Copper (Cu) is an essential component of biological redox reactions and its deficiency is fatal to the body. At the same time, Cu is extremely toxic when present in excess. In this regard, several groups of Cu-regulating proteins in the body act to regulate the concentration of Cu within a certain range. However, the overall mechanism underlying the maintenance of Cu homeostasis in the body and cells remains poorly understood. In this review, recent research tools, such as animal models and gene-modified animals, and techniques, such as speciation and imaging of Cu, are highlighted.

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The aromatic α,β-unsaturated carbonyl compounds are known to possess biological and pharmacological activities, such as antioxidative, antibacterial, and antiviral effects. We tested the affinities of six new compounds to bind to the catalytic site of the dihydrofolate reductase enzyme. The binding modes of 1-aryl-(3-substitutedquinolyl)-prop-2-en-1-one analogues to the active site of the dihydrofolate reductase are investigated with ligand docking calculations. Docking simulations indicated that these compounds have the same binding modes for dihydrofolate reductase as methotrexate.

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Alfalfa (Medicago sativa L.) is a genus distributed widely throughout the world and considered as the richest land source of nature products. In this study, we obtained a purified saponin mixture (PSM) through a simple, repeatable and controllable separation method from alfalfa. The compounds of the saponin mixture were identified as Azukisaponin V and Soyasaponin I, which was determined as 79.26 and 20.74% respectively by HPLC-evaporative light scattering detection (ELSD) method. The effects of PSM on plasma lipid metabolism were studied in hyperlipidemic mice induced by Triton WR-1339. Results showed that PSM could significantly inhibit the increase of total cholesterol (TC), triacylglycerol (TG) and low density lipoprotein cholesterol (LDL-C) levels, and the decrease of plasma high density lipoprotein cholesterol (HDL-C) level in plasma of hyperlipidemic mice induced by Triton WR-1339. Further study demonstrated that PSM not only enhanced the activities of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) in liver, but also increased their mRNA levels in hyperlipidemic mice. The results above indicate that PSM from alfalfa exerts effects on plasma lipid metabolism through elevating the activity of liver lipases in hyperlipidemic mice. The obtained data would be a good contribution to the development and utilization of alfalfa in the world.

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Whilst accumulating reports have shown the beneficial effects of coffee against chronic liver diseases, the effects of coffee against fatty liver have not yet been reported. In this cross-sectional and the follow-up studies, we investigated the effects of coffee on the production of fatty liver in healthy males, using ultrasonography. In the follow-up study, annual changes in daily coffee intake during the 5-year study period were compared between subjects who appeared to have fatty liver (fatty liver group) and those who did not appear to have fatty liver (non-fatty liver group) in the matched study according to age, body mass index (BMI) and daily exercise level. The effects of changes in daily coffee intake between both groups on the development of fatty liver were investigated after adjustments for related factors, such as BMI, daily coffee and alcohol intake and exercise level. In the cross-sectional study, the subjects with fatty liver were revealed to have a lower daily coffee intake than those without fatty liver. In the follow-up study, a significant difference in the changes in daily coffee intake was observed between the fatty liver and non-fatty liver groups with a decrease in daily coffee intake observed in the former group, compared with the change in the latter group. Further, daily coffee intake was negatively correlated with the development of fatty liver after adjustments for related factors. These findings suggest a protective effect of coffee against fatty liver.

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Strain differences in the induction of cytochrome P450 (CYP) affect drug actions and side effects. Strain differences in the induction of CYP are important to evaluate drug-drug interactions in CYPs. We clarified strain differences in the induction of CYP3A1/3A2 and nuclear receptors by evaluating mRNA levels and metabolic activities in Sprague-Dawley (SD) rats and Dark Agouti (DA) rats (models for extensive and poor metabolism of CYP2D6, respectively). To clarify strain differences in CYP levels, we examined nuclear receptors such as the constitutive androstane receptor (CAR) and pregnane X receptor, which regulate the transcription of CYPs and transporters. We investigated CYP3A inductions in the liver after repeated intraperitoneal injections of phenobarbital (PB) or dexamethasone (DEX) into SD rats and DA rats for 3d. mRNA levels of CYP and nuclear receptors were determined by real-time reverse transcriptase-polymerase chain reaction. Metabolic activities of CYP3A were also determined. Increased CYP3A mRNA levels were observed in both rat strains after treatment with PB or DEX compared with the respective rat strains treated with vehicle alone. Induction of CYP3A mRNAs by DEX was higher in SD rats than in DA rats, suggesting that SD rats could be more susceptible to DEX than DA rats. Inductions of CAR by PB differed between strains. The increase in mRNA levels and activity of CYP3A by PB in SD rats and DA rats were similar. However, there were strain differences in CYP3A1/3A2 inductions after DEX treatment.

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Mutanase is an α-1,3-glucanase that cleaves the α-1,3-glucosidic linkages of glucan polysaccharides termed mutan. There has been interest in mutanase as it has the potential to be considered in preventive medicine. Previously, we identified the mutanase of Paenibacillus humicus from fermented soybeans and observed that the enzyme hydrolyzed insoluble glucans and mutan in vitro. Here we focused on the ability of mutanase in preventing dental caries. We proposed that the enzyme participates in the degradation of insoluble glucan biofilms in microplates, which were used as a model of biofilms on tooth surface. In this study, we investigated the potential of mutanase to hydrolyze insoluble glucan biofilms synthesized by a typical cariogenic streptococcus, Streptococcus mutans 10449, in 96-well microplates. The mutanase specifically hydrolyzed insoluble glucan biofilms composed of insoluble glucans synthesized by S. mutans. The mutanase hydrolyzed approximately 60% of S. mutans glucan biofilms in the wells and liberated oligosaccharides from the glucan films. We observed that recombinant mutanase could hydrolyze mutan biofilms, and the enzyme was characterized as an endo-α-1,3-glucanase because its hydrolysis of mutan biofilms, resulted in the predominant liberation of α-1,3-glucan tetrasaccharides. The present study suggests that Paenibacillus mutanase from fermented soybeans has potential application in preventive medicine because of its ability to degrade oral insoluble glucan biofilms.

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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.

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The memory enhancing effects of orally administered citidine-5-diphosphocholine or citicoline (CDP-choline) on memory impairment without neuronal cell death are still unknown. We investigated the effects of CDP-choline on memory disturbance induced by scopolamine using an eight-armed radial maze task. Orally repeated administration of CDP-choline or histidine significantly restored the errors impaired by scopolamine. CDP-choline may increase hippocampal and neocortical acetylcholine levels. These results suggest that CDP-choline has ameliorative effects on the impairment of spatial memory induced not only by neuronal cell death but also by impaired cholinergic signal.

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Coffee is the beverage consumed during pregnancy that is most commonly associated with adversely affecting the developing fetoplacental unit. Since placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD-2) is an important determinant for maintenance of fetal glucocorticoid levels during pregnancy, we investigated the effect of coffee on 11β-HSD-2 expression in the human placental choriocarcinoma cell line, JEG-3. Addition of coffee to the culture medium of JEG-3 cells increased 11β-HSD-2 gene expression after 24hr in a dose-dependent manner. Cellular 11β-HSD-2 activity, which was calculated by the amount of cortisol that was converted to cortisone in the culture medium, also increased in the coffee-treated JEG-3 cells. None of the major constituents of coffee, such as caffeine, caffeic acid or chlorogenic acid, showed any ability to induce 11β-HSD-2 gene expression. The 11β-HSD-2-inducing activity in coffee was extractable with ethyl acetate. Increasing the degree of roasting improved the ability of the coffee extract to induce 11β-HSD-2 gene expression indicating that the active constituent(s) is produced during the roasting process.

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The application of an optimized Fenton reaction and UV irradiation to the degradation of highly chlorinated biphenyls (PCBs) was performed. Recommended conditions were: Fe²⁺/Fe³⁺ concentration (16mM), H₂O₂ concentration (12%), temperature (70°C), pH (2.0), reaction time under stirring (≥2hr), re-addition of iron solution (1 hr intervals) and H₂O₂ (30 min intervals) and UV irradiation (254 nm, intensity 0.4mW cm^-2). When the system was applied to mixtures of PCBs at 5 ppm [PCBs congeners Kanechlor (KC)-400/600], low chlorine substituted biphenyls (Cl: 2-5) were completely decomposed after 2 hr, and even >90% of high chlorine substituted biphenyls (Cl: 6-8) could be degraded. When the present system was first applied to PCBs present in polluted electric insulating oil (25 ppm), >80% of PCBs were successfully degraded after 8 hr. These results suggest that the present UV-Fe²⁺/Fe³⁺-H₂O₂ system could be employed as a new technique for combating persistent organic pollutants such as PCBs.

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