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T Ono

Publications and source records attributed to T Ono.

At least 397 records · Page 22Linked to original sources
PubMed1997

The calpain proteolytic system in neonatal hypoxic-ischemia.

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (HI, unilateral occlusion of the common carotid artery +7.70% O2 for 100 min) and allowed to recover for up to 14 days. Calpain caseinolytic activity was found to increase in both hemispheres for at least 20 hr. Hypoxic exposure per se increased the activity of calpains, more pronounced in a membrane-associated fraction, probably through interaction with cellular components, whereas HI introduced a loss of activity, most likely through consumption and loss of proteases. Consecutive tissue sections were stained with antibodies against calpastatin, alpha-fodrin, the 150-kDa breakdown product of alpha-fodrin (FBDP, marker of calpain proteolysis) or microtubule-associated protein 2 (MAP-2, marker of dendrosomatic neuronal injury). Areas with brain injury displayed a distinct loss of MAP-2, which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally, and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. The cytosolic fraction (CF) and the membrane and microsomal fraction (MMF) of cortical tissue were subjected to Western blotting and stained with antibodies against calpain, calpastatin and the 150-kDa breakdown product of alpha-fodrin (FBDP). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62-68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This translocation of calpains coincided with the appearance of FBDP in the ipsilateral, HI hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204-292% of controls). No significant changes in FBDP were found in the contralateral, undamaged hemisphere, despite translocation of calpains in both hemispheres, a prerequisite for calpain activation. This discrepancy may be related to changes in the endogenous inhibitor, calpastatin. Calpastatin protein was found to decrease during and shortly after HI in the ipsilateral, but not the contralateral, hemisphere. The inhibitory activity of calpastatin also tended to decrease after HI, indicating that a reduction of calpastatin may be necessary for extensive calpain activation to occur. The mRNA of m-calpain increased in the HI hemisphere 48 hr after the insult (167%, p < 0.001), a time point when the protein was also increased. In summary, our findings indicate that calpains are activated during HI and in the early phase of reperfusion after HI, preceding neuronal death.

Animals↗
PubMed1997

A simplified squalene epoxidase assay based on an HPLC separation and time-dependent UV/visible determination of squalene.

A novel and highly simplified enzyme assay for squalene epoxidase (EC 1.14.99.7) has been developed. The assay relies on the UV/visible determination of squalene at 195 nm, as it elutes from an octadecylsilane HPLC column. An acetonitrile/water (95.5/0.5, v/v) mixture was found to provide an ideal mobile phase, into which aqueous enzyme reaction mixture aliquots could be injected. Squalene, the natural substrate for squalene epoxidase, may be quantitatively determined within the concentration range 0-30 microM, with a calibration curve exhibiting an r2 (where r2 is the square of the Pearson correlation coefficient r) of 0.995. The HPLC retention time for squalene was significantly longer (> 15 min) than that for any other component required to prepare an enzyme assay reaction mixture, so facilitating its identification and quantification. In this way HPLC was used to follow enzymic squalene consumption within aliquots taken over a 30-min period. Previously reported squalene epoxidase assays rely on the radiolabeling and subsequent monitoring of squalene as it is metabolized by the enzyme. A highly simplified enzyme assay for squalene epoxidase is therefore reported.

Chromatography, High Pressure Liquid↗
PubMed1997

Protective protein in the bovine lysosomal beta-galactosidase complex.

Cathepsin A [EC 3.4.16.1], so called protective protein, occurs as an enzyme complex with lysosomal beta-galactosidase [3.2.1.23] and is involved in the stable enzymic expression of lysosomal sialidase [3.2.1.18]. In this study we investigated the enzymatic properties of cathepsin A in the bovine beta-galactosidase complex and how it is involved in the molecular multiplicities of the beta-galactosidase and sialidase complexes. Bovine protective protein homologous to the human protein had a molecular weight of 48 kDa on SDS-PAGE and cathepsin A activity optimum around pH 6.0. It hydrolyzed dipeptide substrates composed of hydrophobic amino acids much faster than any other type of substrate tested. This specificity was found to be conserved from human to a non-mammal, chicken. Immunoprecipitation using an anti beta-galactosidase antibody demonstrated that cathepsin A is a component of both the sialidase and beta-galactosidase complexes. The over 700 kDa sialidase complex depolymerized by a brief incubation at pH 7.5 and the sialidase was inactivated irreversibly via formation of an enzyme active smaller species of sialidase. The 669 kDa beta-galactosidase complex dissociated reversibly into a 120 kDa beta-galactosidase and a 170 kDa cathepsin A, but the 120 kDa beta-galactosidase, free from the cathepsin A, formed a 260 kDa aggregate under the same conditions. Inactivation of cathepsin A by heat treatment did not affect its complex forming activity. The 170 kDa protective protein dissociated into a 50 kDa one at pH 7.5, which no longer formed the complex. These findings indicate that the 170 kDa protective protein could be the minimum unit required for in vitro reconstitution of the complex, and that its complex forming activity is carried in a heat-stable domain. Both beta-galactosidase and cathepsin A activities were labile under the dissociated condition, indicating that it physiologically stabilizes not only beta-galactosidase but also itself by forming the complex.

Animals↗
PubMed1997

Localization of the squalene epoxidase gene (SQLE) to human chromosome region 8q24.1.

Squalene epoxidase (EC 1.14.99.7) catalyzes the first oxygenation step in sterol biosynthesis and is suggested to be one of the rate-limiting enzymes in this pathway. We previously isolated the mammalian squalene epoxidase cDNAs and demonstrated the transcriptional regulation of human squalene epoxidase by sterols and inhibitors. The present study was undertaken to determine the chromosomal mapping of the human squalene epoxidase gene (SQLE). PCR evidence localizes human SQLE to chromosome 8 by using the NIGMS (National Institute of General Medical Sciences) Human/Rodent Somatic Cell Hybrid Mapping Panel 2 as template. To refine the localization of human SQLE further, PCR on the Stanford G3 Radiation Hybrid Panel was performed. The result shows that human SQLE is most tightly linked to D8S508, which is reported to be located at 8q24.13-qter (lod score 7.87). Moreover, fluorescence in situ hybridization also maps human SQLE to 8q24.1.

Chromosome Mapping↗
PubMed1997

Simple detection of in vivo genotoxicity of pyrimethamine in rodents by the modified alkaline single-cell gel electrophoresis assay.

We tested the genotoxicity of pyrimethamine in 5 mouse and rat organs (liver, lung, kidney, spleen, and bone marrow) using a modified alkaline single-cell gel electrophoresis (SCG) (Comet) assay. Mice and rats were sacrificed 1, 3, 6, and 24 h after oral administration of the drug at 50 and 120 mg/kg, respectively. Nuclei were isolated from each tissue and evaluated for DNA migration. Pyrimethamine induced DNA damage in cells of the liver, kidney, and lung in both species. For mice, DNA damage persisted in the liver for 24 h, while it peaked in the lung and kidney at 6 and 24 h, respectively. For rats, DNA damage in the liver peaked at 1 h and returned to almost control level at 24 h. Genotoxicity in the spleen was only observed in mice. Our results suggest that the SCG technique, using isolated nuclei can be applied to rats and mice and that the optimal sampling time is different for different organs and species.

Animals↗
PubMed1997

Superlattice Formation on Star Polymer Solutions

Polyisoprene (PI) stars (arm number f = 4 approximately 237) were prepared by coupling of PI monoanions with tetrachlorosilane or by cross-linking PI anions with divinylbenzene. The structural ordering of such stars was investigated through small-angle X-ray scattering. PI stars (f > ca. 90) formed a body-centered cubic (bcc) structure near the overlap threshold. This structure changed to a mixed lattice of bcc and face-centered cubic structures with increasing polymer concentration.

Journal Article↗
PubMed1997

Parallel polarization electron paramagnetic resonance studies of the S1-state manganese cluster in the photosynthetic oxygen-evolving system.

Magnetic properties of the S1-state manganese cluster in the oxygen-evolving photosystem II were studied by parallel polarization electron paramagnetic resonance spectroscopy. Dark minus light spectra gave rise to a broad S1-state signal with a g value of about 4.9 [Dexheimer, S. L., Klein, M. P. (1992) J. Am. Chem. Soc. 114, 2821-2826]. Temperature variation of the signal intensity between 1.9 and 10 K observed in PS II with a sucrose buffer indicates that the signal originates from an excited state with a spin S of 1 with separation from the ground state (S = 0) of about 2.5 K. The S1-state signal was also observed in the sucrose buffer supplemented by 50% glycerol. However, no S1-state signal was detected by addition of 3% methanol or 30% ethylene glycol in the sucrose buffer, although illumination at 200 K in the presence of these alcohols induced the normal multiline S2 signal. Furthermore, modification of the Mn cluster by Cl- or Ca2+ depletion from PS II membranes failed to produce a detectable S1-state signal. A possible magnetic structure of the Mn cluster responsible for the generation of the S1-state signal is discussed on the basis of these observations.

Buffers↗
PubMed1997

Activity-dependent survival of rat cerebellar granule neurons is not associated with sustained elevation of intracellular Ca2+.

Ca2+ plays a pivotal role for the activity-dependent survival of neurons. In primary culture of cerebellar granule neurons, we found that there is no significant difference in intracellular Ca2+ level in the survival-promoting condition (cultures in the presence of 25 mM KCl) and that in the apoptosis-inducing condition (cultures in the presence of 5 mM KCl). This was not due to the inactivation of voltage-dependent L-type Ca2+ channels in the survival-promoting condition, but due to the enhanced rate of the influx and the efflux of Ca2+ in the survival-promoting condition compared to that in the apoptosis-inducing condition. These results suggest that the activity-dependent survival of the granule neurons is not associated with sustained rise of intracellular Ca2+ but associated with the enhanced turnover rate of Ca2+.

Animals↗
PubMed1997

Activity-dependent expression of parathyroid hormone-related protein (PTHrP) in rat cerebellar granule neurons. Requirement of PTHrP for the activity-dependent survival of granule neurons.

To identify genes whose expression is neuronal activity-dependent, we used an mRNA differential display technique and discovered that parathyroid hormone-related protein (PTHrP) is expressed in an activity-dependent manner in primary cultures of rat cerebellar granule neurons. PTHrP mRNA was expressed as early as 1 h by the addition of KCl to a final concentration of 25 mM to the culture medium. This expression was induced by Ca2+ influx through voltage-dependent L-type Ca2+ channels and regulated at the transcriptional step. PTHrP mRNA was persistently expressed before and after the time of commitment of granule neurons to apoptosis when they are cultured in the presence of 25 mM KCl or both 150 microM N-methyl-D-aspartic acid and 15 mM KCl, both of which promote the survival of these neurons. PTHrP was rapidly secreted into the culture medium in a depolarization-dependent manner. Parathyroid hormone/PTHrP receptor mRNA was also expressed in the primary cultures, and its expression was up-regulated by KCl and/or N-methyl-D-aspartic acid. The addition of anti-PTHrP antiserum to the culture medium resulted in a reduction of the activity-dependent survival of the granule neurons. These results suggest that PTHrP is involved in an autocrine loop and required for the survival of granule neurons.

Animals↗
PubMed1997

Two forms of avian(chicken) TATA-binding protein mRNA generated by alternative polyadenylation.

We have isolated and sequenced the cDNA encoding avian(chicken) TATA-binding protein (cTBP). The cTBP protein shows a significant homology to those of the other species, and especially its C-terminal region (180 amino acid residues) is identical to those of the vertebrates. By Northern blot analysis, we found that two transcripts with about 2.1 kb (cTBP0) and 2.7 kb (cTBP1) were expressed in various chicken tissues, though only one type of the TBP transcript was reported in vertebrates. A primer extension study demonstrated a single transcription start site. The analysis of the genomic structure of cTBP with the sequences of the two types of cTBP cDNAs(cTBP0 and cTBP1) revealed that the alternative polyadenylation generates two transcripts with different 3'untranslated regions (3'UTRs), indicating a putative role of the different 3'UTRs on the stability of cTBP mRNAs.

Amino Acid Sequence↗
PubMed1997

Single neuron responses in the monkey anterior cingulate cortex during visual discrimination.

Single neuron activity was recorded from the monkey anterior cingulate cortex during operant behavior based on discrimination of rewarding, aversive, and neutral objects. Of 550 neurons recorded, 116 responded during the task; 36, during visual discrimination; 40, during bar pressing for operant responding. Of these, 26 vision-related neurons responded differentially to rewarding, aversive and neutral objects, and 11 bar press-related neurons differentiated bar pressing to avoid shock from bar pressing to obtain reward. Responses of these neurons depended on associative meaning (aversive or rewarding) of the objects since these neuronal responses were modulated by the reversal learning. The results provide neuronal bases for involvement of the anterior cingulate cortex in emotional and motivational processes.

Animals↗
PubMed1997

El Tor hemolysin of Vibrio cholerae O1 forms channels in planar lipid bilayer membranes.

We investigated the channel formation by El Tor hemolysin (molecular mass, 65 kDa) of Vibrio cholerae O1 biotype El Tor in planar lipid bilayers. The El Tor hemolysin channel exhibited asymmetric and hyperbolic membrane current with increasing membrane potential, meaning that the channel is voltage dependent. The zero-current membrane potential measured in KCI solution showed that permeability ratio PK+/PCl- was 0.16, indicating that the channel is 6-fold more anion selective over cation. The hemolysin channel frequently flickered in the presence of divalent cations, suggesting that the channel spontaneously opens and closes. These data imply that the El Tor hemolysin damages target cells by the formation of transmembrane channels and, consequently, is the cause of osmotic cytolysis.

Bacterial Toxins↗
PubMed1997

Species-specificity of pyrimethamine in the rodent bone marrow micronucleus test.

An antiprotozoal agent pyrimethamine is a potent clastogen that induces structural chromosome aberrations and micronuclei in cultured Chinese hamster (CHL) cells in vitro. Our previous study on the compound, however, demonstrated no significant induction of micronuclei in the mouse bone marrow micronucleus test, even with dosings on 4 consecutive days. In the present study, we investigated the clastogenicity of pyrimethamine in the rat bone marrow micronucleus test. An obvious dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs) was observed when the compound was given 3 times by oral gavage at 20-120 mg/kg/day. The MNPCE frequency at the highest dose was 80 times that of the control group. Single dose of pyrimethamine at 80 and 160 mg/kg also significantly induced MNPCEs. Thus, pyrimethamine showed species-specific effects in rodent bone marrow micronucleus assays. Our finding implies that micronucleus tests using not only mouse, but also rat, may be necessary for the evaluation of clastogens detected in in vitro cytogenetic studies.

Administration, Oral↗
PubMed1997

The relationship between monkey hippocampus place-related neural activity and action in space.

To solve complex spatial problems like visual scanning and spatial navigation, animals must explore and actively sense an array of environmental stimuli. Recent studies have led to an agreement that the hippocampal formation (HF) is essential to the internal representation of spatial relation in animals. In the present study, neural activity was recorded from the HF of three monkeys, which steered a cab to various locations by pressing the appropriate bars (spatial moving task). Place-related activity of most HF neurons persisted even if the direction the monkey faced was rotated during the task. However, when the experimenter, rather than the monkey, controlled the device, the place-related neural activity of most HF neurons turned out to be obscure. The results suggest that the HF represents space effectively in situations in which the animal acts in space.

Animals↗
PubMed1997

The effect of azelastine on acute radiation dermatitis in mice models.

PURPOSE: In our previous report we described the clinical value of azelastine, an oral antiallergic agent, as an inhibitor of radiation dermatitis. Here we studied the effect of azelastine on normal skin and tumor size after irradiation in a mouse model. METHODS AND MATERIALS: The modifying effects of azelastine on both the degree of radiation dermatitis and antitumoral effect of radiation therapy were investigated in the normal skin as well as in SCC VII tumors of C3H/He mice. The right hind legs, with or without tumors, were irradiated with 20-60 Gy at 0.62 Gy/min. Azelastine was administered via the mouse chow, and acute skin reactions and tumor growth curves were compared between the azelastine and control groups. RESULTS: The acute skin reactions of the azelastine group were significantly less prominent than those of the control group (p < 0.01). At a dose of 40 Gy the dose modification factors were 1.19-1.25. The tumor growth curves of the azelastine and control groups were almost identical, indicating that the treatment response of irradiation was not affected by administration of azelastine. CONCLUSIONS: Application of azelastine reduces the degree of acute radiation dermatitis without affecting the antitumoral effect of radiation therapy.

Animals↗
PubMed1997

Granule cell disinhibition in dentate gyrus of genetically seizure susceptible El mice.

Paired-pulse inhibition was investigated electrophysiologically in the dentate gyrus using hippocampal slices from epileptic El mice. At short interpulse intervals (IPIs), the inhibition was 30% in the El, and 90% in the control ddY mice at the ages of 10 and 15 weeks. No difference in inhibition was observed at the age of 5 weeks. Bicuculline, a GABAA receptor antagonists, attenuated the inhibition during short IPIs n the ddY mice, while in the El mice, phenobarbital and flunitrazepam, which enhance GABAA receptor function, restored the inhibitory activity comparable to that of the ddY. The disinhibition progressed with growth, closely correlating with seizure development in El mice. These results suggest that decrease in the GABAergic inhibition occurs in the dentate gyrus of the El mice with growth. GABA concentration in the hippocampus was also quantified using HPLC. In El mice, GABA level was significantly lower than that in ddY mice at the ages of 5 and 15 weeks. Thus, the disinhibition observed in the El dentate gyrus at 15 weeks of age does not appear to be directly related to the content of GABA. GABAergic disinhibition suggests possible loss of unknown inhibition control factor(s) in the El dentate gyrus as growth progresses. The growth-dependent disinhibition in the granule cells may be prerequisite for epileptogenesis in El mice.

Aging↗
PubMed1997

Septal neuronal responses related to spatial representation in monkeys.

Neuronal activity in the monkey septal nuclei was recorded during performance of a place-dependent go/no-go task in which reward contingencies of the objects were variable with reference to the spatial location of a monkey's cab in one of four places in an experimental room. Of 430 septal neurons recorded, 58 responded differentially to views outside the cab at the four locations of the monkey (place-differential neurons). To investigate the possibility that an ensemble of place-differential neurons represents a space by encoding different scenes (views), responses of the 58 place-differential neurons were analyzed by multidimensional scaling (MDS). The MDS transformed relationships among the four places, expressed as correlation coefficients between all possible pairs of two places based on the 58 place-differential responses, into geometrical relationships in a two-dimensional virtual space. The four places distributed at relative positions in a two-dimensional virtual space derived from the MDS were similar to those in the real experimental room. Furthermore, these correlation coefficients derived from 58 place-differential responses significantly and negatively correlated to behavioral performance in the discrimination of the four places. The results suggest that the ensemble of place-differential responses in the septal nuclei may predict behavioral performance to discriminate places and may represent a space based on the scenes viewed from different locations.

Acoustic Stimulation↗