Phylloquinone (PK) is a major form of dietary vitamin K; however, the most prevalent form of vitamin K in humans and animals is menaquinone-4 (MK-4). Despite its high concentrations, the origin of MK-4 is yet to be defined. It is postulated that PK is converted into MK-4 and accumulates in extrahepatic tissues. To clarify this, PK with a deuterium-labelled 2-methyl-1,4-naphthoquinone ring was administered orally to mice and their cerebra were collected for deuterium (D) NMR and liquid chromatography (LC)-MS/MS analyses. We identified labelled MK-4 formed by conversion of the given PK, and this conversion occurred following oral or enteral administration but not parenteral or intracerebroventrical administration. Through the oral route, PK with the deuterium-labelled side chain in addition to the labelled 2-methyl-1,4-naphthoquinone was clearly converted into labelled MK-4 with a non-deuterium-labelled side chain, implying that PK was converted into MK-4 via the removal of an integral side chain. Our results suggest that cerebral MK-4 originates from PK intake, comprising the release of menadione (K₃) from PK in the intestine followed by prenylation of K₃ into MK-4 in the intestine or other tissues. We recently demonstrated that deuterium-labelled PK (PK-d₇) and deuterium-labelled K₃ (K₃-d₈) are converted to deuterium-labelled MK-4 (MK-4-d₇) in human osteoblast-like MG-63 cells. Moreover, we identified that the side chain substrate involved in the conversion of PK or K₃ to MK-4 is geranylgeranyl diphosphate deriving from the mevalonate pathway. Therefore, MK-4 biosynthesis likely plays an important role in biological functions of the brain and bones.

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High-tech products made by nano-materials are tightly connected to people's life. However environmental impacts and health effects of nanoparticles are widely getting attention. In this paper, we focused on the potential size-dependent cytotoxicity of nanomaterials. Silica (SiO₂) nanoparticles were used as model material. The ion exchange method and modified StÖber method were used to controllably prepare monodisperse uniform silica nanoparticles with six types of size (20, 50, 80, 140, 280 and 760 nm). And the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cellular morphology were introduced to evaluate the effect of the six sizes SiO₂ nanoparticles on pheochromocytoma (PC12) and human embryonic kidney (HEK293) cells viability. The results indicated that nanosized-SiO₂ (20, 50 and 80 nm) caused more cell damage when compared with microsized-SiO₂ (140, 280 and 800 nm). IC₅₀ of 24 hr exposure in PC12 cells were 118.2±7.3, 320.4±9.8, and 380.7±10.5 μg/ml, and in HEK293 cells were 80.2±6.4, 140.3±9.6 and 309.2±11.3 μg/ml for 20, 50- and 80-nm SiO₂ particles, respectively. Additionally, the SiO₂ particles of the other three sizes over 80-nm had little influence on the cell viability at the concentrations below 2000 μg/ml for two cultured cells. The results suggested that the exposure of SiO₂ nanoparticles with different sizes leaded to cellular morphological modifications and apoptosis in a size-dependent manner. Further studies on the molecular mechanisms of apoptosis and the toxicity in vivo are necessary.

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The present study was designed to investigate diabetes induced germ cell apoptosis in testis and its protection by treatment with hexane fraction of hydro-methanolic extract of root of Musa paradisiaca and leaf of Coccinia indica in composite manner. Testicular oxidative stress injury was reflected by diminution in the activities of antioxidant enzymes such as catalase, peroxidase, superoxide dismutase and glutathione-s-transferase along with elevation in levels of conjugated diene and thiobarbituric acid reactive substances in diabetic condition. Serum testosterone and insulin levels were decreased in association with elevation in apoptosis of pancreatic islet cells in diabetic rats. Giant cells number along with elevation in the number of apoptotic cells were noted in seminiferous tubules in diabetic model animals. Significant improvements in the levels of blood glucose, serum insulin and testosterone and testicular oxidative stress parameters were noted versus control after treatment of said fraction at a dose of 2 mg (1:3)/0.2 ml olive oil per 100 g body weight per day for 45 days to diabetic rats. Numbers of giant cells in seminiferous tubules, apoptotic germ cells and apoptotic pancreatic islet cells were decreased significantly in fraction treated diabetic group versus control. From UV-spectroscopic and TLC studies in connection with phytochemical screening of the said fraction, phenol, flavonoid and alkaloid types of compound were found. From these results it may be concluded that the active ingredient(s) present in hexane fraction of root of Musa paradisiaca and leaf of Coccinia indica have the potential to correct diabetes-induced testicular germ cell apoptosis.

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Neuroinflammatory responses are involved in Alzheimer's disease (AD) pathogenesis. In the present study, the protective effects of resveratrol, a polyphenolic compound, on cognitive deficits induced by lipopolysaccharide (LPS) were examined in rats, and the possible mechanisms were explored. The data showed that resveratrol administration by intraperitoneal injection (i.p.) may inhibit cognitive deficits induced by bilateral intracerebroventricular injection of 5 μg of LPS in rats. Subsequently, resveratrol afforded beneficial actions on the inhibition of tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and amyloid-β protein precursor (APP) generation, as well as the inhibition of phospho-transcription factors of nuclear factor κB p65 (p-NF-κB p65) activation followed by the presence of LPS in rat hippocampus. The results of the present study indicate that resveratrol may attenuate LPS-induced direct neuroinflammation in rats, and its mechanisms are, at least partly, due to inhibition of the generation of TNF-α, COX-2, and APP, and the related phosphorylation of NF-κB. These findings suggest that resveratrol might be a potential agent for treatment of neuroinflammation-related diseases, such as AD.

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Amberlite XAD-2 resin extracts of river and drinking water sampled in each month during the period from January to December 2008 from the Northwest district of Chiba Prefecture were investigated to characterize and determine their cholinesterase (ChE)-inhibiting potentials and pesticide levels. The XAD-2 extracts from river water collected during the mid-spring to mid-summer periods exhibited strong inhibition effect to horse serum ChE, reflecting the application of organophosphorus and carbamate pesticides to paddy fields. Gas chromatographicmass spectrometric (GC/MS) determinations of the XAD-2 extracts of the river water collected during spring to summer periods also showed to be comparatively high levels of agricultural chemicals, such as herbicides, insecticides and fungicides, as compared with those detected in the drinking water. Although a considerable reduction in the ChE-inhibiting potentials and in the GC/MS detectable compound levels was observed for the river water samples, it is particularly interest that ChE-inhibiting potentials still remained in the drinking water.

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The effect of sunlight on the development of allergic diseases is not well understood. In this study, we show that increased production of the proinflammatory mediators interleukin (IL)-10, tumor necrosis factor (TNF)-α, and nitric oxide (NO) induced by ultraviolet B (UVB) is mediated via the mitogen-activated protein kinase (MAPK) signaling pathway in human keratinocyte (HaCaT) cells. Cells were exposed to UVB irradiation (0.1-1 kJ/m²) either with or without specific inhibitors of NO [carboxy-2-pheryl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO)], extracellular signal-regulated kinase (ERK; U0126), c-Jun NH2-terminal kinase (JNK; SP600125), and p38 MAPK (SB203580). The levels of IL-10, TNF-α, and NO were then measured. The NO donor [(±)-N-[(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridinecarboxamide (NOR4)] was used to assess the involvement of NO in cytokine production. The activation of p38 MAPK was investigated in UVB-irradiated cells treated with p38 MAPK inhibitor, SB203580. The production of IL-10, TNF-α, and NO by HaCaT cells increased upon exposure to UVB. The NO inhibitor, carboxy-PTIO suppressed NO production induced by UVB. NOR4 increased the production of TNF-α, but not that of IL-10. The UVB-induced production of IL-10 and TNF-α were significantly suppressed by the specific inhibitors U0126, SP600125, and SB203580. In conclusion, UVB induced the production of proinflammatory mediators via activation of the p38 MAPK signaling pathway, suggesting that sunlight might promote the development of allergic diseases (such as dermatitis) through an augmented inflammatory response involving the increased production of proinflammatory cytokines and NO.

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TS-1 is an oral anticancer agent comprising three components: two biochemical modulators of 5-fluorouracil (5-FU) and tegafur (FT), a prodrug of 5-FU. TS-1 displays potent anti-tumor activity by maintaining effective 5-FU concentrations in serum for a prolonged period. FT is gradually converted to 5-FU in vivo via 5'-hydroxylation mediated by cytochrome P450s. As a result, genetic polymorphisms can cause wide individual differences in serum concentration of 5-FU after administration of TS-1, requiring monitoring of serum concentration of 5-FU for each patient. Chemotherapy with TS-1 plays a major role in the treatment of oral cancer. In cases where a patient with oral cancer shows dysphagia, TS-1 may need to be tube-administered. Although tube administration by the simple suspension method has been recommended from a biosafety perspective, particularly for anti-cancer agents, its efficacy remains unclear. We established a simple high-performance liquid chromatography method for simultaneous determination of FT and 5-FU levels at clinical sites by modifying a method reported in the literature.¹⁾ Unlike the original method, this simplified method does not require extraction procedures to separate FT and 5-FU, and requires only 250 μl of serum. Using this method, we compared FT concentrations in the sera of oral cancer patients administered TS-1 orally or via a tube-assisted simple suspension method. No significant differences in FT concentrations were apparent between these two modes of administration. TS-1 treatment by tube administration using the simple suspension method thus seems useful for patients with dysphagia as an alternative to oral dosage.

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We have reported that methylamine dichloramine (CH₃NCl₂) causes colitis in mice and that in addition to its oxidative potentials, its cell membrane permeability is important for the onset of ulcerative colitis (UC). The aim of the present study was to determine if CH₃NH₂, a typical low-molecular weight biological amine, aggravates experimental UC in mice through in vivo formation of its chloramines. The biological oxidation potentials of low-molecular chloramines (50-200 μM) were evaluated by hemolysis and methemoglobin formation in sheep erythrocytes (1×10⁸ cells/ml). ICR-strain mice were administered drinking water containing 1.5% dextran sulfate sodium (DSS), a potent UC inducer in mice, for 6 days. The mice were intraperitoneally administered CH₃NH₂ (5-40mg/kg per day) for 5 days. The colonic lesions were characterized by visible parameters and microscopic analysis of histological alterations and the number of infiltrating and myeloperoxidase positive neutrophils, respectively. Methylamine chloramines showed considerably higher potentials for both hemolysis and methemoglobin formation than the other chloramines tested. The administration of CH₃NH₂ increased the excretion of CH₃NH₂ itself into feces in a dose-dependent manner and markedly aggravated experimental UC accompanying the increased neutrophil infiltration. These results strongly support the possibility that CH₃NH₂ causes serious aggravation in UC via the formation of its chloramines and suggest the participation of low-molecular weight biological amines in deteriorating colitis.

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Although selenium (Se) is not an essential element in plants, Se metabolism in plants is remarkable, mainly for the purpose of phytoremediation and nutritional supplementation of Se. Brassica juncea (Indian mustard) is known as an Se accumulator. In addition to Indian mustard, unique Brassicaceae plants are consumed in Japan. Brassica rapa var. hakabura, commonly known as nozawana, and Brassica rapa var. peruviridis, commonly known as komatsuna, are typical Brassicaceae plants. In this study, we evaluated the Se-accumulating ability of these unique Brassicaceae plants. There were no significant differences in the Se concentrations in the roots and the leaves of the three Brassicaceae plants. However, nozawana most efficiently accumulated Se among the three Brassicaceae plants because it showed the most rapid growth, resulting in the highest biomass. Speciation revealed that the Se species accumulated in the three plants were identical. In addition, nozawana contained easily extractable essential minerals, such as iron, copper, and zinc. Potentialities of nozawana as a source of minerals and the phytoremediation of soil and water contaminated with Se were discussed.

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Nobiletin enhances differentiation and lipolysis of 3T3-L1 adipocytes and improves hyperglycemia and insulin resistance in obese (ob) diabetic ob/ob mice. We investigated the effects of nobiletin on lipid metabolism and accumulation of body fat in rats. The control group was fed a 20% high-fat diet and 1% cholesterol, and the nobiletin group was fed same diet supplemented with 0.1% (w/w) nobiletin. The rats were fed for 4 weeks. Weights of epididymal, perirenal, total white adipose tissues (WAT: mesenteric, perirenal, and epididymal), and the subcutaneous WAT in the nobiletin group were significantly lower than those in the control group. This decrease was brought about by nobiletin without affecting triglyceride (TG) levels in the liver and skeletal muscle. Plasma TG levels tended to be decreased by nobiletin. The size and diameter of WAT adipocytes in the nobiletin group were significantly lower than those in the control group. This decrease may be partly due to lower lipoprotein lipase (a major determinant for the development of obesity) levels in WAT of the nobiletin group than that of the control group. Plasma levels of high density lipoprotein cholesterol and apolipoprotein A-I increased significantly with administration of nobiletin. These results suggested a beneficial effect of nobiletin on lipid metabolism. However, no significant differences were observed between the nobiletin and the control groups in proteins such as ATP-binding cassette transporter A1, and sterol regulatory element-binding protein-1 in the liver, PPARγ and tumor necrosis factor-α (TNF-α) in WAT, and adiponectin and TNF-α in plasma.

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Black rice vinegar (kurosu) made from a jar is a traditional vinegar in Japan. Kurosu has been commonly used as a healthcare supplement; however, it is not clear whether kurosu has a treatment effect on the expression of drug metabolism enzymes and transporters, whose expression alterations may induce food-drug interaction. Alteration of the principal drug metabolism enzymes and expression following kurosu and moromi, the residue from black vinegar brewing, for 30-day treatment was evaluated. Water, 0.23% acetic acid, concentrated kurosu (containing 0.23% acetic acid) and moromi was administered to Wister rats. The treatment did not significantly affect the biochemical parameters of serum, alanine aminotransferase, alkaline phosphatase, total protein, creatinine in the serum, and body weight. The treatment also did not affect the expression levels of cytochrome P450 (Cyp) 1a2, Cyp2b1/2, Cyp3a1, glutathione S-transferase, p-glycoprotein, multidrug resistance protein 2, breast cancer resistance protein, organic anion transport polypeptide 2, and organic anion transport 2 and 3 in the liver. In conclusion, the present study suggests that kurosu from a jar does not have toxic effect and does not alter expression levels of principal drug metabolism enzymes and transporters.

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The in vitro physicochemical properties of 6 bottled water products (as drinks), advertised as having cluster sizes of H₂O molecules smaller than those of regular water, were studied. No significant differences in the boiling temperature (BT, near 100°C) or freezing temperature (FT, near 0°C) were observed between all 8 aqueous solutions examined, including deionized water (DW) and tap water as controls, although DW had the lowest mean BT and highest mean FT values. The specific gravity of all the examined aqueous solutions, including the controls, changed in a temperature-dependently manner, showing the greatest value (near 1.000) at 4°C. Analyses of inorganic components revealed that various mineral components were dissolved in aqueous solutions examined other than DW, suggesting that the trends of the lowest BT and highest FT for the DW control were due to the absence of these solutes. These results did not demonstrate the presence of an abnormality in the hydrogen bonds between H₂O molecules in the sample solutions compared to the controls. In other words, the claims by the manufacturers in their advertisements concerning the cluster sizes of H₂O molecules for their water products were not confirmed by the findings of this study.

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The primary purpose of this study was to investigate the chronic effects of practicing 30 min of basic karate exercises (BKEs) for 10 weeks on maximal oxygen uptake (VO₂max) in sedentary collegiate women who had no previous karate experience. The secondary purpose of this study was to investigate physiological responses and intensities of BKEs to examine the intensity of exercise. Nine women practiced 30 min of BKEs, 4 days·week^-1, for 10 weeks. The six other women acted as controls. The 30 min of BKEs consisted of 9 min of stationary basics in a parallel stance (S-Basics I), 12 min of stationary basics in a front stance (S-Basics II), and 9 min of movement basics in the front stance (M-Basics). For S-Basics I, the mean percent of maximum VO₂ reserve (%VO₂R) was much lower than the accepted threshold, while the mean percent of maximum heart rate reserve (%HRR) was slightly lower than the accepted threshold for increasing VO₂max, i.e., 40% of VO₂R or HRR. For S-Basics II, the mean %VO₂R was marginal, while the mean %HRR was above the accepted threshold for increasing VO₂max. The mean %VO₂R and %HRR for M-Basics were above the threshold for increasing VO₂max. VO₂max in both l·min^-1 and ml·kg^-1·min^-1 in the experimental group significantly increased at the end of the 10 weeks of training (from 1.80±0.30 to 2.00±0.34 l·min^-1 and 32.3±4.1 to 36.0±4.4 ml·kg^-1·min^-1, respectively), while neither value changed significantly in the control group. In conclusion, 30 min of BKEs can reach the minimal threshold level to increase cardiovascular fitness and can improve cardiovascular fitness in sedentary women.

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This study was conducted to clarify whether phosphoenolpyruvate (PEP), an intermediate substance of glycolysis, has the potential to attenuate cellular injury induced by oxidative stress or dysfunctions in energy metabolism in vitro. PEP (0.5-10 mM) attenuated hydrogen peroxide (H₂O₂)-induced cellular injury in the porcine proximal kidney tubular cell line, LLC-PK₁ in a dose-dependent manner. PEP also prevented cellular injury in LLC-PK₁ cells induced by the glycolysis inhibitor, 2-deoxy-D-glucose (2-DG). In addition, PEP significantly enhanced the degradation of H₂O₂. The prevention of H₂O₂-induced cellular injury mediated by PEP was more potent than that of the carbohydrates, glucose and trehalose, which are used as components of organ preservation solutions for clinical transplantation. In conclusion, we demonstrated that PEP is a bifunctional carbohydrate with anti-oxidant properties and suggest that PEP is potentially useful as an organ preservation agent in clinical transplantation.

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The relationship between protein and mRNA levels of mevalonate pyrophosphate decarboxylase (MPD) in rat tissues remains to be clarified. In this study, we examined the distribution of the mRNA in Wistar rat tissues by real-time PCR. When the relative expression of MPD in 1 mg of tissue was quantified using glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as an internal control, the mRNA level was found to be markedly higher in the spleen and liver than in other organs. The correlation coefficient between protein and mRNA levels of MPD was 0.847, indicating that the protein level of MPD in Wistar rat is distributed in the tissues almost entirely dependent on the mRNA level of MPD. We previously reported that the protein levels of MPD in the liver and brain of spontaneously hypertensive rats, stroke-prone (SHRSP) are reduced. Thus, we compared mRNA levels of MPD in the liver and brain between Wistar Kyoto rats (WKY) and SHRSP. The levels in liver of SHRSP were significantly decreased, but not in brain, as compared with WKY. Also, mRNA levels of sterol regulatory element binding protein-2, which is transcription factor of cholesterol, in liver of SHRSP was similar to those of WKY. These findings indicate that the reduced protein level of MPD in the liver of SHRSP is caused by a decrease in mRNA level of MPD, and that in the brain of SHRSP is caused by increased degradation of the MPD protein.

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Tissue engineering for skin regeneration is closely related to advances in carrier materials for fibroblast. To investigate the optimal fibroblast scaffold, we developed a silk protein containing multiple (Ala-Gly-Ala-Gly-Ser-Gly)_n sequences, and used it for in vitro evaluations of cell shape, adhesion and matrix production. NIH-3T3 fibroblasts were cultured on silk scaffold and on control culture plates. At 2 hr, fibroblasts cultured on silk scaffold showed round-rose shape, but cells spread on control plates. The numbers of adhesive fibroblasts were the same in both scaffolds, indicating that silk scaffold affects cell shape but not adhesive efficiency. To examine the effects of silk scaffold on cytoskeletal changes of fibroblasts, cells were applied to actin staining. Fibroblasts cultured for 2 hr on silk scaffold showed dense actin fibers which formed ring-like structures, while actin stress fibers were formed in cells cultured on control plates. In a long-term culture of 14 days, piled matrices produced by fibroblasts have been shown distinctly in cells cultured on silk scaffold compared with cells cultured on control plates. The expression of fibronectin mRNA was elevated in fibroblasts cultured on silk scaffold compared with control, but the expression of collagens, type I and type III, mRNAs was similar in fibroblasts cultured on both scaffold. These results indicate that silk scaffold influences cell shape and actin fiber, and enhances matrix production with increased fibronectin. Therefore, silk protein may be an useful scaffold for regenerative therapy in various skin wounds.

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Long-term ingestion of coffee polyphenols (CPP) reduces body fat in humans. The aim of the present study was to investigate the effect of daily consumption of CPP on energy metabolism in humans. Seven healthy male subjects, with a mean (±SEM) age of 34.7±2.0 y and a body mass index (kg/m²) of 21.8±0.6, participated in a placebo-controlled, double-blind, crossover intervention study with two different test beverages. The subjects consumed 185 g of a test beverage with or without CPP (359 mg) daily for 1 week. Energy metabolism was evaluated by indirect calorimetry before and after the test period after fasting and up to 3.5 hr postprandially, and during cycle ergometry. Indirect calorimetry showed that, compared to 1-week ingestion of the control beverage, 1-week ingestion of the CPP beverage led to significantly higher oxygen consumption in the sedentary period and during the cycle ergometry. CPP beverage consumption led to a higher fat utilization in the sedentary period and a higher anaerobic threshold during exercise compared with control beverage consumption. In conclusion, daily consumption of CPP increases postprandial fat utilization in healthy humans.

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