dog blood, whereas no stereoselective difference was found in human blood. 3. The concentration in the heart showed no significant difference between two enantiomers after administration of 14C-esmolol to rats. 4. The chiral inversion was found neither in blood after administration of 14C-d-esmolol or 14C-l-esmolol to dogs nor in human and dog blood after the in vitro incubation. 5. There was no significant difference in protein bindings of each enantiomer after the in vitro incubation with human, dog and rat serum, and the bound fraction was the highest in human, followed by dog and rat serum. In conclusion, these findings suggest that pharmacokinetics of enantiomers slightly differ in dog, whereas there are no stereoselective differences in human blood kinetics.

The present study investigated the involvement of nitric oxide (NO) in phencyclidine (PCP)-induced place aversion and preference in the place conditioning paradigm. PCP-induced place aversion in naive mice was dose-dependently attenuated by administration of N(G)-nitro-L-arginine methyl ester (L-NAME), a NO synthase (NOS) inhibitor, during the conditioning. The NOS activity and dopamine (DA) turnover in the hippocampus in mice showing PCP-induced place aversion were decreased, such changes being restored by administration of L-NAME during the conditioning. On the other hand, PCP-induced place preference in mice pretreated with PCP for 28 days was not attenuated by administration of L-NAME during the conditioning. Although NOS activity was not changed, the DA turnover in the cerebral cortex was increased in mice showing PCP-induced place preference. In mice pretreated with L-NAME and PCP for 28 days before the place conditioning paradigm, PCP neither induced place preference, nor changed the NOS activity or DA turnover. These results suggest that NO is involved in the acquisition of PCP-induced aversive effects, and in the development of PCP-induced preferred effects. Further, the functional change of the DAergic neuronal system mediated by NO in the hippocampus and cerebral cortex may be necessary for the expression of aversive effects and development of preferred effects, respectively, induced by PCP.

A new biflavonoid named pancibiflavonol (1) was isolated from an EtOH extract of the stem bark of Calophyllum panciflorum, along with six known biflavonoids, and its structure was elucidated by spectroscopic methods. These biflavonoids all exhibited significant inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus early antigen activation in Raji cells.

Primer pairs and nucleic acid preparations were used with RT-PCR to detect peach latent mosaic viroid (PLMVd) from stone fruits collected across Japan. The viroid was detected from 94.3% of the peach samples, 5.3% of Japanese plum samples, 4.4% of mume samples and 3.7% of sweet cherry samples, but was not detected from European plums and apricots in local orchards in Japan. It was also detected from many peach cultivars at the National Institute of Fruit Tree Science (NIFTS), including all peach yellow mosaic- and oil blotch-infected cultivars. Symptom expression was also monitored on the RT-PCR-assayed stone fruit trees maintained at NIFTS. Only one of the peach cultivars positive for PLMVd expressed mosaic symptoms on leaves; however, the other cultivars had no symptoms. Two PLMVd isolates from peach cultivars Nakatsu Hakutou and Akatsuki in Japan were sequenced; they consisted of 337 nucleotides. The Nakatsu Hakutou isolate differed from a French isolate at 24 sites in the sequence, including 24 base exchanges, whereas the Akatsuki isolate differed at 28 sites, including 26 base exchanges, one insertion and one deletion. Two isolates showed 91 and 92% sequence homology with the PLMVd from France.