Biomedical subjects

L Chikahisa

Publications and source records attributed to L Chikahisa.

At least 19 recordsLinked to original sources

PubMed1998

Protective actions of 5'-n-alkylated curcumins on living cells suffering from oxidative stress.

To elucidate the roles of 5'-n-alkyl chains of curcumin derivatives in their protective actions on cells against oxidative stress, we compared the efficacy of curcumin and 5'-alkylated curcumins to inhibit the peroxidation of linoleic acid and to protect rat thymocytes suffering from H2O2-induced oxidative stress with their permeation into cells. The inhibitory action of 5'-n-alkylated curcumins on lipid peroxidation increased as the length of hydrocarbon chains of 5'-n-alkylated curcumins was prolonged. This potency order was not confirmed for the protective actions of 5'-n-alkylated curcumins on cells against oxidative stress. Among 5'-alkylated curcumins, the most potent protective action was observed for 5'-n-C3H7-curcumin because it had the greatest permeation into the cells. Further increases in the length of hydrocarbon chains (up to n-C19H39) of 5'-n-alkylated curcumins greatly attenuated their protective actions by reducing their permeation into the cells.

Animals↗

PubMed1998

Cytotoxic actions of FTY720, a novel immunosuppressant, on thymocytes and brain neurons dissociated from the rat.

Effects of FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol HCl), a novel immunosuppressant, were examined on neurons and thymocytes respectively dissociated from rat brains and thymus glands using a flow cytometer to see if FTY720 exerts cytotoxic actions not only on spleen cells as previously reported but also on the other cells. FTY720 at a concentration of 10 microM deteriorated almost all of the thymocytes, while it was not the case for brain neurons. FTY720 increased the intracellular concentration of Ca2+ ([Ca2+]i) of thymocytes in both the presence and absence of external Ca2+, although the [Ca2+]i increased by FTY720 in the presence of external Ca2+ was much greater than that in the absence of external Ca2+. Thus, FTY720 may increase the membrane permeability of Ca2+ and release Ca2+ from intracellular Ca2+ stores in thymocytes. Furthermore, the number of thymocytes stained with ethidium, a dye impermeant to intact membranes, time-dependently increased after drug application. Therefore, FTY720 at concentrations of 3 - 10 microM non-specifically increases the membrane permeability of thymocytes, resulting in necrotic cell death, although FTY720 at micromolar concentrations was reported to induce apoptosis of spleen cells.

Animals↗

PubMed1997

Flow-cytometric analysis on kainate-induced decrease in the cellular content of non-protein thiols in dissociated rat brain neurons.

In order to study the kainate-induced oxidative stress on brain neurons, the effect of kainate on cellular content of glutathione in rat cerebellar neurons were examined using a flow cytometer and 5-chloromethylfluorescein, a fluorescent dye for cellular non-protein thiols (mainly glutathione). Kainate at concentrations ranging from 30 microM to 1 mM produced a dose-dependent decrease in cellular content of glutathione. Exposure of neurons to kainate at concentrations of 300 microM or greater seemed to deplete cellular glutathione. Potency of kainate in reducing cellular content of glutathione was greater than those of glutamate and N-methyl-D-aspartate (NMDA). Kainate-induced decrease in cellular content of glutathione was partly attenuated by 6-nitro-7-cyano-quinoxaline-2,3-dione, a blocker of non-NMDA receptors and removal of external Ca2+. Results indicate that kainate causes Ca2(+)-dependent oxidative stress that decreases the cellular content of glutathione via activation of non-NMDA type of glutamate receptors.

Animals↗

PubMed1997

New curcuminoids isolated from Zingiber cassumunar protect cells suffering from oxidative stress: a flow-cytometric study using rat thymocytes and H2O2.

Effects of new complex curcuminoids (cassumunin A and cassumunin B) isolated from tropical ginger, Zingiber cassumunar, were examined in dissociated rat thymocytes suffering from oxidative stress induced by 3 mM hydrogen peroxide by using a flow cytometer and ethidium bromide. The effects were compared with those of curcumin, a natural antioxidant, whose chemical structure is included in those of cassumunins A and B. Pretreatment of rat thymocytes with the respective cassumunins at concentrations ranging from 100 nM to 3 microM dose-dependently prevented the hydrogen peroxide (H2O2)-induced decrease in cell viability. It had the same action, although less effective, against the treatment with cassumunin A or B (3 microM) immediately after or 60 min after start of the oxidative stress. Respective potencies of cassumunins A and B in protecting the cells suffering from H2O2-induced oxidative stress were greater than that of curcumin. It is suggested that cassumunins A and B may possess a potent protective action on living cells suffering from oxidative stress.

Animals↗

PubMed1997

Modulation of tumor cell motility by plasmin.

Using a pure chemotactic model, we investigated the effect of plasmin on tumor cell motility. In the presence of various extracellular matrix proteins, plasmin facilitated motility of human melanoma LOX and lung cancer Lu-99 cells. Laminin contributed most to the action of plasmin. The cell motility induced by plasmin and laminin was chemokinetic in nature and was almost completely suppressed by alpha2-antiplasmin. To further characterize the action of plasmin, various signal transduction kinase inhibitors were tried out. The results suggested that plasmin may modulate cell motility through protein kinase C and mitogen-activated protein kinase cascades in cooperation with laminin.

Cell Movement↗

PubMed1996

Flow-cytometric estimation on glutamate- and kainate-induced increases in intracellular Ca2+ of brain neurons: a technical aspect.

Effects of glutamate and kainate on the intracellular Ca2+ concentration ([Ca2+]i) in a large population (several thousand) of dissociated cerebellar granule cell neurons were evaluated using a flow-cytometer and a combination of two fluorescent dyes, fluo-3-AM for estimating [Ca2+]i and ethidium bromide for removing neurons that had compromised membranes from the cell population examined. The number of neurons responding to glutamate or kainate in augmenting the fluo-3 fluorescence increased in a dose-dependent manner. The number of neurons responding to kainate was much greater than that to glutamate. CNQX, a blocker of non-NMDA receptors, completely blocked the response elicited by kainate while the complete blockade of this glutamate-induced response was made by a combination of MK-801, a NMDA receptor blocker, and CNQX. Nicardipine, a calcium antagonist, decreased the number of neurons responding to glutamate and kainate, suggesting involvement of voltage-dependent calcium channels. These results indicate that the flow-cytometric measurement of glutamate and kainate responses has the potential to provide answers to such questions as what percentage of the population of neurons respond to these amino acids and what is the resulting distribution of [Ca2+]i.

Aniline Compounds↗

PubMed1996

Ginkgo biloba extract protects brain neurons against oxidative stress induced by hydrogen peroxide.

Effect of Ginkgo biloba extract was examined on dissociated rat cerebellar neurons suffering from oxidative stress induced by hydrogen peroxide using a flow cytometer and ethidium bromide. Hydrogen peroxide at a concentration of 3 mM increased the number of neurons stained with ethidium (presumably dead neurons) in a time-dependent manner. Pretreatment of neurons with G. biloba extract (10 micrograms/ml) greatly delayed a time-dependent increase in number of dead neurons during exposure to hydrogen peroxide. It was true, but less effective, in the case of treatment with G. biloba extract immediately or 60 min after start of oxidative stress. Results implicate G. biloba extract as a potential agent in protecting the neurons suffering from oxidative stress induced by hydrogen peroxide.

Animals↗

PubMed1996

Flow cytometric analysis of the H2O2-induced increase in intracellular Ca2+ concentration of rat thymocytes.

The effect of hydrogen peroxide (H2O2) on the intracellular Ca2+ concentration ([Ca2+]i) of rat thymocytes was examined by a flow cytometer and two fluorescent dyes, fluo-3-AM and ethidium bromide, a dye impermeant to intact membranes, to characterize the H2O2-induced increase in [Ca2+]i. H2O2 at concentrations greater than 30 microM dose-dependently increased the [Ca2+]i of thymocytes which were not stained with ethidium. Removal of external Ca2+ greatly reduced the degree of H2O2-induced increase in [Ca2+]i. However, H2O2 still increased the [Ca2+]i under the external Ca(2+)-free condition. Diethylmaleate, which is known to produce a chemical depletion of cellular nonprotein thiol, significantly increased the [Ca2+]i. Dithiothreitol, which is used to protect cellular nonprotein thiol, slightly decreased the [Ca2+]i, but greatly reduced the H2O2-induced increase in [Ca2+]i. Therefore, it is considered that H2O2 may increase the [Ca2+]i through a mechanism related to the effects of H2O2 on the cellular nonprotein thiol.

Aniline Compounds↗

PubMed1996

Fluorescent estimation of H2O2-induced changes in cell viability and cellular nonprotein thiol level of dissociated rat thymocytes.

We have developed a procedure to simultaneously estimate cell viability and the cellular level of nonprotein thiol (presumably glutathione) using two fluorescent dyes, 5-chloromethylfluorescein (5CMF) and ethidium, and rat thymocytes. Diethylmaleate and N-ethylmaleimide reduced, respectively, the intensity of 5CMF fluorescence to 0.23 and 0.1, relative to the control. Incubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, decreased the intensity of 5CMF fluorescence to 0.61. Results indicate that 5CMF fluorescence can be attenuated by agents that decrease the level of cellular nonprotein thiols, suggesting that 5CMF fluorescence is utilized for estimating the level of cellular glutathione. Hydrogen peroxide (10 microM to 3 mM) reduced the intensity of 5CMF fluorescence in a dose-dependent manner and increased the number of thymocytes stained with ethidium (presumably dead cells or cells with compromised membranes) at concentrations of 300 microM or greater. Reduction of cellular glutathione level seems to precede cell death in which oxidative stress is involved.

Animals↗

PubMed1996

Oxidative stress-induced increase in intracellular Ca2+ and Ca(2+)-induced increase in oxidative stress: an experimental model using dissociated rat brain neurons.

In order to study the oxidative stress-induced change in intracellular concentration of Ca2+ ([Ca2+]i) and Ca(2+)-induced oxidative stress, effects of hydrogen peroxide and ionomycin, a calcium ionophore, on rat cerebellar neurons were examined using a flow cytometer and fluorescent dyes: fluo-3 for monitoring [Ca2+]i; 2',7'-dichlorofluorescin, for reactive oxygen species; and 5-chloromethylfluorescein, for cellular nonprotein thiols. Oxidative stress induced by hydrogen peroxide dose-dependently increased [Ca2+]i and decreased the content of nonprotein thiols. Ionomycin increased oxidative metabolism and decreased the content of nonprotein thiols. Results suggest that oxidative stress induces an increase in [Ca2+]i while an increase in [Ca2+]i increases oxidative stress in neurons.

Aniline Compounds↗

PubMed1995

Change in membrane permeability induced by amyloid beta-protein fragment 25-35 in brain neurons dissociated from rats.

Effects of amyloid beta-protein fragment 25-35, A beta P(25-35), on the membrane permeability of organic molecules were examined in the brain neurons dissociated from rats by using an argon laser (equipped in flow cytometer and laser microscope) and a combination of two fluorescent dyes, fluo-3-AM and ethidium bromide. A beta P(25-35) at concentrations of 1 microM or greater induced both leakage of fluo-3 from the neurons and permeation of ethidium across the membrane in a dose-dependent manner, although both dyes are highly impermeant to the intact plasma membrane. Thus, A beta P(25-35) seems to increase not only membrane permeability of inorganic ions such as Ca2+, Na+ and K+, as previously suggested, but also that of organic molecules. Therefore, the brain neuron membrane is suggested to lose its integrity in the presence of A beta P(25-35) that leads to neuronal death.

Amyloid beta-Peptides↗

PubMed1994

Attenuation of Ca(2+)-induced increase in oxidative metabolism by cooling and calmodulin antagonist in mammalian brain neurons: a flow-cytometric study.

Effects of cooling and calmodulin antagonist on ionomycin-induced increase in oxidative metabolism (or formation of reactive oxygen species) of rat cerebellar neurons was examined using a flow cytometer and 2',7'-dichlorofluorescin diacetate, a fluorescent dye for intracellular hydrogen peroxide. Cooling neurons to temperatures below 16 degrees C greatly attenuated ionomycin-induced augmentation of oxidative metabolism without affecting the Ca2+ influx produced by ionomycin. Rewarming neurons to 36 degrees C in presence of ionomycin increased the oxidative metabolism, indicating a temperature-sensitive metabolic process. Substitution of Ca2+ with Ba2+ or Sr2+ completely abolished an ionomycin-induced increase in the oxidative metabolism. Pretreatment with W-7, a calmodulin antagonist, at concentrations of 10 microM or higher (up to 100 microM) produced a dose-dependent attenuation of ionomycin-induced increase in oxidative metabolism. Results suggest that calmodulin is involved in the ionomycin-induced increase in oxidative metabolism of dissociated cerebellar neurons.

Animals↗

PubMed1994

Flow cytometric analysis on cytotoxic action of amyloid beta protein fragment 25-35 on brain neurons dissociated from the rats.

Effects of amyloid beta protein fragment 25-35, A beta P(25-35), on membrane permeability and cell viability were examined in the brain neurons dissociated from the rats using a flow cytometer and two fluorescent dyes, fluo-3 to monitor intracellular Ca2+ concentration ([Ca2+]i) of neurons and ethidium which is impermeant to membranes of intact neurons to stain dead and dying neurons. A beta P(25-35) augmented fluo-3 fluorescence of some neurons at concentrations greater than 1 microM, indicating an increase in [Ca2+]i although other neurons (about 80% of total neurons) did not respond to A beta P(25-35) even at 10 microM. A beta P(25-35) at 1 microM or greater increased dose-dependently the number of ethidium-stained neurons, suggesting a dose-dependent increase in number of dead and dying neurons. Results suggest that A beta P(25-35) increases the membrane permeability of brain neurons, resulting in a destabilized intracellular homeostasis that leads to neuonal death.

Amyloid beta-Peptides↗

PubMed1994

Methylmercury-induced augmentation of oxidative metabolism in cerebellar neurons dissociated from the rats: its dependence on intracellular Ca2+.

Effect of methylmercury chloride on oxidative metabolism of cerebellar neurons dissociated from the rats was examined using 2',7'-dichlorofluorescin (DCFH) which is oxidized by cellular hydrogen peroxide to be a fluorescent compound (DCF) and fluo-3, an indicator for intracellular Ca2+ concentration ([Ca2+]i). Methylmercury at 1 microM or less did not affect DCF fluorescence of cerebellar neurons. Further increase in concentration of methylmercury (up to 30 microM) induced changes in DCF fluorescence. Thus, DCF fluorescence was slightly attenuated during 5 min after applying methylmercury to the neurons, indicating a decrease in oxidation of DCFH. Thereafter, DCF fluorescence was time-dependently augmented in continued presence of methylmercury, indicating an increase in DCFH oxidation. Although methylmercury-induced augmentation of DCF fluorescence was greatly suppressed under external Ca(2+)-free condition, it was not the case for methylmercury-induced attenuation of DCF fluorescence. Methylmercury at 3 microM or more dose-dependently increased the [Ca2+]i. Results suggest that methylmercury increases intracellular Ca2+ in cerebellar neurons, resulting in an increase in formation of reactive oxygen species that may contribute to cell injury.

Aniline Compounds↗

PubMed1994

Effect of N,N-diethyldithiocarbamate on ionomycin-induced increase in oxidation of cellular 2',7'-dichlorofluorescin in dissociated cerebellar neurons.

Effect of N,N-diethyldithiocarbamate (DDC), an inhibitor for cytosolic superoxide dismutase, on an ionomycin-induced increase in oxidative metabolism was examined in cerebellar neurons dissociated from the rats, using a flow cytometer and 2',7'-dichlorofluorescin diacetate and fluo-3-AM, fluorescent dyes for intracellular hydrogen peroxide and Ca2+, respectively. DDC reduced the ionomycin-induced augmentation of 2',7'-dichlorofluorescin fluorescence in a dose-dependent manner. DDC did not affect cellular content of 2',7'-dichlorofluorescin and ionomycin-induced increase in intracellular Ca2+ concentration. Results indicate that ionomycin increases the formation of superoxide anion in brain neuron.

Aniline Compounds↗

PubMed1994

Effect of tri-n-butyltin on intracellular Ca2+ concentration of mouse thymocytes under Ca(2+)-free condition.

Effect of tri-n-butyltin at concentrations ranging from 100 nM to 1 microM on the intracellular Ca2+ concentration of mouse thymocytes was examined under Ca(2+)-free conditions in comparison with those of 50 nM A23187, 100 nM thapsigargin and 10 microM cyclopiazonic acid, using the fluorescent dye for intracellular Ca2+, fluo-3. Tri-n-butyltin persistently increased the intensity of fluo-3 fluorescence while A23187, thapsigargin and cyclopiazonic acid produced a transient augmentation of the fluorescence. Pretreatment with A23187 greatly decreased the fluorescence responses induced by 1 microM tri-n-butyltin. However, the effect of thapsigargin and cyclopiazonic acid on the tri-n-butyltin-induced response was much weaker than that of A23187. In the presence of tri-n-butyltin, the transient response produced by A23187 was greatly prolonged. Results may suggest that tri-n-butyltin increases the membrane Ca2+ permeability of the intracellular organelles (cellular calcium stores) and decreases the Ca2+ pump activity of thymocyte membrane, resulting in a sustained increase in the intracellular Ca2+ concentration under Ca(2+)-free concentration.

Aniline Compounds↗

PubMed1993

Fluorescent estimation on the effect of Ca2+ antagonists on the oxidative metabolism in dissociated mammalian brain neurons.

Effect of organic Ca2+ antagonists on the oxidative metabolism or the formation of reactive oxygen species was estimated on dissociated mammalian neurons using a flow cytometer and 2',7'-dichlorofluorescin which is oxidized to be fluorescent by cellular oxidants. Of the organic Ca2+ antagonists used, flunarizine and nifedipine decreased the intensity of fluorescence at 1 microM or more while it was not the case for verapamil and diltiazem, suggesting one of the favourable actions of flunarizine and nifedipine on ischemic brain damage.

Animals↗

PubMed1992

Tri-n-butyltin increases intracellular Ca2+ in mouse thymocytes: a flow-cytometric study using fluorescent dyes for membrane potential and intracellular Ca2+.

Effects of tri-n-butyltin (TBT) on mouse thymocytes were examined using a flow-cytometer and fluorescent dyes for membrane potential and intracellular Ca2+ ([Ca2+]i). TBT at concentrations from 1 x 10(-7) M to 3 x 10(-7) M caused hyperpolarization in thymocytes during 30 min. after drug application in a time- and dose-dependent manner. Further increase in TBT concentration (to 1 x 10(-6) M) made hyperpolarization of thymocytes more profound within 5 min. after application, thereafter gradually depolarized them during the next 25 min. TBT at 3 x 10(-8) M or more (up to 1 x 10(-6) M) increased the [Ca2+]i of thymocytes. After reaching maximum [Ca2+]i at the various TBT concentrations used within 5 min. after drug application, the [Ca2+]i slightly decreased in a time-dependent manner. Effects of TBT on membrane potential and the [Ca2+]i were greatly reduced under nominal external Ca(2+)-free condition. Results suggest that TBT can promote Ca(2+)-influx to thymocytes, resulting in hyperpolarization by activation of Ca(2+)-dependent K+ current. The increase in [Ca2+]i by TBT may be related to its cytotoxic action on thymocytes.

Animals↗