Hiroaki Ohya-Nishiguchi

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.

Copper biosorption by chemically treated Micrococcus luteus cells

In order to clarify the binding states of copper in microbial cells, copper biosorption from aqueous systems using the chemically treated Micrococcus luteus IAM 1056 cells (hot water-treated, diluted NaOH-treated, chloroform–methanol-treated, and chloroform–methanol/concentrated KOH-treated cells) was examined. The intact cells of M. luteus adsorbed 527 μmol of copper per g cells, and its copper adsorption was very rapid and was affected by the solution pH. The chloroform–methanol/concentrated KOH-treated cells showed higher copper biosorption capacity than the intact and the other chemically treated cells. The electron paramagnetic resonance (EPR) parameters, g∥ and |A∥|, of Cu(II) ion in microbial cells indicate that Cu(II) ion in the intact and all the chemically treated cells have coordination environments with nitrogen and oxygen as donor atoms, being similar to those of type II proteins. The parameter g∥ also indicated that the coupling between Cu(II) ion and the cell materials in the CHCl3–MeOH/concentrated KOH-treated cells is rather more stable than those between Cu(II) ion and the cell materials in the other treated cells.

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.

Direct evidence of uranium(V) intermediates by electron spin resonance in photo- and electrolytic reduction processes of uranyl complexes in organic solutions

Abstract ESR and optical spectra of pentavalent uranium have been observed in both processes of photo- and electrolytic reduction of UO2(dimethylsulphoxide)5(ClO4)2, UO2(NO3)2 · 5dimethylsulphoxide and UO2(NO3)2 · 2triethylphosphate in each corresponding organic ligand solution. The optical spectra measured in both reductions of these complexes indicate the formation of uranium(V) (λmax; 770, 970 and 1400 nm). Uranyl perchlorate shows a nearly symmetric ESR signal with g1 = 2.5 and the linewidth of 150 mT (type 1) in both photo- and electrolytic reduction processes. Nitrates show the type 1 signal in electrolytic reduction but an asymmetric signal with g1 = 1.97 (type 2) in photoreduction. These results reflect the difference in the first coordination spheres of perchlorate and nitrate complexes.

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.

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 or 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 b 558. The results indicate that cytochrome b 558 in resting neutrophils is a hexa‐coordinated ferric hemoprotein in a low‐spin state. The obtained g??? and g??? values for the hemichrome were consistent with that of bis(imiduzole)‐coordinated hemoprotein.

A possible model of hemoprotein-peroxide complexes formed in an iron-tetraphenylporphyrin system

A ferric low-spin species with an anomalously small g3-g1 separation generated by the reaction of Fe(III)tetraphenylporphyrin with t-butylhydroperoxide in the presence of tatramethylammonium hydroxide was characterized by ESR spectroscopy. The reaction kinetics, investigated by monitoring ESR intensity, indicated that this low-spin complex is highly reactive and easily changed to non-heme type iron complexes. The rhombic parameters of this complex are very similar to those of heme-peroxide adducts such as [Fe(II)-hemoglobin-O2]- and [Fe(II)-horseradish peroxidase-O2]-.

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.

Important role of Fe(III)TPP-oxygen-skatole ternary complex in tryptophan dioxygenase model reaction system

Abstract The mechanism of the dioxygenation of 3-methyl- indole occurring in the presence of Fe(III)TPPCl and alkaline reagents was studied by means of product analyses and spectroscopic measurements. The results of GC-MS measurements indicated that 3-methyl- indole (skatole) was converted to o-formamidoacetophenone (FA), in which two oxygen atoms derived from atmospheric oxygen were involved. The optical spectrum recorded for the mixture at −78°C demonstrated the presence of a new iron complex as characterized by absorption maxima at 421, 550 and 586 nm. The ESR spectrum recorded for the same reaction mixture revealed the formation of two types of ferric low-spin complexes (g1 = 2.32, g2 = 2.17, g3 = 1.95; g1 =2.24, g2 =2.16, g3 = 1.96) with anomalously small g anisotropy. From comparison of ESR parameters of the complexes with those of previously reported heme-butyl peroxide complexes, the present complexes were assumed to be the six- coordinate Fe(III)TPP-peroxide complex. Based on superhyperfine splittings (27 gauss) derived from 17O2 enriched oxygen (17O2, I= 5/2), these complexes were concluded to be the Fe(III)TPP(−OCH3)- (−OO-skatole) and Fe(III)TPP(−OO-skatole)2 complexes, having 3-methyl-3-hydroperoxo-indolenine at the axial position. The Fe(III)TPP-oxygen-substrate ternary complex will be an important intermediate species generated in the dioxygenation processes of skatole.

Proton Magnetic Resonance in Some Triarylaminium Salt Free Radicals

The static magnetic susceptibilities from 77 to 300 K and the proton NMR spectra from 1.5 to 77 K have been measured on powder samples of tris-p-chlorophenylaminium perchlorate and chloroantimonate, and tris-p-tolylaminium perchlorate and chloroantimonate. The Weiss constants have been determined to be −2.0, −40, −0.5, and −10 K respectively. A broad maximum which indicates an antiferromagnetic interaction has been observed in the electron susceptibilities of the chloroantimonates obtained from the paramagnetic shift. The magnetic behavior is interpreted assuming the linear-chain model. On the other hand, the perchlorates obeyed the Curie-Weiss law down to 1.5 K. The isotropic hyperfine coupling constants of these four aminium salts have been estimated from the proton NMR spectra, from which the cations of these crystalline aminium salts can be expected to have similar configurations.