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.

Detection of vanadyl-nitrogen interaction in organs of the vanadyl-treated rat: electron spin echo envelope modulation study.

ESEEM spectroscopy was applied for the first time to organs of an animal, viz. the kidney and liver of the rat treated with vanadyl sulfate. The aim of this study is to investigate the in vivo coordination structure of vanadyl ions administrated, and to gain information concerning the insulin-mimic activity of vanadium. ESEEM measurements for kidney and liver performed at 77 K have established nitrogen coordination to a certain percentage of vanadyl ion in the organs. The rotios of nitrogen-coordinating vanadyl ion were estimated as 70-80% in the liver, and 50-55% in the kidney. Isotropic portions of the 14N HFC were estimated as magnitude of Aiso approximately 5.0 MHz for liver, and approximately 5.2 MHz for kidney, indicating that the coordinating nitrogen is an amino nitrogen. Coordination of the Lys epsilon-amine or the N-terminal alpha-amine of a protein or (a peptide) to vanadyl ion in vivo is suggested.

Characterization of neutrophil b-type cytochrome in situ by electron paramagnetic resonance spectroscopy.

Electron paramagnetic resonance spectroscopy at 4.2 K was successfully used to characterize neutrophil b-type cytochrome in situ. The spectra of resting neutrophils taken under aerobic conditions gave a set of characteristic signals in a high magnetic field (g = 2.85, 2.21 and 1.67) beside signals for myeloperoxidase and others. From the g values, shapes and the results of other experiments, these signals were attributed to those of cytochrome b558. The results indicate that cytochrome b558 in resting neutrophils is a hexa-coordinated ferric hemoprotein in a low-spin state. The obtained gz and gx values for the hemichrome were consistent with that of bis(imidazole)-coordinated hemoprotein.

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.

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.

Purification and characterization of two types of fumarase from Escherichia coli.

Two distinct types of fumarase were purified to homogeneity from aerobically grown Escherichia coli W cells. The amino acid sequences of their NH2-terminals suggest that the two enzymes are the products of the fumA gene (FUMA) and fumC gene (FUMC), respectively. FUMA was separated from FUMC by chromatography on a Q-Sepharose column, and was further purified to homogeneity on Alkyl-Superose, Mono Q, and Superose 12 columns. FUMA is a dimer composed of identical subunits (Mr = 60,000). Although the activity of FUMA rapidly decreased during storage, reactivation was attained by anaerobic incubation with Fe2+ and thiols. Studies on the inactivation and reactivation of FUMA suggested that oxidation and the concomitant release of iron inactivated the enzyme in a reversible manner. While the inactivated FUMA was EPR-detectable, through a signal with g perpendicular = 2.02 and g = 2.00, the active enzyme was EPR-silent. These results suggested FUMA is a member of the 4Fe-4S hydratases represented by aconitase. After the separation of FUMC from FUMA, purification of the former enzyme was accomplished by chromatography on Phenyl-Superose and Matrex Gel Red A columns. FUMC was stable, Fe-independent and quite similar to mammalian fumarases in enzymatic properties.

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.

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.

In vivo longitudinally detected ESR measurements at microwave regions of 300, 700, and 900 MHz in rats treated with a nitroxide radical.

A signal detector of longitudinally detected ESR (LODESR) is independent of the resonant frequency. We developed an in vivo LODESR spectrometer operating in the regions of 300, 700, and 900 MHz. Using this apparatus, we estimated signal intensities at different operating frequencies obtained from non- or high-dielectric loss phantoms that contained nitroxide radical solutions and from live rats that had received a nitroxide radical. Our result, higher signal intensities in the high-dielectric loss samples (such as physiological saline solution and animals) at a lower frequency, shows that the influence of a decrease in dielectric loss dominates over the signal reduction caused by smaller Zeeman splitting. We believe that this finding strongly supports an in vivo ESR resonant frequency that tends to be low.

Spin-trapping and direct EPR investigations on the hepatotoxic and hepatocarcinogenic actions of luteoskyrin, an anthraquinoid mycotoxin produced by Penicillium islandicum Sopp. Generations of superoxide anion and luteoskyrin semiquinone radical in the redox systems consisted of luteoskyrin and liver NADPH- or NADH-dependent reductases.

Luteoskyrin is a hepatotoxic and hepatocarcinogenic bisdihydroanthraquinone produced by Penicillium islandicum Sopp. By observing the EPR spectra of DMPO-spin adducts and luteoskyrin semiquinone radical, we investigated in vitro whether luteoskyrin is reduced to its semiquinone radical leading to the generation of active oxygen species in redox systems catalyzed by NADPH-dependent cytochrome reductases of the liver. We found (1) the formation of luteoskyrin semiquinone radical in the NADPH-cytochrome P-450 reductase system under anaerobic conditions, (2) the generation of O2- in the systems composed of luteoskyrin, NAD(P)H, and either rat liver microsomal NADPH-cytochrome P-450 reductases or submitochondrial particles and (3) dicoumarol showed no effect on the O2- generation in the case of submitochondrial particles. From these results we proposed that luteoskyrin liver injuries are induced by the active oxygen species generated in the process of autoxidation of luteoskyrin semiquinone radical which is produced in the one-electron redox systems catalyzed by the liver NAD(P)H-dependent cytochrome reductases.

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.

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.

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.

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.

A CT using longitudinally detected ESR (LODESR-CT) of intraperitoneally injected nitroxide radical in a rat's head.

We have developed an in vivo longitudinally detected ESR (LODESR) imaging system operating at 700 MHz based on a loop-gap resonator and a pair of saddle-type pickup coils. A good linear relationship between the LODESR signal intensity and the applied power in a range up to 15.8 W was obtained. The detection of LODESR signals was barely affected by variations in the resonant properties. The characteristic of LODESR is suitable for in vivo examination. Using this system, we succeeded in obtaining LODESR-CT images of the head region of a rat after the intraperitoneal injection of a nitroxide radical.

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.

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.

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.

Effect of 'bakumondo-to', a Chinese-Japanese herbal medicine, on cultured and dispersed salivary gland cells.

'Bakumondo-to', a Chinese-Japanese herbal medicine, has been used for patients with xerostomia in Japan. Although the efficacy of this medicine for xerostomia has been reported, the pharmacological basis was only partially understood. The aim of this study was to clarify the direct effect of Bakumondo-to on salivary gland cells using isolated and cultured cells. In the physiological experiment using the fluorescent dye fura-2, Bakumondo-to showed no direct effect on isolated parotid gland cells. On the other hand, Bakumondo-to, when applied to cultured salivary gland cells, showed enhancement effects on cell proliferation. After inspection by transmission electron microscopy, we concluded that Bakumondo-to did not show an increase in the number of secretion granules but did increase the mean size of secretion granules in parotid gland cells. These mechanisms, together with other in vivo effecters, may contribute to clinical efficacy.