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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.

Staphylococcus aureus (S. aureus) commonly colonizes the human skin and nostrils. However, it is also associated with a wide variety of diseases. S. aureus is frequently isolated from the skin of patients with atopic dermatitis (AD), and is linked to increased disease severity. S. aureus impairs the skin barrier and triggers inflammation through the secretion of various virulence factors. S. aureus secretes phosphatidylinositol-specific phospholipase C (PI-PLC), which hydrolyses phosphatidylinositol and cleaves glycosylphosphatidylinositol-anchored proteins. However, the role of S. aureus PI-PLC in the pathogenesis of skin diseases, including AD, remains unclear. In this study, we sought to determine the role of S. aureus PI-PLC in the pathogenesis of skin diseases. PI-PLC was observed to enhance the invasion and persistence of S. aureus in keratinocytes. Besides, PI-PLC promoted the penetration of S. aureus through the epidermal barrier in a mouse model of AD and the human organotypic epidermal equivalent. Furthermore, the loss of PI-PLC attenuated epidermal hyperplasia and the infiltration of Gr-1+ cells and CD4+ cells induced by S. aureus infection in the mouse model of AD. Collectively, these results indicate that PI-PLC eases the entry of S. aureus into the dermis and aggravates acanthosis and immune cell infiltration in infected skin.

We examined the initial attachment of E. coli to abiotic surfaces conditioned with cereal extracts. The extracts were water-soluble fractions prepared from flours of barley, quinoa, rice, and wheat. Strains used were E. coli ATCC 8739, E. coli NBRC 3301, E. coli NBRC 3302, E. coli NBRC 13168, E. coli NBRC 13891, and E. coli O157:H7 sakai. When surfaces of glass and stainless steel were conditioned at 25 °C for 30 min with 0.5% cereal extracts, significantly lower numbers of E. coli cells attached to the conditioned surfaces than unconditioned ones, irrespective of strains used. The highest activity in reduction of the number of E. coli cells attached to the abiotic surfaces was found in the wheat extract. The suppressive activity was stable after treatments of the extract by autoclave and enzymatic digestion with α-amylase and proteinase K. We purified the active compound by ammonium sulfate fractionation and gel filtration with HiPrep 16/60 Sephacryl S-200 HR after the enzymatic treatments. The purified compound showed an average molecular mass of about 300 kDa by light-scattering measurements. Analyses of its components indicated that the active compound was arabinoxylan; the molar ratios were 1.0 (arabinose) to 2.46 (xylose). Commercially available arabinoxylan (average molecular mass: 370 kDa) also showed the similar activity. To our knowledge, this is the first report on a dietary fiber from cereals which suppresses the initial attachment of E. coli to abiotic surfaces.

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.

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.

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.

Irritant contact dermatitis (ICD) is one of the most common inflammatory skin diseases caused by exposure to chemical irritants. Since chemical irritants primarily damage keratinocytes, these cells play a pivotal role in ICD. One of the phosphoinositide-metabolizing enzymes, phospholipase C (PLC) δ1, is abundantly expressed in keratinocytes. However, the role of PLCδ1 in ICD remains to be clarified. Here, we found that croton oil (CrO)-induced ear swelling, a feature of ICD, was attenuated in keratinocyte-specific PLCδ1 knockout mice (PLCδ1 cKO mice). Dendritic epidermal T cells (DETCs), which have a protective role against ICD, were activated in the epidermis of the PLCδ1 cKO mice. In addition, the skin of CrO-treated PLCδ1 cKO mice showed increased infiltration of Gr1+CD11b+ myeloid cells. Of note, elimination of Gr1+CD11b+ myeloid cells restored CrO-induced ear swelling in PLCδ1 cKO mice to a similar level as that in control mice. Taken together, our results strongly suggest that epidermal loss of PLCδ1 protects mice from ICD through induction of Gr1+CD11b+ myeloid cells and activation of DETCs.

Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P2, and synthesis of PI(4,5)P2 in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P2 in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P2 regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics.

Phospholipids are distributed asymmetrically in the plasma membrane (PM) of mammalian cells. Phosphatidylinositol (PI) and its phosphorylated forms are primarily located in the inner leaflet of the PM. Among them, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a well-known substrate for phospholipase C (PLC) or phosphoinositide-3 kinase, and is also a regulator for the actin cytoskeleton or ion channels. Although functions of PI(4,5)P2 in the inner leaflet are well characterized, those in the outer leaflet are poorly understood. Here, PI(4,5)P2 was detected in the cell surface of non-permeabilized cells by anti-PI(4,5)P2 antibodies and the pleckstrin-homology (PH) domain of PLCδ1 that specifically binds PI(4,5)P2. Cell surface PI(4,5)P2 signal was universally detected in various cell lines and freshly isolated mouse bone marrow cells and showed a punctate pattern in a cholesterol, sphingomyelin, and actin polymerization-dependent manner. Furthermore, blocking cell surface PI(4,5)P2 by the addition of anti-PI(4,5)P2 antibody or the PH domain of PLCδ1 inhibited cell attachment, spreading, and migration. Taken together, these results indicate a unique localization of PI(4,5)P2 in the outer leaflet that may have a crucial role in cell attachment, spreading, and migration.

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.

Many businesses thrive by producing health supplements from agricultural products, as exemplified by the production of functional (or health) foods using plants traditionally cultivated in 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 on 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 Ca(2+), but not Zn(2+) levels. The cellular content of superoxide anions increased and that of glutathione decreased. Indirubin depolarized the cellular plasma and mitochondrial membranes. It did not potentiate or attenuate the cytotoxicity of A23187 (Ca(2+) overload) and H(2)O(2) (oxidative stress). The results suggested that indirubin induces both apoptotic and non-apoptotic cell death. It may be difficult to predict and prevent the 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.

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.

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.

We screened edible seaweeds in the ability to adsorb Shiga toxin (Stx) by an equilibrated dialysis method. Although water insoluble fractions of fourteen dry seaweeds did not adsorb Stx, most water soluble fractions were found to adsorb it to one degree or another. Among the seaweed tested, the extract of the Ulva linza Linnaeus [Enteromorpha linza (Linnaeus) J. Agardh] was found to well adsorb both Stx1 and Stx2. We purified the Stx-adsorbing substance from the U. linza extract by DEAE-Toyopearl column chromatography and gel filtration with HiPrep 16/60 Sephacryl S-300 HR column. The purified substance showed an average molecular mass of about 800 kDa by polyacrylamide gel electrophoresis. Analyses of its components indicated that the substance was a highly rhamnose-containing polysaccharide with sulfate esters of 18%. Apparent dissociation constants (Kd) of the polysaccharide to Stx1 and Stx2 were calculated to be 1.9 and 3.5 μM, respectively. To our knowledge, this is the first report on Stx-adsorbing dietary fibers.

Keratinocytes undergo a unique type of programmed cell death known as cornification, which leads to the formation of the stratum corneum (SC), the main physical barrier of the epidermis. A defective epidermal barrier is a hallmark of the two most common inflammatory skin disorders, psoriasis, and atopic dermatitis. However, the detailed molecular mechanisms of skin barrier formation are not yet fully understood. Here, we showed that downregulation of phospholipase C (PLC) δ1, a Ca2+-mobilizing and phosphoinositide-metabolizing enzyme abundantly expressed in the epidermis, impairs the barrier functions of the SC. PLCδ1 downregulation also impairs localization of tight junction proteins. Loss of PLCδ1 leads to a decrease in intracellular Ca2+ concentrations and nuclear factor of activated T cells activity, along with hyperactivation of p38 mitogen-activated protein kinase (MAPK) and inactivation of RhoA. Treatment with a p38 MAPK inhibitor reverses the barrier defects caused by PLCδ1 downregulation. Interestingly, this treatment also attenuates psoriasis-like skin inflammation in imiquimod-treated mice. These findings demonstrate that PLCδ1 is essential for epidermal barrier integrity. This study also suggests a possible link between PLCδ1 downregulation, p38 MAPK hyperactivation, and barrier defects in psoriasis-like skin inflammation.

Hematopoietic stem cells (HSCs) maintain the entire blood system throughout life and are utilized in therapeutic approaches for blood diseases. Prospective isolation of highly purified HSCs is crucial to understand the molecular mechanisms underlying regulation of HSCs. The zebrafish is an elegant genetic model for the study of hematopoiesis due to its many unique advantages. It has not yet been possible, however, to purify HSCs in adult zebrafish due to a lack of specific HSC markers. Here we show the enrichment of zebrafish HSCs by a combination of two HSC-related transgenes, gata2a:GFP and runx1:mCherry. The double-positive fraction of gata2a:GFP and runx1:mCherry (gata2a+runx1+) was detected at approximately 0.16% in the kidney, the main hematopoietic organ in teleosts. Transcriptome analysis revealed that gata2a+runx1+ cells showed typical molecular signatures of HSCs, including upregulation of gata2b, gfi1aa, runx1t1, pbx1b, and meis1b. Transplantation assays demonstrated that long-term repopulating HSCs were highly enriched within the gata2a+runx1+ fraction. In contrast, colony-forming assays showed that gata2a−runx1+ cells abundantly contain erythroid- and/or myeloid-primed progenitors. Thus, our purification method of HSCs in the zebrafish kidney is useful to identify molecular cues needed to regulate self-renewal and differentiation of HSCs.

Differentiation and proliferation of keratinocyte are controlled by various signalling pathways. The epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Inhibition of EGFR signalling disturbs keratinocyte proliferation, differentiation and migration. Previous studies have revealed that one of the EGFR downstream signalling molecules, phospholipase Cγ1 (PLCγ1), regulates differentiation, proliferation and migration of keratinocytes in in vitro cell culture system. However, the role of PLCγ1 in the regulation of keratinocyte functions in animal epidermis remains unexplored. In this study, we generated keratinocyte-specific PLCγ1 knockout (KO) mice (PLCγ1 cKO mice). Contrary to our expectations, loss of PLCγ1 did not affect differentiation, proliferation and migration of interfollicular keratinocytes. We further examined the role of PLCγ1 in irritant contact dermatitis (ICD), in which epidermal cells play a pivotal role. Upon irritant stimulation, PLCγ1 cKO mice showed exaggerated ICD responses. Further study revealed that epidermal loss of PLCγ1 induced sebaceous gland hyperplasia, indicating that PLCγ1 regulates homeostasis of one of the epidermal appendages. Taken together, our results indicate that, although PLCγ1 is dispensable in interfollicular keratinocyte for normal differentiation, proliferation and migration, it is required for normal ICD responses. Our results also indicate that PLCγ1 regulates homeostasis of sebaceous glands.

We screened foods containing indigestible ingredients in the ability to adsorb Shiga toxin (Stx). When 5 mg of foods and dietary fibers such as dry vegetables and inulin were mixed and incubated with 0.5 mL of Stx solution (100 ng/mL) containing 0.5% bovine serum albumin, both Stx1 and Stx2 seemed to be adsorbed by only a fermented food, natto (a traditional Japanese food prepared from steamed soybeans by the biological action of Bacillus subtilis). We purified the Stx-adsorbing substance from natto by extraction with H2 O, acid treatment, Proteinase K treatment, and an ion exchange chromatography. The purified substance showed an average molecular mass of about 600 kDa. We identified it as poly-γ-glutamate (PGA) by amino acid analysis of its hydrolysate and peptide analysis after its treatment with Proteinase K. Purified PGA (MW: molecular weight = about 600 kDa) was considered to adsorb both Stx1 and Stx2 when we separated adsorbed and unadsorbed Stxs (MW = about 72 kDa) by an ultrafiltration method with a centrifugal filter unit (MWCO: molecular weight cut-off = 100 K). However, PGA with the ability to adsorb Stx was an insoluble form precipitated in the filter unit during centrifugation. PGA precipitated beyond the saturated density was also confirmed to well adsorb both Stx1 and Stx2 by an equilibrated dialysis method. To the best of our knowledge, this is the 1st report on food-adsorbing Stx.

Methyl cinnamate (MC) and essential oils containing MC possess beneficial antimicrobial, antifungal, and insecticidal effects, among ohters. 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 poteniation 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.

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 Zn

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.

Hematopoietic stem cells (HSCs) are maintained in the specific microenvironment, termed the HSC niche. Some essential cellular components of HSC niches have been identified in the murine bone marrow, such as sinusoidal endothelial cells, perivascular cells, and mesenchymal stem cells. The zebrafish is an excellent model for the study of HSCs due to its many unique advantages, including valuable tools and experimental methods (e.g. transgenic/mutant animals, transplantation assays, cell culture assays, etc.). Moreover, the major hematopoietic organ in zebrafish is the kidney, so-called the “kidney marrow”, providing a parallel view of the HSC niche over evolution. Little is known, however, regarding which cell types plays a role in HSC niches in the kidney. This is due in part to the lack of robust methods to purify HSCs from the zebrafish kidney. Here, we show that zebrafish HSCs are highly enriched in the double-positive fraction of gata2a:GFP and runx1:mCherry (gata2a+ runx1+), and that gata2a+ runx1+ cells are closely associated with the sinusoidal endothelium in the adult kidney. An in vivo competitive repopulation assay showed that the frequency of HSCs was at least 550 times higher in gata2a+ runx1+ cells than total hematopoietic cells in the zebrafish kidney. Histological analyses revealed that gata2a+ runx1+ cells were mainly observed in the dorsal lateral area of the kidney where sinusoidal capillaries are abundantly observed. Loss of Jam1a, which is expressed in both sinusoidal endothelial cells and hematopoietic cells, led to a remarkable reduction in the sinusoidal area of the kidney and defect in supporting the hematopoietic repopulation. Our data thus suggest that the sinusoidal endothelium is an evolutionarily conserved component of HSC niches in vertebrates.

Previous studies on the cytotoxicity of arachidonic acid (ARA) elucidated the involvement of oxidative stress and Ca(2+). In the present study, the Zn(2+)-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 Zn(2+) by Zn(2+) chelators attenuated the ARA-induced increase in cell lethality. Thus, Zn(2+) is suggested to be involved in ARA cytotoxicity. ARA at 3-10 μM elevated intracellular Zn(2+) level. The Zn(2+) chelators attenuated the ARA-induced increase in intracellular Zn(2+) level while ARA significantly increased intracellular Zn(2+) level in the presence of 3 μM ZnCl2, suggesting the involvement of external Zn(2+). Zn(2+) reportedly exerts cytotoxic action under oxidative stress induced by hydrogen peroxide, via an excessive increase in intracellular Zn(2+) levels. Since ARA induces oxidative stress, the simultaneous administration of zinc and ARA may be harmful.

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.

We examined poly-γ-glutamate from natto, a Japanese fermented food, in the ability to adsorb Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). The polymer was immobilized by direct coupling to EAH-SepharoseTM. The poly-γ-glutamate-Sepharose (about 10 mg of ligand/mL of gel) adsorbed Stx2, but not Stx1: its dissociation constant (Kd) against Stx2 was calculated to be 14.0 μM. To analyze the binding site of poly-γ-glutamate against Stx2, we similarly immobilized glutamate and glutarate. Glutamate- and glutarate-Sepharoses (each 7 μmol of ligand/mL of gel) similarly adsorbed Stx2, but not Stx1; Kd values against Stx2 were calculated to be 14.0 and 30.0, respectively, μM. The common structures of PGA-, glutamate-, and glutarate-Sepharoses were considered to be glutaryl groups. When we added the mixture of Stx2 and poly-γ-glutamate-Sepharose to Caco-2 cells (a human colon epithelial cell line), poly-γ-glutamate-Sepharose was found to reduce the cytotoxicity of Stx2.

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 hr 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.

We screened foods containing indigestible ingredients in the ability to adsorb Shiga toxin (Stx). When 5 mg of foods and dietary fibers such as dry vegetables and inulin were mixed and incubated with 0.5 mL of Stx solution (100 ng/mL) containing 0.5% bovine serum albumin, both Stx1 and Stx2 seemed to be adsorbed by only a fermented food, natto (a traditional Japanese food prepared from steamed soybeans by the biological action of Bacillus subtilis). We purified the Stx-adsorbing substance from natto by extraction with H

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.

We have examined the cytotoxic effect of rhodexin A isolated from the extract of Rhodea japonica on human leukemia K562 cells using a flow cytometer and compared it with that of ouabain. Rhodexin A at 30 nM started to attenuate growth without affecting viability and further increases in the concentration of rhodexin A (100 nM or more) completely inhibited growth with decreasing viability. Rhodexin A at 30-100 nM increased the G(2)M population, but decreased the G(0)G(1) population, suggesting cell cycle arrest in the G(2)M phase. Rhodexin A at 100 nM increased the number of cells with hypodiploid DNA, indicating that rhodexin A induced apoptosis. The potency of rhodexin A to inhibit growth was greater than that of ouabain. The results indicate that rhodexin A exerts a potent inhibitory action on the growth of human leukemia K562 cells by inducing cell cycle arrest and apoptosis. Rhodexin A may also be a candidate for cancer treatment because there have been clinical reports of tumor regression in patients taking cardiac glycosides.

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 extra-cellular factor that influenced cytokinesis.

Effects of hydrogen peroxide (H(2)O(2)) on rat thymocytes were examined, using a flow cytometer and three fluorescent probes, annexin V-fluorescein isothiocyanate (annexin V-FITC) for detecting phosphatidylserine expressed on the membrane surface, ethidium bromide for estimating dead cells, and fluo-3-acetoxymethyl ester (fluo-3-AM) for monitoring changes in intracellular Ca(2+) concentration ([Ca(2+)](i)), to characterize H(2)O(2)-induced cytotoxicity. Exposure to H(2)O(2) (30 microM or more) increased the number of annexin V-positive live cells dose- and time-dependently while the number of dead cells increased at concentrations of 1 mM or more. H(2)O(2) (30 microM or more) increased [Ca(2+)](i) in a dose-dependent manner. Threshold concentration of H(2)O(2) to increase [Ca(2+)](i) was similar to that to increase annexin V binding to membranes. The H(2)O(2)-induced change in cell membranes was attenuated under Ca(2+)-free conditions. Therefore, it is likely that Ca(2+) is involved in the H(2)O(2)-induced cytotoxicity. Deferoxamine was effective to protect the cells suffering from H(2)O(2)-induced oxidative stress, suggesting a contribution of hydroxyl radicals generated by the Fenton reaction. Quercetin also exerted a potent protective action on cells suffering from H(2)O(2)-induced oxidative stress. The results indicate that the exposure of rat thymocytes to H(2)O(2) at micromolar concentrations increases annexin V binding to cell membranes in a Ca(2+)-dependent manner, suggesting the possibility that the oxidative stress caused by H(2)O(2) (and/or hydroxyl radicals) induces apoptosis via increasing [Ca(2+)](i).

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.

In order to reveal one of possible mechanisms for neuronal protective action of extract of Ginkgo biloba leaves (EGBL), the effect of EGBL on kainate- and KCl-induced increases in intracellular Ca(2+) concentration ([Ca(2+)]i) of rat cerebellar neurons was examined using a confocal laser microscope with appropriate fluorescent probes. EGBL at 3 microg/ml started to attenuate kainate-induced increase of [Ca(2+)]i and further increase in EGBL concentration (up to 30 microg/ml) concentration-dependently and significantly inhibited the kainate response. The complete inhibition by EGBL was observed in some neurons when the concentration was 10-30 microg/ml. The kainate-induced increase in [Ca(2+)]i was mainly due to Ca(2+) influx through voltage-dependent Ca(2+) channel opened by membrane depolarization via activation of kainate receptor-channel. However, the increase in [Ca(2+)]i by KCl was not significantly affected by EGBL at concentrations where the kainate response was greatly inhibited. EGBL consisting of flavone glycosides and terpene lactones is known to be an antioxidant. Furthermore, in this study, it is shown that EGBL exerts an inhibitory action on kainate receptor (a subtype of glutamate receptor). Since some of neurodegenerative diseases are due to cell death induced by glutamate excitotoxicity and oxidative stress, EGBL may be very suitable for preventing and/or treating such diseases.

Phospholipase C (PLC) is a key enzyme in phosphoinositide turnover. Among 13 PLC isozymes, PLCδ1 and PLCδ3 share high sequence homology and similar tissue distribution, and are expected to have functional redundancy in many tissues. We previously reported that the simultaneous loss of PLCδ1 and PLCδ3 caused embryonic lethality because of excessive apoptosis and impaired vascularization of the placenta. Prenatal death of PLCδ1/PLCδ3 double-knockout mice hampered our investigation of the roles of these genes in adult animals. Here, we generated PLCδ1/PLCδ3 double-knockout mice that expressed PLCδ1 in extra-embryonic tissues (cDKO mice) to escape embryonic lethality. The cDKO mice were born at the expected Mendelian ratio, which indicated that the simultaneous loss of PLCδ1 and PLCδ3 in the embryo proper did not impair embryonic development. However, half of the cDKO mice died prematurely. In addition, the surviving cDKO mice spontaneously showed cardiac abnormalities, such as increased heart weight/tibial length ratios, impaired cardiac function, cardiac fibrosis, dilation, and hypertrophy. Predating these abnormalities, excessive apoptosis of their cardiomyocytes was observed. In addition, siRNA-mediated simultaneous silencing of PLCδ1 and PLCδ3 increased apoptosis in differentiated-H9c2 cardiomyoblasts. Activation of Akt and protein kinase C (PKC) θ was impaired in the hearts of the cDKO mice. siRNA-mediated simultaneous silencing of PLCδ1 and PLCδ3 also decreased activated Akt and PKCθ in differentiated-H9c2 cardiomyoblasts. These results indicate that PLCδ1 and PLCδ3 are required for cardiomyocyte survival and normal cardiac function.

Phospholipase C is a key enzyme in phosphoinositide turnover. Although its functions have been extensively studied at the cellular level, many questions remain concerning its functions at the organ and individual animal levels. Here we demonstrate that mice lacking phospholipase Cδ1 develop granulocytosis associated with elevated serum levels of the granulopoietic cytokine interleukin-17. Re-introduction of phospholipase Cδ1 into keratinocytes of phospholipase Cδ1-deficient mice reverses this phenotype, whereas conditional ablation of phospholipase Cδ1 in keratinocytes recreates it. Interleukin-17 and its key upstream regulator interleukin-23 are also upregulated in epidermis. Loss of phospholipase Cδ1 from keratinocytes causes features of interleukin-17-associated inflammatory skin diseases. Phospholipase Cδ1 protein is downregulated in the epidermis of human psoriatic skin and in a mouse model of psoriasis. These results demonstrate that phosphoinositide turnover in keratinocytes regulates not only local inflammatory responses but also serum cytokine levels and systemic leukocyte counts, and affects distant haematopoietic organs. Phospholipase C is a signalling molecule with many cellular functions, but its physiological role at the organismal level is largely unknown. Kanemaruet al.show that phospholipase Cδ1 in the mouse epidermis influences interleukin and leukocyte concentrations in the blood.

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 (YCl(3)) on the intracellular Zn(2+) level was examined in rat thymocytes using a flow cytometer with FluoZin-3-AM and propidium iodide. The application of YCl(3) significantly decreased the intensity of the FluoZin-3 fluorescence, suggesting a decrease in the intracellular Zn(2+) level or quenching of the FluoZin-3 fluorescence by Y(3+). However, since Y(3+) 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 Zn(2+) into the cells. The application of YCl(3) attenuated the FluoZin-3 fluorescence augmented by externally applied ZnCl(2) in a concentration-dependent manner. This suggested that Y(3+) inhibited the Zn(2+) influx, resulting in the decrease in the intracellular Zn(2+) level. Yttrium may induce dyshomeostasis of intracellular Zn(2+), leading to some cytotoxic actions.

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.

Spatial and temporal activation of phosphoinositide turnover enables eukaryotic cells to perform various functions such as cell proliferation/differentiation, fertilization, neuronal functions, and cell motility. In this system, phospholipase C (PLC) is a key enzyme, which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) into two second messengers, inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG). Ins(1,4,5)P(3) triggers the release of calcium from intracellular stores, and DAG mediates the activation of protein kinase C (PKC). In parallel, PI(4,5)P(2) also directly regulates a variety of cellular functions, including cytoskeletal remodeling, cytokinesis, phagocytosis, membrane dynamics, and channel activity, in addition to its role as a substrate for PLC and phosphatidylinositol 3-kinase (PI3K), which generates PI(3,4,5)P(3). An imbalance of these phosphoinositides contributes to the pathogeneses of various human diseases. Therefore, strict regulation of the levels of PI(4,5)P(2) and PI(3,4,5)P(3) by PLC or other interconverting enzymes is necessary for cellular functions. In this review, we focus on the roles of PLC as a calcium-regulating enzyme and as a modulator of the phosphoinositide balance.

Phospholipase C (PLC), a key enzyme involved in phosphoinositide turnover, hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate two second messengers, inositol 1,4,5-triphosphate and diacylglycerol. PLCeta2 (PLCeta2), a neuron-specific isozyme of PLC, is abundantly expressed in the postnatal brain, suggesting the importance of PLCeta2 in the formation and maintenance of the neuronal network in the postnatal brain. However, the detailed expression patterns of PLCeta2 in the brain and other neuronal tissues remain to be clarified. Here, we generated PLCeta2 knockout/LacZ knockin (plch2(lacZ)(/)(lacZ)) mice-the first mice to lack full-length PLCeta2. Although the plch2(lacZ)(/)(lacZ) mice exhibited no obvious abnormalities, the LacZ reporter revealed unexpected and abundant expressions of PLCeta2 in the habenula and retina. We confirmed these PLCeta2 expression patterns by in situ hybridization and immunohistochemical analyses. In the retina, strong PLCeta2 expression was detected in the photoreceptor (rod/cone), outer nuclear layer, outer plexiform layer, and inner nuclear layer, suggesting that PLCeta2 is expressed in rods and cones, and also in horizontal, bipolar, and amacrine cells, but not in ganglion cells. Interestingly PLCeta2 exhibited a dynamic expression pattern during postnatal retinal development, strongly suggesting that this isozyme might be involved in the development and maturation of the retina. Since both the habenula and retina are thought to play important roles in the regulation of circadian rhythms, our results suggest that PLCeta2 may be involved in the function of habenula and retina.

The inhibitory effects of mustard and its major pungent compound, allyl isothiocyanate (AIT) on the growth of five species of bacteria and the relation between their inhibitory activities were studied. Brown mustard extract was prepared as 20% mustard in 70% ethanol after myrosinase treatment. AIT was dissolved in 70% ethanol to form a concentration equivalent to that in the mustard extract. Bacteria were cultured in nutrient broth containing the mustard extract or AIT at 30°C on a reciprocal shaker, and bacterial growth was determined by turbidimetry. The duration of the lag phase of growth was proportional to the concentration of mustard or AIT in the medium, and the turbidity of the stationary phase was sometimes decreased by these inhibitors. The concentrations of mustard in the medium that inhibited bacterial growth for 24h were 0.138%, 0.104%, 0.064%, 0.043% and 0.089%, and those of AIT were 14.5, 12.3, 6.5, 3.6 and 7.2ppm for Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Pseudomonas fragi and Ps. aeruginosa, respectively. These results suggested that the inhibitory effect of mustard was mainly due to AIT. Furthermore, it was recognized that the effect of mustard on S. aureus and E. coli was bacteriostatic at 0.8% whereas that on Ps. aeruginosa was bactericidal at 0.2%.

A reverse-phase high performance liquid chromatography (HPLC) was developed to separate and quantitate allyl isothiocyanate (AIT), a pungent principle of brown mustard, and cinnamaldehyde (CA), an aromatis principle of cinnamon. These compounds were extracted from ground spices with 70% (v/v) ethanol, separated on a Hitachi-gel # 3011-O by reverse-phese HPLC using methanol as a mobile phase with a flow rate of 1.00ml/min and detected by ultraviolet absorption at (190-400)nm. (1) AIT had a UV absorption maximum at 245nm and RT was 2.39min. AIT content of mustard extract (20% w/v) was 1.808±0.019mg/ml and in terms of defatted mustard, it was equivalent to 9.040±0.095mg/g. (2) CA had a UV absorption maximum at 286nm and RT 2.80min. CA content of cinnamon extract (20% w/v) was 6.810±0.014mg/ml, and in cinnamon, it was 34.050±0.070mg/g.