非微生物素材表面への大腸菌の初期付着を抑制する穀類抽出液の分析

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

Rhamnan sulfate attenuates methylmercury cytotoxicity in rat thymic lymphocytes

Rhamnan sulfate, one of sulfated polysaccharides from seaweeds, is considered to have various characteristics such as antioxidant, anticoagulant, anti-inflammatory, antitumor, contraceptive, and antiviral activities, for the treatment of several diseases. We examined the effect of rhamnan sulfate on thymic lymphocytes treated simultaneously with methylmercury chloride, a toxic organometallic compound, using a flow-cytometric techniques with fluorescent probes, fluo-3-AM (an indicator for intracellular Ca2+) and propidium iodide (an indicator for dead cells). Rhamnan sulfate attenuated the methylmercury-induced increase in cell lethality. This effect of rhamnan sulfate is supposed to be due to the attenuation of methylmercury-induced elevation of intracellular Ca2+ levels. Rhamnan sulfate may be useful for the prevention of organometallic intoxication

Adsorption of Shiga Toxin to Poly-γ-Glutamate Precipitated

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 2 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

ヒト白血病K562細胞でクロトリマゾールにより誘発される細胞死に対するマイクロモル濃度の亜鉛の影響

Our recent study showed that the simultaneous application of clotrimazole with CdCI2 or PbCI2 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, ZnCI2, 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

Binding affinity of poly-γ-glutamate to Shiga toxin

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

ヒト白血病K562細胞におけるアドリアマイシン作用のクレモフォールELによる修飾

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 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

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

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 3-20mg/100g (wet weight). On the other hand, PC1P was present in limited vegetables which show higher GIPC-PLD activity, such as inner cabbage leaves (5.2mg/100g). 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