Continuous microwave-assisted step-by-step extraction of bioactive water-soluble materials and fucoidan from brown seaweed Undaria pinnatifida waste


Nonspecific phospholipase C3 of radish has phospholipase D activity toward glycosylinositol phosphoceramide

Glycosylinositol phosphoceramide (GIPC) is a major sphingolipid in the plasma membranes of plants. Previously, we found an enzyme activity that produces phytoceramide 1-phosphate (PC1P) by hydrolysis of the D position of GIPC in cabbage and named this activity as GIPC-phospholipase D (PLD). Here, we purified GIPC-PLD by sequential chromatography from radish roots. Peptide mass fingerprinting analysis revealed that the potential candidate for GIPC-PLD protein was nonspecific phospholipase C3 (NPC3), which has not been characterized as a PLD. The recombinant NPC3 protein obtained by heterologous expression system in Escherichia coli produced PC1P from GIPC and showed essentially the same enzymatic properties as those we characterized as GIPC-PLD in cabbage, radish and Arabidopsis thaliana. From these results, we conclude that NPC3 is one of the enzymes that degrade GIPC.


Characterization of uptake and metabolism of very long-chain fatty acids in peroxisome-deficient CHO cells

Fatty acids (FAs) longer than C20 are classified as very long-chain fatty acids (VLCFAs). Although biosynthesis and degradation of VLCFAs are important for the development and integrity of the myelin sheath, knowledge on the incorporation of extracellular VLCFAs into the cells is limited due to the experimental difficulty of solubilizing them. In this study, we found that a small amount of isopropanol solubilized VLCFAs in aqueous medium by facilitating the formation of the VLCFA/albumin complex. Using this solubilizing technique, we examined the role of the peroxisome in the uptake and metabolism of VLCFAs in Chinese hamster ovary (CHO) cells. When wild-type CHO cells were incubated with saturated VLCFAs (S-VLCFAs), such as C23:0 FA, C24:0 FA, and C26:0 FA, extensive uptake was observed. Most of the incorporated S-VLCFAs were oxidatively degraded without acylation into cellular lipids. In contrast, in peroxisome-deficient CHO cells uptake of S-VLCFAs was marginal and oxidative metabolism was not observed. Extensive uptake and acylation of monounsaturated (MU)-VLCFAs, such as C24:1 FA and C22:1 FA, were observed in both types of CHO cells. However, oxidative metabolism was evident only in wild-type cells. Similar manners of uptake and metabolism of S-VLCFAs and MU-VLCFAs were observed in IFRS1, a Schwan cell-derived cell line. These results indicate that peroxisome-deficient cells limit intracellular S-VLCFAs at a low level by halting uptake, and as a result, peroxisome-deficient cells almost completely lose the clearance ability of S-VLCFAs accumulated outside of the cells.

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids


Isolation of glycosylinositol phosphoceramide and phytoceramide 1-phosphate in plants and their chemical stabilities.

Glycosylinositol phosphoceramide (GIPC) is a sphingophospholipid in plants. Recently, we identified that GIPC is hydrolyzed to phytoceramide 1-phosphate (PC1P) by an uncharacterized phospholipase D activity following homogenization of certain plant tissues. We now developed methods for isolation of GIPC and PC1P from plant tissues and characterized their chemical stabilities. Hydrophilic solvents, namely a lower layer of a mixed solvent system consisting of isopropanol/hexane/water (55:20:25, v/v/v) was efficient solvent for extraction and eluent in column chromatography. GIPC was isolated by Sephadex column chromatography followed by TLC. A conventional method, such as the Bligh and Dyer method, was applicable for PC1P extraction. Specifically, PC1P was isolated by TLC following mild alkali treatment of lipid extracts of plants. The yields of GIPC and PC1P in our methods were both around 50-70%. We found that PC1P is tolerant against heat (up to 125 °C), strong acid (up to 10 M HCl), and mild alkali (0.1 M KOH). In contrast, significant degradation of GIPC occurred at 100 °C and 1.0 M HCl treatment, suggesting the instability of the inositol glycan moiety in these conditions. These data will be useful for further biochemical and nutritional studies on these sphingolipids.

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences


Analysis of cereal extracts as conditioning solutes to suppress the initial attachment of Escherichia coli to abiotic surfaces


Suppressive effect of edible seaweeds on SOS response of Salmonella typhimurium induced by chemical mutagens


Quantitative Analysis of Glycosylinositol Phosphoceramide and Phytoceramide 1-Phosphate in Vegetables

Previously, we found an unidentified sphingolipid in cabbage, and determined it as phytoceramide 1-phosphate (PC1P). PC1P is found to be produced from glycosylinositol phosphoceramide (GIPC) by the action of phospholipase D (PLD) activity. Although GIPC is abundant sphingolipid, especially in cruciferous vegetables, amount of daily intake, digestibility and nutritional activity of GIPC are not well understood. Here, we investigated amounts of GIPC and PC1P in vegetables. GIPC was found in all vegetables examined (13 kinds) at levels 3-20 mg/100 g (wet weight). On the other hand, PC1P was present in limited vegetables which show higher GIPC-PLD activity, such as inner cabbage leaves (5.2 mg/100 g). Because PC1P is formed during homogenization by activated GIPC-PLD, level of PC1P in boiled cabbage leaves was very low. Although digestibility of GIPC is unknown at present, a portion of dietary GIPC is considered to be converted to PC1P during mastication by plant-derived GIPC-PLD activity in some vegetables.

Journal of Nutritional Science and Vitaminology


Glycosylinositol phosphoceramide-specific phospholipase D activity catalyzes transphosphatidylation

Glycosylinositol phosphoceramide (GIPC) is the most abundant sphingolipid in plants and fungi. Recently, we detected GIPC-specific phospholipase D (GIPC-PLD) activity in plants. Here, we found that GIPC-PLD activity in young cabbage leaves catalyzes transphosphatidylation. The available alcohol for this reaction is a primary alcohol with a chain length below C4. Neither secondary alcohol, tertiary alcohol, choline, serine nor glycerol serves as an acceptor for transphosphatidylation of GIPC-PLD. We also found that cabbage GIPC-PLD prefers GIPC containing two sugars. Neither inositol phosphoceramide, mannosylinositol phosphoceramide nor GIPC with three sugar chains served as substrate. GIPC-PLD will become a useful catalyst for modification of polar head group of sphingophospholipid.


Cytometrical analysis of the adverse effects of indican, indoxyl, indigo, and indirubin on rat thymic lymphocytes

Many businesses thrive by producing health supplements from agricultural products, as exemplified by the production of functional (or health) food using plants traditionally cultivated in the rural areas. Dyes, such as indican, indigo, indoxyl, and indirubin, present in dye plants, possess antibacterial, antifungal, and antiproliferative activities. However, these effects may also lead to cytotoxicity. Thus, studies in normal mammalian cells are necessary to identify cytotoxicity and prevent adverse effects of functional foods that contain these dyes. In this study, the effects of indican, indigo, indoxyl, and indirubin were evaluated by flow cytometry using appropriate fluorescent probes in rat thymic lymphocytes. Among the dyes analyzed, indirubin exerted distinct cellular activities. Treatment with indirubin (10 30 µM) increased the population of shrunken dead cells. The side scatter, but not forward scatter, increased in indirubin-treated living cells. It increased the population of annexin V-bound living and dead cells and that of dead cells without annexin V. Indirubin elevated intracellular Ca2+, but not Zn2+ levels. The cellular content of superoxide anions and increased glutathione decreased. Indirubin depolarized the cellular plasma and mitochondrial membranes. It did not potentiate or attenuate the cytotoxicity of A23187 (Ca2+ overload) and H2O2 (oxidative stress). The results suggested that indirubin induces both apoptotic and non-apoptotic cell death. It may be difficult to predict and prevent adverse effects of indirubin due to its diverse activities on normal mammalian cells. Therefore, indirubin should be removed from products that contain dye plant extracts.


Zinc-dependent and independent actions of hydroxyhydroquinone on rat thymic lymphocytes

Coffee contains hydroxyhydroquinone (HHQ). HHQ is one of by-products released during bean roasting. Therefore, it is important to elucidate the bioactivity of HHQ to predict its beneficial or adverse effects on humans. We studied zinc-dependent and independent actions of commercially-procured synthetic HHQ in rat thymocytes using flow cytometric techniques with propidium iodide, FluoZin-3-AM, 5-chloromethylfluorescein diacetate, and annexin V-FITC. HHQ at 10¬50 µM elevated intracellular Zn2+ levels by releasing intracellular Zn2+. HHQ at 10 µM increased cellular thiol content in a Zinc-dependent manner. However, HHQ at 30 50 µM reduced cellular thiol content. Although the latter actions of HHQ (30 50 µM) were suggested to increase cell vulnerability to oxidative stress, HHQ at 0.3 100 µM significantly protected cells against oxidative stress induced by H2O2. The process of cell death induced by H2O2 was delayed by HHQ, although both H2O2 and HHQ increased the population of annexin V-positive living cells. However, HHQ at 10 30 µM promoted cell death induced by A23187, a calcium ionophore. HHQ at 10 30 µM exerted contrasting effects on cell death caused by oxidative stress and Ca2+ overload. Because HHQ is considered to possess diverse cellular actions, coffee with reduced amount of HHQ may be preferable to avoid potential adverse effects.


Determination of binding affinity of poly-gamma-glutamate to Shiga toxin


N-(3-oxododecanoyl)-L-homoserine-lactone, a quorum sensing molecule, affects cellular content of nonprotein thiol content in rat lymphocytes: Its relation with intracellular Zn2+

Cellular actions of N-(3-oxododecanoyl)-L-homoserine-lactone (ODHL), a quorum sensing molecule of bacteria, were studied on rat thymocytes using a flow cytometer with appropriate fluorescent dyes to elucidate the effects of ODHL on host cells. A bell-shaped concentration-response relation was observed in the ODHL-induced changes in cellular glutathione content ([GSH]i). ODHL concentration-dependently increased intracellular Zn2+ levels ([Zn2+]i) and cellular O2- content ([O2-]i). The bell-shaped relation induced by ODHL can be explained as follows: a low concentration of ODHL is expected to induce moderate oxidative stress that intracellularly releases Zn2+ by converting thiols to disulfides. A slight elevation of [Zn2+]i may increase the [GSH]i. On the other hand, it is likely that a high concentration of ODHL causes severe oxidative stress that further causes both the decrease in [GSH]i and the increase in [Zn2+]i. Excessive increase in [Zn2+]i may augment oxidative stress that further decreases the [GSH]i. Other notable actions induced by ODHL included the elevation of [Zn2+]i by Zn2+ influx and the increase in [GSH]i under Zn2+-free conditions. Therefore, it is suggested that ODHL elicits diverse actions on host cells.


Diverse cellular actions of tert-butylhydroquinone, a food additive, on rat thymocytes

Tertiary butylhydroquinone (TBHQ) is a food additive that possesses antioxidant activity. Its alternative applications have been explored in recent studies. However, there is controversy regarding safety. In this study using rat thymocytes, the cellular actions of TBHQ at sublethal concentrations were examined. TBHQ at concentrations of 3 µM or more elevated intracellular Zn2+ concentration ([Zn2+]i) in a dose-dependent manner, by increasing membrane Zn2+ permeability and releasing Zn2+ from cellular stores. TBHQ at 30 µM significantly increased side scatter (cell density) and the exposure of phosphatidylserine (PS) on cell membrane surfaces. It also decreased cellular glutathione (GSH) content without affecting cell lethality. Forward scatter was attenuated by 100 µM TBHQ. Thus, it is considered that TBHQ at sublethal concentrations (30 µM or less) exerts some adverse actions on cells. TBHQ at 10 30 µM attenuated the increase in cell lethality induced by hydrogen peroxide (H2O2), while potentiation of H2O2 cytotoxicity by 100 µM TBHQ was observed. The range of concentrations of TBHQ from benefit to toxicity under in vitro conditions may be 10 30 µM. Although TBHQ exhibits antioxidative actions at concentrations that are lower than those which elicit adverse cellular effects, sublethal levels of TBHQ cause some adverse actions that may be clinically concerned.


Screening and analysis of edible seaweeds in the ability to adsorb Shiga toxin.


Change in plasma membrane potential of rat thymocytes by tert-butylhydroquinone, a food additive: Possible risk on lymphocytes

Tertiary butylhydroquinone (TBHQ) is a food additive and has various beneficial actions under in vitro and in vivo experimental conditions. Therefore, it is necessary to collect additional data on the toxicity of TBHQ in order to avoid adverse effects during clinical applications. Changes in plasma membrane potential are associated with changes in physiological functions even in non-excitable cells such as lymphocytes. Thus, compounds that affect membrane potential may modify some lymphocytic functions. The effect of TBHQ on plasma membrane potential was examined in rat thymocytes using flow cytometric techniques. Treatment of rat thymocytes with TBHQ caused hyperpolarization and then depolarization. The TBHQ-induced hyperpolarization was due to the activation of Ca2+-dependent K+ channels. TBHQ elevated intracellular Ca2+ levels. The depolarization by TBHQ was caused by a nonspecific increase in membrane ionic permeability. Both the sustained depolarization and elevation of intracellular Ca2+ level by TBHQ are thought to be adverse for thymocytes because such changes disturb membrane and intracellular signaling. The thymus is most active during neonatal and pre-adolescent periods. If TBHQ exerts adverse actions on thymocytes, it may result in an immunotoxic effect in neonates and adolescents.


Adsorption of Shiga Toxin to Poly-γ Glutamate Precipitated.


Zinc increases vulnerability of rat thymic lymphocytes to arachidonic acid under in vitro conditions

Previous studies on the cytotoxicity of arachidonic acid (ARA) elucidated the involvement of oxidative stress and Ca2+. In the present study, the Zn2+-related cytotoxicity of ARA was studied by a flow cytometric technique with appropriate fluorescent probes in rat thymocytes. Addition of 10 µM ZnCl2 enhanced the increase in cell lethality induced by 10 µM ARA. The removal of Zn2+ by Zn2+ chelators attenuated the ARA-induced increase in cell lethality. Thus, Zn2+ is suggested to be involved in ARA cytotoxicity. ARA at 3 10 µM elevated intracellular Zn2+ level. The Zn2+ chelators attenuated the ARA-induced increase in intracellular Zn2+ level while ARA significantly increased intracellular Zn2+ level in the presence of 3 µM ZnCl2, suggesting the involvement of external Zn2+. Zn2+ reportedly exerts cytotoxic action under oxidative stress induced by hydrogen peroxide, via an excessive increase in intracellular Zn2+ levels. Since ARA induces oxidative stress, the simultaneous administration of zinc and ARA may be harmful.


Methyl cinnamate increases cell vulnerability to oxidative stress induced by hydrogen peroxide in rat thymocytes

Methyl cinnamate (MC) and essential oils containing MC possess beneficial antimicrobial, antifungal, and insecticidal effects, among others. Such effects are related to the biocidal action of MC. The antioxidant activity of MC has also been reported elsewhere. It has been suggested that MC may be cytotoxic to cells exposed to oxidative stress. To test this possibility, the effect of MC on rat thymocytes was examined while the cells were subjected to oxidative stress induced by hydrogen peroxide (H2O2). Flow cytometric techniques with appropriate fluorescent probes were used for quantification. MC increased cell vulnerability to oxidative stress via acceleration of the cell death process and/or potentiation of oxidative stress. The use of MC is widespread because of its beneficial actions, and thus further attention should be paid to whether MC is effective under oxidative stress.


Survey of Annual Events and Their Special Foods in Tokushima Prefecture

徳島県における行事食の現状

We carried out survey as to what people in Tokushima prefecture eat at various annual events except on New Year's Day from 2009 to 2010.The subjects of this survey were residents of Tokushima prefecture who have lived there for more than 10 years. The subjects were divided into three age groups; 1) those younger than 30 years old; 2) those between 30 and 50; and 3) those over 50.Our findings showed that eating cakes on Christmas Day and buckwheat noodles at night of the New Year's Eve is fairly common. This tendency is rampant among the people over 30 and fewer among younger generations, though the contrast is not very sharp.People over 50 who used to have a habit of cooking by themselves the above food for special events, tend to buy the ready cooked ones instead of preparing them in their houses.

日本の調理文化の地域性の特別研究のうち，平成21年から平成22年に「行事食・儀礼食」についてアンケート調査を行った．徳島県に10年以上在住する人を対象に，正月を除く年中行事について30歳未満，30歳以上50歳未満，50歳以上の年代区分に分類し検討した．人日，端午の節句，七夕，土用の丑，盂蘭盆，重陽の節句，春分の日，秋分の日，冬至，秋祭りでは，認知率および経験率の双方に年代区分で有意差がみられた．節分「巻き寿司・のり巻き」，土用の丑「うなぎの蒲焼き」は行事食としての喫食率が高かった．春分の日，秋分の日の「ご飯・だんご」，春祭り，秋祭りの「ご飯・すし」は，若い世代への伝承が薄れていることが推察された．全ての年代区分で年末のクリスマス，大みそかは行事として定着しており，「ケーキ」や「年越しそば」を多く食べていた．多くの行事食は以前には家庭で作ったが，現在は買う入手方法へ変化しており，特に50歳以上で顕著であった．


A Survey of Foods for Rites of Passage in Tokushima Prefecture

徳島県における通過儀礼食の現状


Yttrium decreases the intracellular Zn2+ concentration in rat thymocytes by attenuating a temperature-sensitive Zn2+ influx

Yttrium is used in the production of various electronic devices because the alloy it contains enhances or modifies the properties of other elements. In order to study the cytotoxic action of yttrium, the effect of yttrium chloride (YCl3) on the intracellular Zn2+ level was examined in rat thymocytes using a flow cytometer with FluoZin-3-AM and propidium iodide. The application of YCl3 significantly decreased the intensity of the FluoZin-3 fluorescence, suggesting a decrease in the intracellular Zn2+ level or quenching of the FluoZin-3 fluorescence by Y3+. However, since Y3+ did not attenuate the FluoZin-3 fluorescence under cell-free conditions, the latter suggestion was ruled out. Rat thymocytes possess a temperature-sensitive membrane pathway that carries Zn2+ into the cells. The application of YCl3 attenuated the FluoZin-3 fluorescence augmented by externally applied ZnCl2 in a concentration-dependent manner. This suggested that Y3+ inhibited the Zn2+ influx, resulting in the decrease in the intracellular Zn2+ level. Yttrium may induce dyshomeostasis of intracellular Zn2+, leading to some cytotoxic actions.

Environmental Toxicology and Pharmacology


Cytometric analysis on cytotoxicity of curcumin on rat thymocytes: Proapoptotic and antiapoptotic actions of curcumin

Curcumin exhibits various pharmacological actions including anti-inflammatory, anti-infectious, and anticancer actions. Furthermore, the supplements containing curcumin are supplied for persons consuming alcoholic beverage. A primary criterion for an ingredient ingested by general population is that it exerts no harmful effect. In this study, we examined the effect of curcumin on rat thymocytes to see if curcumin exerts cytotoxicity on normal cells. The incubation with 10 µM curcumin for 24 h increased the population of dead cells while it was not the case for 5 µM or less. Curcumin at 5-10 µM increased the populations of shrunken cells and the cells positive to annexin V, phenomena for early stage of apoptosis. However, the incubation with 10 µM curcumin suppressed the increase in population of cells with hypodiploid DNA, a phenomenon for late stage of apoptosis. Thus, curcumin at 10 µM may show both proapoptotic and antiapoptotic actions. The simultaneous incubation with 5 µM, but not 3 µM, curcumin and 0.5 % ethanol increased the population of shrunken cells. It is likely that curcumin at 5 µM or more exerts cytotoxic action on normal cells although many studies show some anticancer actions of curcumin at 10 µM or more on cancer cells.


Cytotoxic characteristics of two isomeric dimers produced by oxidation of sesamol, an antioxidant in sesame oil

Antioxidants themselves are oxidized to prevent oxidation of other molecules. One may ask if the oxidized antioxidants are safe for humans. However, there is very little information on the toxicity of oxidized antioxidants. We previously identified cytotoxic compounds in the products from oxidation of sesamol, a potent antioxidant in sesame oil. In this study using a flow cytometer with fluorescent probes, we revealed cytotoxic characteristics of two isomeric dimers (dimer A and B) in rat thymocytes. Increase in cell lethality by dimer A was more profound than those by sesamol and dimer B. The incubation of cells with dimer A increased the populations of shrunken cells and the cells with phosphatidylserine exposed on outer surface of cell membranes. Since these phenomena are parameters for early stage of apoptosis, the results indicate that dimer A may promote the process of apoptosis. However, the population of the cells containing hypodiploid DNA, a parameter for late stage of apoptosis, was decreased by the incubation with dimer A. It was not the case for dimer B. Results indicate that dimer A may inhibit the degradation of DNA during apoptosis. Taken together, it is likely that dimer A exerts both proapoptotic action and inhibitory action on late stage of apoptosis in rat thymocytes.


Attempt of Student Volunteers in the Special Needs Education

特別支援教育における学生ボランティアの活用の試み

障害を持つ児童生徒への教育は特別支援教育に移行し，学習障害， ADHD，高機能自閉症(広汎性発達障害)などの新しい障害領域が加えられた．制度的にはこのような変化があるが，学校現場の支援体制は物的環境，人的環境において整っていない．大学が県・市の教育委員会と連携し，大学生を学習支援ボランティアとして小中学校に派遣し，教員の補助的役割を担うことは，特別支援教育にとってどのような影響があるかを報告することを目的とした．県・市の教育委員会から各10名程度の大学生の募集があったので， 3・4年生を対象として募集した．募集に応じた学生は事前オリエンテーションを各教育委員会から受け，小中学校に4時間/1日，派遣された．派遣校教員と学生ボランティアへの面接，質問紙調査から「教室に支援の手と目が増えた(学習支援ボランティアがいることで子どもをみてくれる人が増え，支援してくれる人が増えた)」， 「子どもの反応から対応を考えるカがついた」などの評価が得られた．

Journal of University Education Research


Cytometric analysis of the cytotoxic action of adenosine 5'-monophosphate on rat thymocytes

The cascade of adenosine 5'-monophosphate -activated protein kinase (AMPK) is known to be a sensor of cellular energy charge. In this context, a paradigm for obesity treatment via the activation of AMPK has attracted the suppliers of complementary medicines and supplements. It is possible that products that increase the concentration of adenosine 5'-monophosphate (AMP) in the body would be efficacious in reducing body weight. However, since there is currently little information concerning the toxicity of AMP, the cytotoxic action of AMP on rat thymocytes was examined by flow cytometry. Incubation of cells with AMP at a concentration of 30 µM or more for 24 h significantly increased the populations of dead cells, shrunken cells, and cells containing hypodiploidal DNA in a concentration-dependent manner. Z-VAD-FMK, a pan-inhibitor of caspases, attenuated the AMP-induced changes in cell populations. It is concluded that AMP at a concentration of 30 µM or more exerts a cytotoxic action, which is dependent on the activation of caspases.


災害時のメニュー開発 -中国・四国支部報告-

災害時のメニュー開発 -中国・四国支部報告-


Reciprocal effects of glucose on the process of cell death induced by calcium ionophore or H2O2 in rat lymphocytes

We have examined the effects of glucose at high concentrations on the process of cell death induced by excessive increase in intracellular Ca2+ concentration ([Ca2+]i) or oxidative stress in rat lymphocytes. The cell death elicited by the excessive increase in [Ca2+]i seemed to be induced by an activation of Ca2+-dependent K+ channels because the inhibitors for Ca2+-dependent K+ channels attenuated the decrease in cell viability. Glucose at 30-50 mM augmented the decrease in cell viability by the excessive increase in [Ca2+]i. It was not specific for glucose because it was the case for sucrose or NaCl, suggesting an involvement of increased osmolarity in adverse action of glucose. On the contrary, glucose protected the cells suffering from oxidative stress induced by H2O2, one of reactive oxygen species. It was also the case for fructose or sucrose, but not for NaCl. The process of cell death induced by H2O2 started, being independent from the presence of glucose. Glucose delayed the process of cell death induced by H2O2. Sucrose and fructose also protected the cells against oxidative stress. The reactivity of sucrose to reactive oxygen species is lower than those of glucose and fructose. The order in the reactivity cannot explain the protective action of glucose. Glucose at high concentrations exerts reciprocal actions on the process of cell death induced by the oxidative stress and excessive increase in [Ca2+]i.


Increase in intracellular Cd2+ concentration of rat cerebellar granule neurons incubated with cadmium chloride: Cadmium cytotoxicity under external Ca2+-free condition

In order to examine the cadmium cytotoxicity unrelated to external Ca(2+), the effects of micromolar CdCl(2) on intracellular Cd(2+) concentration, cellular content of glutathione, and cell viability of rat cerebellar granule neurons were examined under normal Ca(2+) and external Ca(2+)-free conditions, using a laser confocal microscope with fluorescent probes, fluo-3-AM, 5-chloromethylfluorescein (CMF) diacetate, and propidium iodide. CdCl(2) (10-300muM) dose-dependently increased the intensity of fluo-3 fluorescence. Exposure to CdCl(2) equally enhanced the fluo-3 fluorescence under both Ca(2+) conditions and MnCl(2) did not quench the CdCl(2)-enhanced fluorescence. The results indicate that the enhancement of fluo-3 fluorescence is due to the increase in intracellular Cd(2+) concentration. CdCl(2) at 100-300muM decreased the intensity of CMF fluorescence, indicating the decrease in cellular content of glutathione. The population of cells stained with propidium (dead cells) was increased by 100-300muM CdCl(2). Similar results described above were also observed under external Ca(2+)-free condition. It is suggested that some of cytotoxic actions of CdCl(2) on neurons are unrelated to external Ca(2+), one of main sources for increasing intracellular Ca(2+) concentration.

カドミウム毒性はカルシウムイオンの細胞機能の阻害との関連で示唆されることが多い．そこでカルシウムイオン除去条件下で，カドミウムイオンそれ自体が細胞レベルで同様な細胞毒性を示すことを明らかにした．細胞内でカドミウムイオン濃度の上昇を画像化して，カルシウムイオンの有無に関わらず，カドミウムイオンは同程度の細胞毒性を示した．


Cytometric analysis of lidocaine-induced cytotoxicity: a model experiment using rat thymocytes

Lidocaine is a local anesthetic possessing both lipophilic and hydrophilic properties. It also acts as a surfactant. Thus, the disruption of membranes, resulting in necrosis, is one of possible mechanisms for lidocaine-induced cytotoxicity. However, lidocaine is reported to induce apoptosis. Therefore, in order to compare two mechanisms for cell death induced by lidocaine, the effects of millimolar lidocaine were examined on rat thymocytes by a flow cytometer with appropriate fluorescent probes. Lidocaine decreased the population of living cells with phosphatidylserine-exposed membranes, one of markers for early stage of apoptosis, and increased the population of dead cells without increasing that of cells with hypodiploidal DNA. Lidocaine at millimolar concentrations may deteriorate the membranes of such apoptotic living cells rather than those of intact living cells, resulting in necrosis. It is suggested that the process of apoptosis is not completed in the presence of millimolar lidocaine.

局所麻酔薬リドカインはアポトーシスあるいはネクローシスを起こすという報告がある．我々はリドカインがアポトーシス様細胞膜変化を起こした細胞に作用して，ネクローシスを起こさせることを明らかにした．よって，リドカインがアポトーシスを初期的に起こしても，最終的にはネクローシスという結果になる．


Extract of Ginkgo biloba leaves attenuates kainate-induced increase in intracellular Ca2+ concentration of rat cerebellar granule neurons

イチョウ葉エキスがグルタミン酸興奮毒性を抑制する可能性をラット脳神経細胞にて証明した．これにより，イチョウ葉エキスは認知症(痴呆症)の予防には有効である可能性が示唆された．


Cytotoxic activity of maytanprine isolated from Maytenus diversifolia in human leukemia K562 cells

We examined the cytotoxic effect of maytanprine isolated from the methanol extract of Maytenus diversifolia on human leukemia K562 cells using a flow cytometer and compared its cytotoxicity with that of maytansine, a potent cytotoxic maytansinoid. Maytanprine at concentrations of 0.03 nM or more (up to 1 nM) attenuated cell growth with decreasing cell viability and increased the population of shrunken cells in a concentration-dependent manner. Complete inhibition of growth by maytanprine was observed at concentrations of 0.3 nM or more. The compound at 0.03 nM markedly decreased the population at G0G1 phase in the cell cycle, but only slightly decreased that in the G2M phase, suggesting the possibility that it inhibits or delays cell division, and increased the population of cells with hypodiploidal DNA (apoptotic cells). The potency of maytanprine in inhibiting cell growth was greater than that of maytansine, although the inhibitory action of maytanprine was similar to that of maytansine. The results suggest that maytanprine exerts a potent inhibitory action on the growth of human leukemia K562 cells. M. diversifolia is one natural source of maytanprine, which is more cytotoxic than maytansine.


Flow cytometric estimation of cytotoxic activity of rhodexin A isolated from Rhodea japonica in human leukemia K562 cells


Cytokinesis arrest and nuclear fission in low density populations of Trichomonad Protozoan

Cell growth of anaerobic protozoan Tritrichomonas foetus was analyzed. This protozoan usually proliferates in extremely high density, but protozoan parasites were dispersed uniformly in F-bouillon medium and cell division stopped temporarily. However, nuclear fission continued and giant polynucleated cells formed. Later, cell division resumed and cells returned to normal form. In conditioned medium, cytokinesis of the dispersed parasites did not stop. Results indicated that T. foetus cells secreted an extracellular factor that influenced cytokinesis.


Tri-n-butyltin-induced decrease in cellular level of glutathione in rat thymocytes: a flow cytometric study using 5-chloromethylfluorescein diacetate


Exposure of rat thymocytes to hydrogen peroxide increases annexin-V binding to membranes: inhibitory action of deferoxamine and quercetin

過酸化水素による酸化ストレスがカルシウムイオン依存性に細胞膜にアポトーシス様変化を起こさせることを明らかにした．実験にはラット胸腺細胞を用い，フローサイトメーターによりアネキシン結合，細胞膜透過性，細胞内カルシウムイオン濃度などの測定を行った．細胞内カルシウムイオン濃度の上昇に伴い，アネキシン結合(フォスファチジルセリン露出)が認められた．これらの実験から上記の結論を得た．


Biochemical and physiological properties of pedicellarial lectins from the toxopneustid sea urchins

Pedicellarial lectins (SUL-I, SUL-II, and TGL-I) were purified from the toxopneustid sea urchins, Toxopneustes pileolus and Tripneustes gratilla using gel filtration chromatography, affinity chromatography, and reverse-phase HPLC. SUL-I (Nakagawa et al., 1996) and SUL-II from the large globiferous pedicellariae of T. pileolus are D-galactose-binding lectins with molecular masses of 32 kDa and 23 kDa, respectively; while TGL-I from the globiferous pedicellariae of T. gratilla is a Ca(2+)-independent heparin-binding lectin with a molecular mass of 23 kDa. SUL-I induced mitogenic stimulation on murine splenocytes but TGL-I did not. At higher dose ranges SUL-I exhibited inhibitory effects on the cells. The dual response to SUL-I was effectively inhibited by D-galactose. SUL-I enhanced norepinephrine-induced contraction of isolated rat mesenteric artery with endothelium. When endothelium was removed from the artery, acetylcholine did not relax the norepinephrine-induced contraction. In the same artery the enhancing effect of the contraction by SUL-I was abolished, suggesting that SUL-I acts on the endothelium of mesenteric artery, and may release prostanoids. The present results suggest an extracellular function for SUL-I that may have wide-ranging effects in physiological processes. The primary role of pedicellarial lectins from T. pileolus and T. gratilla might be defense against a foreign body.

ラッパウニの大型叉棘からゲル濾過，アフィニティークロマトグラフィー及びHPLCを用いて，D-ガラクトース特異的レクチン (SUL-I とSUL-II)ならびにシラヒゲウニの叉棘からヘパリン結合性レクチン(TGL-I)をそれぞれ単離した．SUL-I は強いマイトジェン活性を示し，ラットの血管内皮細胞に対してプロスタノイドの遊離を示唆した．TGL-I はマイトジェン活性を示さなかった．


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


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


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


イソチオシアン酸アリルとその誘導体による細菌増殖阻害

イソチオシアン酸アリルとその誘導体による細菌増殖阻害


Inhibitory effects on the growth of several bacteria by brown mustard and allyl isothiocyanate


Separation and quantitation of allyl isothiocyanate in brown mustard and cinnamaldehyde in cinnamon by reverse-phase high performance liquid chromatography


乳酸菌発酵米飼料を活用した「阿波ポーク」生産技術の開発

乳酸菌発酵米飼料を活用した「阿波ポーク」生産技術の開発

徳島県立農林水産総合技術支援センター畜産研究所研究報告

徳島県立農林水産総合技術支援センター畜産研究所研究報告


Cremophor EL modifies adriamycin-induced action on human leukemia K562 cells

Adriamycin and paclitaxel are simultaneously used for cancer treatment in some cases. The formula of paclitaxel contains cremophor EL as a solvent. Since this solvent exerts diverse biological actions, the modification of adriamycin action by cremophor EL has been studied on human leukemia K562 cells. Cremophor EL did not significantly affect the concentration-response relation for antiproliferative action of adriamycin and the cell cycle changed by adriamycin. However, the induction of morphological change induced by adriamycin was significantly augmented by cremophor EL. The simultaneous application of cremophor EL increased the intensity of fluorescence from adriamycin trapped inside the cells in a concentration-dependent manner, suggesting an increase in intracellular concentration of adriamycin by cremophor EL. Adriamycin alone at concentrations higher than those to completely inhibit the growth induced morphological change in K562 cells. Therefore, cremophor EL may potentiate some of actions induced by adriamycin when adriamycin and paclitaxel are simultaneously applied.


Micromolar zinc modifies the cell death induced by clotrimazole in human leukemia K562 cells

Our recent study showed that the simultaneous application of clotrimazole with CdCl2 or PbCl2 exerted potent cytotoxic action in rat thymocytes although respective agents were ineffective. It was also the case of ZnCl2 and clotrimazole in preliminary study using rat thymocytes. Since clotrimazole is supposed to be a candidate for anticancer drug, we examined the effects of clotrimazole, ZnCl2, and their combination on human leukemia K562 cells. The combination of clotrimazole and ZnCl2 exerted potent cytotoxic effects on the growth and lethality of K562 cells by presumably modifying the process of cell death. The result suggests the possibility that endogenous Zn2+ may modify the action of clotrimazole.


レタス種子の生育に及ぼすナトリウムの影響

レタス種子の生育に及ぼすナトリウムの影響

Natural Science Research, Faculty of Integrated Arts and Sciences, The University of Tokushima

徳島大学総合科学部自然科学研究


Further characterization of buthionine sulfoximine-induced potentiation of As2O3 cytotoxicity in human leukemia K562 cells: a flow cytometric study


Potentiation of As2O3-induced cytotoxicity by buthionine sulfoximine in human leukemia K562 cells


Effects of the agents affecting membrane potassium permeability on growth and viability in human leukemia K562 cells


Cytotoxic action of tri-n-butyltin hydride, a nucleaphilic triorganotin, on rat thymocytes: a flow cytometric study


Protective action of tri-n-butyltin on the cells suffering from oxidative strss: Its implication in bioaccumulation


心拍数を目安とした心肺機能の測定

心拍数を目安とした心肺機能の測定

徳島大学総合科学部自然科学研究

徳島大学総合科学部自然科学研究


簡易な方法で得られる体脂肪率と骨振動数の関連について

簡易な方法で得られる体脂肪率と骨振動数の関連について

徳島大学総合科学部自然科学研究

徳島大学総合科学部自然科学研究


Protection of brain neuronss suffering from oxidative stress by new curcuminoids isolated from Zingiber cassumunar


市販粉ワサビと数種化合物併用による細菌増殖阻害作用

市販粉ワサビと数種化合物併用による細菌増殖阻害作用

大阪市立大学生活科学部紀要

大阪市立大学生活科学部紀要


市販粉ワサビ，カラシ粉の細菌増殖阻害作用

市販粉ワサビ，カラシ粉の細菌増殖阻害作用

大阪市立大学生活科学部紀要

大阪市立大学生活科学部紀要


旧式焼酎の高酸度醪のモデル実験について

旧式焼酎の高酸度醪のモデル実験について

鹿児島大学農学部学術報告

鹿児島大学農学部学術報告


旧式焼酎酒母・醪から分離した乳酸菌の生態学的性質について

旧式焼酎酒母・醪から分離した乳酸菌の生態学的性質について

鹿児島大学農学部学術報告

鹿児島大学農学部学術報告


高酸度焼酎醪における乳酸菌の蛍光抗体法による検出について

高酸度焼酎醪における乳酸菌の蛍光抗体法による検出について

鹿児島大学農学部学術報告

鹿児島大学農学部学術報告


Nonivamide, a natural analog of capsaicin, affects intracellular Ca2+ level in rat thymic lymphocytes

Effect of nonivamide, a natural analog of capsaicin, on intracellular Ca2+ level of rat thymocytes was examined using a flow-cytometric technique with appropriate fluorescent probes in order to further characterize the cytotoxicity because nonivamide can be used as an active intergradient of antifouling paints. Nonivamide at concentrations ranging from 30 µM to 300 µM significantly increased the intensity of Fluo-3 fluorescence. The potency of 100 µM nonivamide to increase the fluorescence was similar to that of 100 µM capsaicin. The increase in Fluo-3 fluorescence by 100 µM nonivamide was attenuated under an external Ca2+-free condition. Nonivamide at 100 µM also increased the intensity of Fluo-3 fluorescence in the continued presence of 100 µM capsaicin. It is suggested that nonivamide at high micromolar concentrations increases intracellular Ca2+ level via the activation of vanilloid receptors. Nonivamide concentrations (30 µM or more) that increase intracellular Ca2+ level in rat thymocytes are comparable to those in algal cells. However, it is something hard to argue the implications in environmental science because nonivamide doesn't seem to be released into environment in such a high concentration, and because bioaccumulation of nonivamide has not been reported.