Search for the Majorana Nature of Neutrinos in the Inverted Mass Ordering Region with KamLAND-Zen.

The KamLAND-Zen experiment has provided stringent constraints on the neutrinoless double-beta (0νßß) decay half-life in ^{136}Xe using a xenon-loaded liquid scintillator. We report an improved search using an upgraded detector with almost double the amount of xenon and an ultralow radioactivity container, corresponding to an exposure of 970 kg yr of ^{136}Xe. These new data provide valuable insight into backgrounds, especially from cosmic muon spallation of xenon, and have required the use of novel background rejection techniques. We obtain a lower limit for the 0νßß decay half-life of T_{1/2}^{0ν}>2.3×10^{26} yr at 90% C.L., corresponding to upper limits on the effective Majorana neutrino mass of 36-156 meV using commonly adopted nuclear matrix element calculations.

Dextrorphan attenuates the behavioral consequences of ischemia and the biochemical consequences of anoxia: possible role of N-methyl-d-aspartate receptor antagonism and ATP replenishing action in its cerebroprotecting profile.

The acute anti-ischemic and anti-anoxic effects of dextrorphan (DX) were compared with those of dizocilpine (MK-801) in a variety of animal models, and in vivo and in vitro testings under anoxic conditions. DX reduced the incidence of death in ischemic mice and improved the rotarod performance of mice with brain ischemia. The ischemically-impaired memory of mice treated with DX markedly improved, as shown in the step-through type passive avoidance test, Morris water maze and in the habituation of exploratory behavior test. MK-801 likewise improved the water maze performance of the ischemically-impaired mice, but to a lesser extent. The step-through type passive avoidance performance of ischemic mice was not improved by MK-801. In the passive avoidance task with normal mice, DX, like MK-801, produced anterograde amnesia at doses higher than those needed to attenuate the behavioral effects of ischemia. DX, intravenously or centrally administered, markedly and dose-dependently reduced the incidence of death in mice receiving potassium cyanide (KCN). DX lessened the reduction in adenosine triphosphate (ATP) and increased lactate contents in mice dosed with KCN and also lessened the reduction in ATP in the TCA cycle and oxidative phosphorylation reactions caused by KCN (0.58 mmol/l), whereas MK-801 failed to show any effect on ATP formation pathways in vivo and in vitro, and failed to protect mice against KCN-induced lethal toxicity in vivo. In the in vitro studies, DX increased the adenylate kinase activity of the rat brain homogenate. DX was found to be a cerebroprotectant with anti-ischemic and anti-anoxic actions, the effects probably stemming from its N-methyl-d-aspartate receptor antagonistic property in cooperation with its ATP replenishing action.

Impulsivity and AMPA receptors: aniracetam ameliorates impulsive behavior induced by a blockade of AMPA receptors in rats.

The study aimed to ascertain the involvement of central AMPA receptors in impulsive behaviors of aged rats and to examine the effects of aniracetam. Premature response in the two-lever choice reaction task was assessed as an index of impulsivity. Intracerebroventricular injection of 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX), an AMPA receptor antagonist, dose-dependently (10.1-1009 ng/rat) increased only premature response without altering responding speed and choice accuracy 30 min after the injection. Aniracetam (30 mg/kg p.o.), a positive allosteric modulator of AMPA receptors, or AMPA (55.9 ng/rat, co-injected with NBQX) completely restored the NBQX-induced increase in impulsivity. These results indicate that AMPA receptors are tonically involved in the regulation of impulsivity.

Anxiolytic effects of aniracetam in three different mouse models of anxiety and the underlying mechanism.

The anxiolytic effects of aniracetam have not been proven in animals despite its clinical usefulness for post-stroke anxiety. This study, therefore, aimed to characterize the anxiolytic effects of aniracetam in different anxiety models using mice and to examine the mode of action. In a social interaction test in which all classes (serotonergic, cholinergic and dopaminergic) of compounds were effective, aniracetam (10-100 mg/kg) increased total social interaction scores (time and frequency), and the increase in the total social interaction time mainly reflected an increase in trunk sniffing and following. The anxiolytic effects were completely blocked by haloperidol and nearly completely by mecamylamine or ketanserin, suggesting an involvement of nicotinic acetylcholine, 5-HT2A and dopamine D2 receptors in the anxiolytic mechanism. Aniracetam also showed anti-anxiety effects in two other anxiety models (elevated plus-maze and conditioned fear stress tests), whereas diazepam as a positive control was anxiolytic only in the elevated plus-maze and social interaction tests. The anxiolytic effects of aniracetam in each model were mimicked by different metabolites (i.e., p-anisic acid in the elevated plus-maze test) or specific combinations of metabolites. These results indicate that aniracetam possesses a wide range of anxiolytic properties, which may be mediated by an interaction between cholinergic, dopaminergic and serotonergic systems. Thus, our findings suggest the potential usefulness of aniracetam against various types of anxiety-related disorders and social failure/impairments.

Apomorphine-induced hypoattention in rats and reversal of the choice performance impairment by aniracetam.

Aging-, disease- and medication-related imbalance of central dopaminergic neurons causes functional impairment of cognition and neuropsychological delirium in humans. We attempted to develop a new delirium model using the direct dopamine agonist, apomorphine, and a choice reaction performance task performed by middle-aged rats. The psychological properties of the model were assessed by determining behavioral measures such as choice reaction time, % correct and % omission. Apomorphine (0.03-0.3 mg/kg s.c.) produced a dose-dependent impairment of task performance. The dose of 0.1 mg/kg prolonged choice reaction time, decreased % correct and increased % omission, indicating that rats had attentional deficits and a reduced arousal or vigilance but no motor deficits or reduced food motivation. This psychological and behavioral impairment of performance resembled that of clinically defined delirium. In this model, the cholinomimetic, aniracetam (10 mg/kg p.o.), reversed the performance impairment induced by apomorphine. Its two metabolites, 2-pyrrolidinone (10 and 30 mg/kg p.o.) and N-anisoyl-gamma-aminobutyric acid (GABA, 10 mg/kg p.o.), effectively reversed the performance impairment as the intact drug did. Another pyrrolidinone derivative, nefiracetam (10 and 30 mg/kg p.o.), tended to worsen the apomorphine effect. The cholinesterase inhibitor, tacrine (10 mg/kg p.o.), markedly worsened all of the behavioral measures. Neuroleptics, haloperidol (0.025 mg/kg s.c.), tiapride (30 mg/kg p.o.) and sulpiride (10 and 30 mg/kg p.o.), antagonized the apomorphine effect. The present results suggest that apomorphine-induced behavioral disturbances in the choice reaction performance task seems to be a useful delirium model and aniracetam may improve delirium through the action of 2-pyrrolidinone and N-anisoyl-GABA, presumably by facilitating dopamine release in the striatum by acting as an AMPA or metabotropic glutamate receptor agonist.

Serotonergic mechanisms involved in the attentional and vigilance task performance of rats and the palliative action of aniracetam.

Central serotonergic systems play an important role in regulating mood/emotion, cognition, sleep and wakefulness, appetite and locomotion and body temperature via multiple receptor subtypes. Among them, 5-HT1A and 5-HT2A/2C receptors have opposite effects with respect to certain functions. The aim of the present study was to compare the effects of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a selective 5-HT1A receptor agonist, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a selective 5-HT2A/2C receptor agonist, on the performance of middle-aged rats in a two-lever choice reaction task that assessed attention and vigilance functions. We also examined the effects of aniracetam, a cognition enhancer, and its major metabolites on the induced performance impairments. 8-OH-DPAT (0.3 mg/kg s.c.) reduced response speed and choice accuracy and increased response omission with a reduction of task-associated motor activity without inducing motor inability or motivational changes. These findings indicate a specific disturbance of attentional and vigilance processes. DOI caused similar impairments at the highest dose tested (3 mg/kg s.c.); at a lower dose (1 mg/kg s.c.), however, it selectively attenuated the response speed, suggesting a selective attention deficit. (-)-Alprenolol, a non-selective 5-HT1A receptor antagonist, and ritanserin, a preferential 5-HT2A receptor antagonist, blocked the 8-OH-DPAT- and DOI-induced performance impairments respectively. Aniracetam ameliorated all the performance deficits, and the metabolites N-anisoyl-GABA and 2-pyrrolidinone partially mimicked the aniracetam effect in the 8-OHDPAT-induced attentional and vigilance impairments. Nefiracetam, another cognition enhancer, improved only the 8-OH-DPAT-induced impairments. Each compound tested alone had no effect on task performance. These results indicate that both serotonergic regulations, possibly via presynaptic 5-HT1A receptors and more likely via postsynaptic 5-HT2A receptors, lead similarly to attention deficits.

Aniracetam restores motivation reduced by satiation in a choice reaction task in aged rats.

This study aims to examine the effects of aniracetam on satiation-induced poor performance in a choice reaction task. Aged rats that mastered the task under food restriction stably maintained the task performance for a long period. Satiation by successive free feeding greatly diminished the performance. Satiation resulted in a decreased % correct, increased % omission and prolonged choice reaction time, indicating a reduction in lever response with low choice accuracy and slow responding speed. Repeated administration of aniracetam (30 mg/kg, po, for 14 days) partially recovered the choice accuracy and lever response, but not the responding speed, task-associated motor activity or impulsivity. In addition, aniracetam did not affect the animals' weights. These results indicate that satiation reduces motivation to perform and attain the task. Aniracetam may restore motivation, probably by improving poor behavioral states (daily attentional and vigilance failures), thereby creating the driving force.

Recovery of diminished mealtime-associated anticipatory behavior by aniracetam in aged rats.

Disease- or age-related neuropsychiatric symptoms and cognitive and chronobiological impairments greatly aggravate the activities of daily living (ADL) in patients. The present study evaluates the effects of aniracetam on a decline in mealtime-associated anticipatory behavior in aged rats, as an animal model of temporally regulated behaviors or habitual daily activities. Aged rats showed a lower but typical nocturnal motor activity rhythm than young rats when the animals were fed ad lib. Mealtime-associated anticipatory behavior emerged in young rats when the rats were fed at a fixed time for 6 days, but the activity in aged rats was diminished. Repeated administration of aniracetam (100 mg/kg PO) or physostigmine (0.1 mg/kg SC) for 7 days ameliorated the impaired anticipatory behavior in aged rats. Nefiracetam (10 mg/kg PO) was ineffective. All compounds tested had no effect on appetite or motor ability. These results indicate that aging disturbs the timing or temporal regulation of anticipatory behavior, probably resulting from dysfunction in a food-entrainable oscillator linked to central cholinergic systems. The restoration of the time-keeping ability by aniracetam may be mediated by the facilitation of reticulothalamic cholinergic neurotransmission, and the action may lead to the improvement of declined ADL in stroke patients.

Assembly of cardiac C-protein during myofibrillogenesis in myogenic cells in culture.

To examine the function of C-protein, a thick filament-associated protein of vertebrate striated muscles, during myofibrillogenesis, the cDNA encoding chicken cardiac C-protein and the truncated cDNA were subcloned into a expression vector and introduced into mouse C2 myogenic cells. The expression and assembly of the C-protein was investigated by immunofluorescence methods. When the cDNA containing the entire open reading frame was introduced, in C2 myoblasts, the transiently expressed exogenous cardiac C-protein existed only diffusely in the cytoplasm, but it became localized in striated structures together with sarcomeric myosin heavy chains (MHC) in myotubes. To clarify the functional domains of C-protein, the cDNA constructs that lack the regions encoding the C-terminal immunoglobulin (Ig) C2 motif or the N-terminal Ig C2 motif were introduced into C2 cells to produce mutant proteins. The truncated chicken cardiac C-protein, which lacked the C-terminal Ig C2 motif, apparently lost the ability to bind to myosin filaments; the protein was not assembled into myofibrils but diffused in the cytoplasm even in the myotubes. The protein without N-terminal Ig C2 motif, however, was assembled into sarcomeric structures just as complete protein molecules. From these results, we conclude that 1) the assembly of sarcomeric MHC into myofibrils in myotubes is accompanied with that of cardiac C-protein, and 2) the C-terminal Ig C2 motif is necessary for assembly of cardiac C-protein in sarcomeric structures in the cytoplasm.

3-O-methyldopa attenuates the effects of Madopar on the haloperidol-induced cataleptic behavior and the locomotor activity in the mouse.

The effects of Madopar (levodopa plus benserazide) on the cataleptic behavioral response to haloperidol and on the locomotor activity in mice were quantitatively compared before and after the administration of 3-O-methyldopa (3OMD). The intraperitoneal administration of 3OMD (200-400 mg/kg) alone did not modify the haloperidol (1.0 mg/kg s.c.)-induced catalepsy. Madopar, depending on the dose regimen, markedly antagonized the haloperidol-induced catalepsy. Pretreatment with 3OMD tended to reverse the antagonistic property of Madopar on the cataleptic behavior in response to haloperidol. The ability of 3OMD to significantly inhibit Madopar effects was observed in the locomotor testing paradigm; the locomotor hyperactivity in Madopar-treated animals was significantly inhibited by a prior intraperitoneal injection of 3OMD. The results from our animal experiments may provide further evidence that impediment of 3OMD formation is meaningful in the treatment of Parkinson's disease with Madopar or levodopa.

Differential expression of C-protein isoforms in developing and degenerating mouse striated muscles.

With the aim of clarifying the roles of C-protein isoforms in developing mammalian skeletal muscle, we cloned the complementary DNA (cDNAs) encoding mouse fast (F) and slow (S) skeletal muscle C-proteins and determined their entire sequences. Northern blotting with these cDNAs together with mouse cardiac (C) C-protein cDNA was performed. It revealed that in adult mice, C, F, and S isoforms are expressed in a tissue-specific fashion, although the messages for both F and S isoforms are transcribed in extensor digitorum longus muscle, which has been categorized as a fast muscle. In addition, although C isoform is expressed first and transiently during development of chicken skeletal muscles, C isoform is not expressed in mouse skeletal muscles at all through the developmental stages; S isoform is first expressed, followed by the appearance of F isoform. Finally, in dystrophic mouse skeletal muscles, the expression of S isoform is increased as it is in dystrophic chicken muscle. These observations suggest that mutations in C isoform (MyBP-C) do not lead to any disturbance in skeletal muscle, although they may lead to familial hypertrophic cardiomyopathy. We also suggest that the expression of S isoform may be stimulated in degenerating human dystrophic muscles.

Cerebral ischemia model with conscious mice. Involvement of NMDA receptor activation and derangement of learning and memory ability.

During anesthesia in mice, both common carotid arteries were tied loosely with an overhand knot suture (an occluder), while two snares (releasers) were placed in the knot so that it could be repeatedly tightened to occlude the arteries and loosened again to allow for reperfusion while the mice were conscious and unrestrained. The incidence of mortality as well as impairment of brain metabolism depended upon the length of cerebral ischemia. Cortical electroencephalogram (EEG) clearly reflected the regional ischemia as evidenced by electrical quiescence. Less mortality was observed with ischemic mice treated with dextrorphan (30 mg/kg p.o.). On day 1 (24 hr after ischemia), there were impairments in complex motor coordination, multichoice swim performance, and step-through or thermal pain-motivated avoidance responses. Thereafter, the battery of tests progressively improved. This improvement depended on the period of resumption of cerebral blood flow; the 7-day, postischemic lapse significantly reduced the deficit observed. Reduction in the degree of habituation of exploratory activity was also clearly observed following ischemic insult. Dextrorphan (1-30 mg/kg i.p.) given to ischemic mice was effective in the habituation and step-through-type passive avoidance test paradigms. In conclusion, 1) the decline in cognition as observed with ischemic mice is due to the temporal and reversible derangement of their neuronal networks; 2) excessively released glutamate is probably of major pathogenic importance in the consequences of cerebral ischemia based on the positive results of the N-methyl-D-aspartate receptor antagonist, dextrorphan; 3) the simple technique could be useful in elucidating the pathophysiologic mechanisms of ischemically elicited derangement of the cerebral organization; and 4) the model could be used to assess the efficiency of drugs with high clinical predictivity.

Immunization with the Candida albicans membrane fraction and in combination with fluconazole protects against systemic fungal infections.

We studied the immunogenicity of a membrane fraction prepared from Candida albicans cells called C. albicans membrane antigen (CMA). The present study revealed that CMA immunization has antifungal activity in mouse models of systemic fungal infection. Immunization of mice by subcutaneous injections of CMA with incomplete Freund adjuvant induced resistance to infections caused not only by C. albicans but also by Aspergillus fumigatus. The level of resistance to candidiasis was as high as that induced by whole-cell immunization. The acquired resistance to candidiasis in the mice immunized with CMA was not diminished by immunosuppressive treatment with cyclophosphamide. The level of resistance to fungal infections was superior to that given by fluconazole (FLC) treatment alone and highly enhanced by the combination with FLC. When CD4(+) cells in CMA-immunized mice were depleted by a monoclonal antibody, the antifungal activity induced by the combination of CMA and FLC was significantly reduced. These results indicate that immunization with CMA is useful for preventing systemic fungal infections and in combination with FLC for increasing resistance after infection.

CD4(+)-T-Cell-mediated resistance to systemic murine candidiasis induced by a membrane fraction of Candida albicans.

We induced resistance to systemic Candida albicans infection through CD4(+)-cell-mediated immunity in mice by immunization with subcutaneous injections of live C. albicans cells emulsified in incomplete Freund adjuvant. Using the resistant mice, we tested subcellular fractions of C. albicans cells for antigenicity. The fractions were derived from digested surface cell walls, insoluble membranes, or soluble and insoluble cytoplasmic materials, which were prepared by treatment with cell wall-digesting enzymes followed by lysis of the consequent protoplasts. Interestingly, the live-cell-immunized mice showed strong cell-mediated immune responses to the membrane fraction (C. albicans membrane antigen [CMA]). In addition, immunization with CMA induced resistance to systemic candidiasis, which disappeared upon administration of anti-CD4 monoclonal antibody. Infusion of splenocytes from the CMA-immunized mice conferred resistance on SCID mice, whereas infusion of CD4(+)-T-cell-depleted splenocytes was unable to induce resistance, indicating the importance of CD4(+) lymphocytes for resistance. These results suggest a potential for the membrane fraction to act as an antigen conferring resistance to systemic candidiasis in place of live cells and also as a source for the isolation of a new antigen.

The histidine interruption of an alpha-helical coiled coil allosterically mediates a pH-dependent ligand dissociation from macrophage scavenger receptors.

Macrophage scavenger receptors uptake modified low density lipoproteins (LDLs) through receptor-mediated endocytosis. In the alpha-helical coiled coil domain, 2 histidines (His168 and His260) disrupt leucine or isoleucine heptad repeats. Substitution of His168 or/and His260 to leucine had no effect on AcLDL binding activities. However, the His260-replaced receptors had their ligand degradation activities diminished. Cell surface ligand release experiments under acidic pH clarified that His260 mutants lost their ligand dissociation activities at 37 degrees C. Furthermore, immunoelectron microscopic experiments using anti-scavenger receptor antibody showed that the His260 replaced receptors were not able to release gold-labeled AcLDL in endosomes. Here we propose an allosterical ligand dissociation mechanism by His260 in macrophage scavenger receptors.