New constraint on the existence of the µ+ → e+ γ decay.

The analysis of a combined data set, totaling 3.6 × 10(14) stopped muons on target, in the search for the lepton flavor violating decay µ(+) → e(+)γ is presented. The data collected by the MEG experiment at the Paul Scherrer Institut show no excess of events compared to background expectations and yield a new upper limit on the branching ratio of this decay of 5.7 × 10(-13) (90% confidence level). This represents a four times more stringent limit than the previous world best limit set by MEG.

New limit on the lepton-flavor-violating decay µ+→e+γ.

We present a new result based on an analysis of the data collected by the MEG detector at the Paul Scherrer Institut in 2009 and 2010, in search of the lepton-flavor-violating decay µ(+)e(+)γ. The likelihood analysis of the combined data sample, which corresponds to a total of 1.8×10(14) muon decays, gives a 90% C.L. upper limit of 2.4×10(-12) on the branching ratio of the µ(+)→e(+)γ decay, constituting the most stringent limit on the existence of this decay to date.

Transplantation of cultured salivary gland cells into an atrophic salivary gland.

Patients with dry mouth have been treated with salivary substitutes and/or medications such as pilocarpine or cevimeline hydrochloride. These treatments temporarily relieve their symptoms and induce salivation from residual tissue. However, no treatment is available for the purpose of regenerating an atrophic gland. In this study, the feasibility of a cell transplantation therapy for the atrophic submandibular glands was investigated in rats. Further, the potential of cell differentiation into a useful phenotype was assessed by immunohistochemistry together with cell tracking with the fluorescent dye PKH 26. Rat submandibular glands were excised, and the salivary gland epithelial cells were cultured for 3 weeks with 3T3 cells as a feeder layer. Ductal ligation of the submandibular gland was employed to generate an atrophic gland. One week after the operation, the ligation was removed, and the cultured cells labeled with PKH 26 were injected into the atrophic submandibular glands. As a control, the cultured cells were also injected into normal submandibular glands. Two weeks after cell transplantation, the transplanted cells were detectable in both the experimental and control groups. The cells were clustered in the connective tissue between the lobules. Four weeks after transplantation, the labeled cells were detectable in the experimental group but not in the control group. In the atrophic glands, the scattered transplanted cells were observed over a broad area of the gland but localized mainly around the acini and ductal region. Immunostaining results showed a possible involvement of the transplanted cells in ductal regeneration, while neither myoepithelial nor acinar differentiations were observed within the 4 weeks since transplantation. This study demonstrated that cell transplantation to the salivary gland is feasible, and that the transplanted cells were selectively attracted to and remained in the damaged area without affecting normal tissue.

Mapping peroxidase in plant tissues by scanning electrochemical microscopy.

Scanning electrochemical microscopy has been firstly used to map the enzymatic activity in natural plant tissues. The peroxidase (POD) was maintained in its original state in the celery (Apium graveolens L.) tissues and electrochemically visualized under its native environment. Ferrocenemethanol (FMA) was selected as a mediator to probe the POD in celery tissues based on the fact that POD catalyzed the oxidation of FMA by H(2)O(2) to increase FMA(+) concentration. Two-dimensional reduction current profiles for FMA(+) produced images indicating the distribution and activity of the POD at the surface of the celery tissues. These images showed that the POD was widely distributed in the celery tissues, and larger amounts were found in some special regions such as the center of celery stem and around some vascular bundles.

Nonstoichiometric La(2 - x)GeO(5 - delta) monoclinic oxide as a new fast oxide ion conductor.

Oxide ion conductivity in La(2)GeO(5)-based oxide was investigated and it was found that La-deficient La(2)GeO(5) exhibits oxide ion conductivity over a wide range of oxygen partial pressure. The crystal structure of La(2)GeO(5) was estimated to be monoclinic with P2(1)/c space group. Conductivity increased with increasing the amount of La deficiency and the maximum value was attained at x = 0.39 in La(2 - x)GeO(5 - delta). The oxide ion transport number in La(2)GeO(5)-based oxide was estimated to be unity by the electromotive force measurement in H(2)-O(2) and N(2)-O(2) gas concentration cells. At a temperature higher than 1000 K, the oxide ion conductivity of La(1.61)GeO(5 - delta) was almost the same as that of La(0.9)Sr(0.1)Ga(0.8)Mg(0.2)O(3 - delta) or Ce(0.85)Gd(0.15)O(2 - delta), which are well-known fast oxide ion conductors. On the other hand, a change in the activation energy for oxide ion conductivity was observed at 973 K, and at intermediate temperature, the oxide ion conductivity of La(1.61)GeO(5 - delta) became much smaller than that of these well-known fast oxide ion conductors. The change in the activation energy of the oxide ion conductivity seems to be caused by a change in the local oxygen vacancy structure. However, doping a small amount of Sr for La in La(2)GeO(5) was effective to stabilize the high-temperature crystal structure to low temperature. Consequently, doping a small amount of Sr increases the oxide ion conductivity of La(2)GeO(5)-based oxide at low temperature.

Collapse of extracellular glutamate regulation during epileptogenesis: down-regulation and functional failure of glutamate transporter function in rats with chronic seizures induced by kainic acid.

We used northern and western blotting to measure the quantity of glutamate and GABA transporters mRNA and their proteins within the hippocampal tissue of rats with epileptogenesis. Chronic seizures were induced by amygdalar injection of kainic acid 60 days before death. We found that expression of the mRNA and protein of the glial glutamate transporters GLAST and GLT-1 were down-regulated in the kainic acid-administered group. In contrast, EAAC-1 and GAT-3 mRNA and their proteins were increased, while GAT-1 mRNA and protein were not changed. We performed in vivo microdialysis in the freely moving state. During the interictal state, the extracellular glutamate concentration was increased, whereas the GABA level was decreased in the kainic acid group. Following potassium-induced depolarization, glutamate overflow was higher and the recovery time to the basal release was prolonged in the kainic acid group relative to controls. Our data suggest that epileptogenesis in rats with kainic acid-induced chronic seizures is associated with the collapse of extracellular glutamate regulation caused by both molecular down-regulation and functional failure of glutamate transport.

In vivo evaluation of hippocampal anti-oxidant ability of zonisamide in rats.

We evaluated the anti-oxidant property of zonisamide (ZNS) in the rat brain under freely moving conditions by means of in vivo microdialysis of two exogenous nitroxide radicals, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) and 3-methoxy carbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM). Time-dependent changes in the signal intensities of these exogenous nitroxide radicals obtained from the hippocampal perfusates were observed using an X-band ESR spectrometer at 20-min intervals. The ESR signal intensities of nitroxide radicals decreased exponentially in all animals, which indicates that their half-life could be used as a parameter to estimate the decay rate of nitroxide radicals. Nitroxide radicals lose their paramagnetism when exposed to reductants in a biological system. Thus, half-life reflects the in vivo reducing ability. Although the half-life of carbamoyl-PROXYL, which could not pass the blood-brain barrier (BBB), was not changed when compared with the controls, pre-treatment with ZNS significantly shortened the half-life of PCAM, which could pass through the BBB. These findings suggest that the ZNS-induced increase in reducing ability did not occur within the extracellular space, but rather mainly at the neural cell membrane. This study is the first in vivo evaluation of the reducing ability of ZNS in freely moving animals.

In vivo electron spin resonance spectroscopy on signal decay of intrastriatal nitroxide radical after acute administration of haloperidol in rats.

Sequential changes in the electron spin resonance (ESR) signal intensity of nitroxide radical perfused in the striatum of rats treated with haloperidol (HPD) were evaluated using a 700-MHz ESR spectrometer. Nitroxide radical was perfused in the striatum by in vivo microdialysis. Nitroxide used was 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl. Following 6-h perfusion of the nitroxide radical by dialysis at the rate of 2 microl/min through the radical introducer that had been stereotaxically implanted in the rat's striatum, HPD or saline was injected intraperitoneally into the rats in the resonator. The sequential changes in the ESR spectrum of the nitroxide radical were then evaluated. Spectra were successively observed in all animals. The half-life, which was estimated on the basis of the exponential decay in signal intensity, was used as a parameter of decay rate of the ESR signal intensity of nitroxide radical. The half-life in the rats injected with HPD was significantly longer than that in controls. This finding suggests that the reducing ability of the striatal extracellular space of a rat acutely treated with HPD was decreased in comparison with that of the control.

Structure, expression, and chromosome mapping of LATS2, a mammalian homologue of the Drosophila tumor suppressor gene lats/warts.

We have cloned and characterized LATS2, a novel mammalian homologue of the Drosophila tumor suppressor gene lats/warts. Northern blot analysis showed ubiquitous expression of mouse LATS2 (MmLATS2) mRNA, whereas expression of human LATS2 (HsLATS2) mRNA was enhanced in skeletal muscle and heart. Immunoblotting analysis of fractionated cell lysates showed HsLats2 to be a nuclear protein. We mapped the MmLATS2 gene to mouse chromosome 14 by interspecific backcross analysis. We also mapped the HsLATS2 gene (by fluorescence in situ hybridization) to the 13q11-q12 region, in which a loss of heterozygosity has been frequently observed in many primary cancers and to which the tumor suppressor genes RB and BRCA2 have also been mapped.

EPR imaging for in vivo analysis of the half-life of a nitroxide radical in the hippocampus and cerebral cortex of rats after epileptic seizures.

Recently, we developed an in vivo temporal electron paramagnetic resonance (EPR) imaging technique to be applied to the brain of a rat, into which a blood-brain barrier (BBB)-permeable nitroxide radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) was injected intraperitoneally. This imaging technique made it possible to measure decay rates of a nitroxide radical in multiple regions of the brain simultaneously. Using this technique, the half-life of PCAM was estimated from the exponential decay of the signal intensity derived from the temporal EPR images in the hippocampus and cerebral cortex of rats after a kainic acid (KA)-induced seizure. The hippocampal half-life of PCAM after KA-induced seizures was significantly prolonged (p < .01), whereas the prolongation of the cortical half-life was not significant. These findings suggest that following a KA-seizure, the intrahippocampal ability to reduce the nitroxide radical is impaired, but the ability is intact in the cerebral cortex. This is the first in vivo quantitative EPR imaging study that has a bearing on the pathogenesis of KA-induced seizures in the brain.

ESR spectroscopy for analysis of hippocampal elimination of a nitroxide radical during kainic acid-induced seizure in rats.

To analyze the balance between free radical production and elimination during seizure, techniques were developed to monitor the sequential changes in the level of an exogenous free radical by ESR in the brains of freely moving rats given kainic acid (KA) to induce seizures (n = 6) and control rats given saline (n = 6). The hippocampus of each rat was perfused with a nitroxide radical solution for 120 min by in vivo microdialysis. Then an intraperitoneal injection of KA or the physiological saline was given to each rat, and the perfusate was changed to Ringer's solution. ESR analysis of sequential samples of dialysate taken after injection revealed an exponential decay in signal amplitude. The median half-life of the nitroxide radical was significantly longer in the KA-treated group than in the control group (P < 0.01). This finding demonstrates a decreased capacity of the hippocampus to eliminate the nitroxide radical from the extracellular space during seizure and may help clarify the mechanism of neuronal cell vulnerability to free radicals during insult or degeneration.

In vivo analysis of hydrogen peroxide and lipid radicals in the striatum of rats under long-term administration of a neuroleptic.

It has been hypothesized that free radicals play a causative role in tardive dyskinesia, which is an inveterate movement disorder caused by chronic administration of neuroleptics. To verify this hypothesis, rats were reared while being regularly treated with water containing a neuroleptic, haloperidol (HPD), for 1 year (HPD group). The changes in the striatal hydrogen peroxide content of the rats in the HPD and control groups were measured by using a Pt-disk microelectrode while the animals were in a freely moving state following intraperitoneal administration of HPD (HPD challenge). We also performed electron spin resonance (ESR) detection of lipid radicals in the striatum before the HPD challenge. HPD challenge led to significant elevation of the intrastriatal hydrogen peroxide in all animals, but the elevation in the HPD group was smaller than that in the control group. However, in the HPD group, marked ESR signals of intrastriatal lipid radicals were observed. We think that these results support the hypothesis on the role of free radicals in tardive dyskinesia.

In vivo 300 MHz longitudinally detected ESR-CT imaging in the head of a rat treated with a nitroxide radical.

In vivo longitudinally detected ESR (LODESR)-CT system operating at 300 MHz was developed, based on the combination of a bridged loop-gap resonator and a pair of saddle-type pickup coils (STPCs). The sensitivity distribution of the STPCs was simulated on the basis of a magnetic dipole model. A LODESR-CT image of a phantom (a physiological saline solution of a nitroxide radical) was satisfactorily corrected by using the simulation result. LODESR-CT images of the head of a rats, in which nitroxide radical was injected intraperitonally, were also obtained through the correction in the same manner. This is the first in vivo CT (i.e. three-dimensional imaging) study with a 300 MHz ESR.

Changes in intracellular Ca2+ concentration produced by the alteration of intracellular pH in rat parotid acinar cells.

The effects of intracellular pH (pH(i)) on intracellular Ca2+ concentration ([Ca2+]i) vary in different cells, and mechanisms underlying these effects are still not clear. In the experiments reported here, the effects of changes in pH(i) produced by ammonium chloride and butyric acid were studied in enzymatically dispersed acinar cells of rat parotid glands. The changes in pH(i) and [Ca2+]i were estimated using the fluorescent dyes biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and fura-2, respectively. pH(i) was altered using NH4Cl, butyric acid, or propionic acid while keeping the external pH constant at 7.4. NH4Cl (20 mM) applied for 4-5 min increased pH(i) from 7.18 to 7.79 (a decrease of proton concentration, [H+]i, from 66 to 16 nM) and produced a transient [Ca2+]i increase followed by a small sustained decrease. On the other hand, butyric acid (20 mM) decreased pH(i) from 7.16 to 6.81 (an increase of [H+]i from 69 to 155 nM) and produced a small sustained increase in [Ca2+]i. Washing out the butyric acid 4 min after application induced the recovery of pH(i) from 6.93 to 7.43 (a decrease of [H+]i from 118 to 37 nM) and a further transient increase in [Ca2+]i. The removal of external Ca2+ had little effect on changes in pH(i) produced by NH4Cl or butyric acid, but markedly reduced both the sustained and transient components of [Ca2+]i response. Cyclopiazonic acid (0.3 microM), an inhibitor of Ca2+ pump in intracellular stores, abolished the transient [Ca2+]i increase produced by the application of NH4Cl or withdrawal of butyric acid. These results suggest that a decrease in [H+]i, not the absolute level of [H+]i may release Ca2+ from intracellular stores.

Temporal brain imaging by a rapid scan ESR-CT system in rats receiving intraperitoneal injection of a methyl ester nitroxide radical.

We performed in vivo ESR-CT (electron spin resonance-computed tomography) on rats' heads, in which the blood-brain barrier-permeable nitroxide radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) was injected intraperitoneally, using a rapid scan ESR-CT system operating at 700 MHz. In a spatiotemporal study we found that different regions of the brain showed differences in the rate of decay of the radical. Repeated injection of PCAM gave clear ESR-CT images of the brain. We think that the present method is useful for evaluating the capacity to eliminate exogenous free radicals in some parts of the brain.

In vivo ESR-CT imaging of the liver in mice receiving subcutaneous injection of nitric oxide-bound iron complex.

ESR measurements and ESR-CT imaging of subcutaneously (SC) injected nitric oxide (NO)-bound iron complexes were conducted on the upper abdomen of live mice. The use of NO-bound iron complex with N-(dithiocarboxy)sarcosine resulted in a clear ESR-CT image showing high intensity areas in the ventral regions, while other NO-bound iron complexes with N-methyl-D-glucamine dithiocarbamate or N,N-diethyl-dithiocarbamate were inappropriate because of low S/N ratios. To investigate the distributions of SC injected NO-bound iron complexes in the abdomens of mice, we measured ESR signals in resected abdominal organs. The signal amplitude was higher in the resected liver than in the resected gastrointestinal organs or the blood samples. The findings suggest that the high intensity areas in the ESR-CT images thus obtained correspond to the liver.

ESR imaging of the rat brain with a nitroxide radical perfused by in vivo microdialysis.

We report here our investigation of the spatial distribution of free radicals using an electron spin resonance (ESR)-imaging system combined with an in vivo brain microdialysis method, which was performed in the resonator of the ESR-imaging system. A nonmagnetic cannula, newly developed in this study, was used for the perfusion of the exogenous free radicals agent. A nitroxide, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl PROYXL), was used as the imaging agent in saline solution at a concentration of 0.3 M, which was perfused into the right caudate putamen of the rat at 2 microliters/min by a microinfusion pump. Two-dimensional ESR projection of the Z-X plane, which was clearly distinguished (about phi 10 mm) from the nonperfused brain area, was obtained 6 h after the beginning of perfusion of carbamoyl PROXYL. The present method is considered to be a useful tool to introduce stable free radicals into a specific area of the brain.

Hydrogen peroxide augmentation in a rat striatum after methamphetamine injection as monitored in vivo by a Pt-disk microelectrode.

We fabricated a Pt-disk microelectrode (diameter 30 microns) to conduct differential double-pulse amperometry (first step: 750 mV, 1 s; second step: 1,100 mV, 1 s) to detect hydrogen peroxide in the brain of a freely moving animal. This measurement determined hydrogen peroxide (detection limit, 0.03 microM) without any observable influence from other oxidizable species, such as dopamine (DA), ascorbic acid, or uric acid. The electrode was implanted into the right striatum of a rat. After intraperitoneal injection of methamphetamine (MAP), hydrogen peroxide concentrations were directly detected while the behavioral changes were monitored. MAP injection led to significant augmentation of hydrogen peroxide, the elevation of which depended on the dose of MAP. This is consistent with a previous report on the increase of DA-release caused by amphetamines and indirect evidence of the production of hydrogen peroxide via DA-metabolism.

Generation of lipid radicals in the hippocampal extracellular space during kainic acid-induced seizures in rats.

We report direct electron spin resonance (ESR) evidence of extracellular free radical formation during kainic acid-induced seizures obtained using in vivo brain microdialysis in freely moving rats. Saline solution containing the spin trap agent alpha-(4-pyridyl-N-oxide)-N-tert-butylnitrone was perfused through the hippocampus. ESR analysis of the dialysate samples revealed a six-line spectra, for which the hyperfine coupling constants corresponded to those of the ESR signal from the lipoxygenase/linoleic acid system, a lipid radical generating system. This result is direct evidence that lipid peroxidation of the neuronal membrane progresses during seizure activity. Increased formation of lipid radicals may participate in the cascade of reactions leading to neuronal damage in the hippocampus following kainic acid-induced seizure activity.