In this study, we investigated whether the inhibition of endogenous phosphorylation of mitogen-activated protein kinase (MAPK) and diallyl disulfide (DADS)-induced phosphorylation of MAPKs with MAPK specific inhibitors, SB203580 and U0126 (for phospho-p38 and phospho-p42/p44, respectively), can induce or enhance apoptosis in human CNE2 nasopharyngeal carcinoma cells. Our data demonstrate that MAPK inhibitors decrease the viability of CNE2 cells, stimulate typical apoptotic morphologic changes, and enhance DADS-induced apoptosis. The present findings indicate that phosphorylation of MAPKs plays an important cytoprotective role in CNE2 cell apoptosis and the DADS-induced apoptotic process.

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Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors worldwide and a leading cause of cancer-related death in the world. The importance of diet in the control of several major cancers is now widely recognized, and it is generally agreed that a plant-based diet may afford a significant degree of protection. The current study was designed to evaluate the effect of genistein on N-nitrosodiethylamine (DEN)-induced (200 mg/kg body weight; by single i.p injection) and Phenobarbital-promoted (0.05% through drinking water for 14 successive weeks) liver cancer in Wistar albino rats. Decreases (p < 0.001) in the activities of Na⁺/K⁺ ATPase and Mg²⁺ ATPase and increases (p < 0.001) in Ca²⁺ ATPase activity were observed in erythrocytes membrane and tissue ATPase of liver cancer-bearing animals when compared with control groups. The change in the activities of these enzymes in membrane and tissue were indicative of the persistent deteriorating effect of DEN in cancer-bearing animals. These enzyme activities were reversed to near normal value in animals treated with genistein. From our results we conclude that genistein may play an important role in preserving membrane asymmetry by suppressing free radicals implying a role for genistein against tumorigenesis.

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The objectives of this study were to determine the extent of smoothness for some selected nonparametric models, and to examine the suitability of the default method for evaluating temperature-mortality relation in Japan. Our analysis was conducted for Japanese aged 65 and older, from 1972 to 1994. The models we selected were smoothing spline and locally-weighted scatterplot smoothing (LOWESS/LOESS). Firstly, we determined the degrees of smoothness by an “a priori” approach. After exhaustively drawing curves of the relation between daily maximum temperature and sex-specific mortality rate for each prefecture using a wide range of smoothing parameters, we selected the degrees of smoothing for each prefecture, based on a priori knowledge. This assumes that we a priori know that the relation between temperature and mortality is V-shaped, i.e., between two temperature extremes the curve should have a minimum mortality rate at a certain temperature (optimum temperature=OT), which is an absolute minimum with no local extremes. For the cases in which no OT was observed for any of the degree of smoothing, we did not assign an OT. Among selected degrees of smoothing, we further selected “best” degrees of smoothing for the models such that the degrees of smoothing yielded OTs for all the prefectures (except for those with non-OT). Next, based on the “best” degrees of smoothing, we examined the generalized cross-validation (GCV) method, which is one of the most successful “default” methods for selecting a smoothing parameter, and which is a default method in R statistical language. For most of the prefectures, the relation between daily maximum temperatures and mortality rates were V-shaped. The OTs varied among prefectures and tended to be higher for southern prefectures. Some of the estimates based on GCV method, in particular for the LOESS models, yielded non-OT type relations even when the “a priori” approach yielded OTs. LOESS model showed more sensitivity to the value of span (the parameter of smoothness); an average difference in OT levels within the “best” selected range of spans was 0.5°C, while that for the smoothing spline model was 0.3°C. This study suggests that, for evaluating the relation between daily mortality and temperature, the smoothing spline model with degree of freedom being 6-7.5 was the most appropriate model for the Japanese data, and that blind use of the default method was problematic in this case.

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In the last decade, extracorporeal bone tissue engineering has found more clinical applications due to the progress and new achievements in the isolation and characterization of stem cells from different sources, as well as, in controlling proliferation and differentiation in vitro. The aim of this study is to evaluate the in vitro behaviour, morphological structure and extracellular matrix synthesis of human dental follicle stem cells (hDFSCs) isolated from human dental bud, when seeded onto a synthetic hydroxyapatite (HA) scaffold (ENGIpore^©). Populations of CD29+, CD90+, CD146+ and CD166+ were sorted by FAC sorter (FACS) analysis and were cultured in osteogenic medium and then, onto the scaffold. These cells were analyzed by optical and electronic microscopy, at week 1 and 6, before and after the differentiation. Light microscopy showed an intense attachment and colonization of the HA scaffold by polygonal-shaped cells. Scanning electron microscopy after six weeks revealed a tri-dimensional organization of the cells and the presence of dense material around the cell clusters. hDFSCs showed participation in protein biosynthesis and demonstrated high proliferation on the synthetic HA scaffold.

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The influence of hydroxyhydroquinone (HHQ) on the antihypertensive effects of chlorogenic acids in coffee was evaluated in a double-blind, randomized controlled trial of high-normotensive and mild hypertensive adult men and women. The subjects were randomly assigned to either an active group that ingested HHQ-reduced coffee (0.05 mg/184 ml) containing chlorogenic acids (299 mg/184 ml) or a control group that ingested coffee containing HHQ (1.69 mg/184 ml) and chlorogenic acids (299 mg/184 ml), corresponding to commercially available products. Each subject was instructed to continuously ingest one can of test beverage (coffee) daily for 12 weeks. A linear mixed-model repeated-measures analysis of covariance of 38 high-normotensives and 60 mild hypertensives showed that systolic blood pressure (SBP) was significantly lower in the active group (n=51) than in the control group (n=47) throughout the intake period (Group effect; p=0.031). A stratified analysis suggested that the antihypertensive effect was greater in the mild hypertensives (Group effect; p=0.013 in SBP and p=0.015 in diastolic blood pressure) than in the high-normotensives. Safety assessment revealed no adverse effects associated with the reduction of HHQ to a level lower than the amounts in commercially available coffee products. These results suggest that HHQ-reduced coffee containing chlorogenic acids can be safely ingested to improve hypertension.

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A cationic sorbent, which bears carboxyl groups of citric acid (CA) derived from esterified wheat straw, was prepared using the solid-phase esterifying technique. Copper (Cu) removal from aqueous solution with modified wheat straw (MWS) was investigated to evaluate its capacities of cation exchange and metal chelation. The effects of various experimental parameters (e.g., solution pH, Cu concentration, sorbent dose, contact time, and presence of light metal) are studied in batch experiments. The maximum value of Cu removal appeared in the range of pH 4 to 5. The percentage of Cu removal decreased with increasing initial Cu concentration. The isothermal data of Cu sorption conformed well to the Langmuir model, and the maximum sorption capacity (Q_m) of MWS for Cuwas 78.13 mg/g. For 100 mg/l of Cu solution, a removal ratio of greater than 96% could be achieved with 2.0 g/l or more of MWS. The equilibrium of Cu removal was reached within 90 min. The presence of univalent and bivalent light metal ions (Na⁺, K⁺, Mg²⁺, and Ca²⁺) did not significantly interfere with Cu removal by MWS.

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We previously reported that chloroquine disrupted lysosomes, but not the shift to low-density lysosomes. In the present study, the effects of primaquine on lysosomal integrity in cultured rat hepatocytes were studied by measuring lysosomal enzyme β-glucuronidase (β-G) or lysosomal-associated membrane glycoprotein (lamp-1) in the cytosolic fraction obtained from cells permeabilized by digitonin, and in the cytosolic fraction obtained by conventional cell fractionation or in Percoll density gradient fractions. The percentage disruption of lysosomes in living cells by 50 μM or 100 μM of primaquine was 1% or 4%, respectively, and lysosomes disrupted by homogenization or centrifugation during cell fractionation by 50 μM or 100 μM of primaquine were 2% or 7%, respectively. The decrease of β-G and lamp-1 in lysosome fractions (fractions 16 to 18) on a Percoll density gradient (1 to 18 fractions) in 50 μM or 100 μM primaquine-treated cells was 9% or 19% for β-G, and 16% or 24% for lamp-1, respectively. The decrease of β-G and lamp-1 in the lysosome fraction was higher than the disruption of lysosomes in living cells or by homogenization or centrifugation during cell fractionation by 50 μM or 100 μM of primaquine. Also, the peak fraction numbers of the subcellular distribution of β-G and lamp-1 on a Percoll density gradient by 50 μM primaquine-treated cells were fraction 17 (high density) and 4 (low density), while those by 100 μM primaquine-treated cells were fraction 6 (low density) or 4 (low density). From these data, we infer that the main effect of primaquine is to cause a shift of lysosomal protein to low density, and then to cause differences in the proportion of membrane and luminal proteins of lysosomes in low-density fractions, although the main effect of chloroquine was the disruption of lysosomes.

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The objective of the present study was to determine if a cystine-cysteine cycle operates between the erythrocytes and the plasma. In the present study we incubated the erythrocytes in krebs ringer phosphate buffer with different osmolarity containing different amounts of cystine. Our results show that erythrocytes do not uptake cystine from the environment when the osmolarity of the buffer is 310 mOsmol/l. Erythrocytes also do not operate a cystine-cysteine cycle in this isoosmolar buffer. However, when exposed to hyperosmolar buffer in the ranges that occur in the kidney medulla which is in between 1200-1400 mOsmol/l erythrocytes start to uptake cystine from the environment and induce a cystine-cysteine cycle. The cystine uptake and cystine-cysteine cycle were characterized by measurement of changes in the free -SH concentrations in erythrocytes and in the buffer. Following incubation of erythrocytes in 1 mM cystine containing 1250 and 1300 mOsmol/l buffer, the free -SH concentrations in the buffer reached to 0.102±0.002 and 0.241±0.013 μmol/ml erythrocyte respectively. Our results demonstrate that erythrocytes display a cystine-cysteine cycle in hyperosmolar environment which is prevailed mainly in the kidney medulla. Our results also display that this process is biologically active and energy dependent. The observed cystine-cysteine cycle is inhibited when the erythrocytes are incubated at lower temperatures and in the absence of glucose. Our results suggest that erythrocytes uptake cystine, intracellulary reduce it to cysteine and release it back to the environment when exposed to hyperosmolar conditions. Erythrocytes may have a role in the regulation of plasma cystine and cysteine concentrations and may contribute to the regulation of plasma redox status.

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Stingless bee (Meliponini) honey is a bioresource used to treat cataracts in traditional medicine. The anticataract activity of twenty flavonoids was explored in an osmotic cataract model, to find a probable link between the putative anti-cataract properties of stingless bee honey eyedrops and their flavonoids. Osmotic cataracts were induced in ovine lenses to produce a model to test anti-cataract drugs in cultured lenses by digital image analysis. Digital images were taken every 4 hr to monitor progressive opacification by measurements of grey level. In 24 hr. the opacification was stable. Osmotic cataracts were induced by incubating ovine lenses in 45% hypotonic HBS for 24 hr to test the anticataract action of twenty synthetic flavonoids at a concentration of 10^-5 M. Luteolin tetramethyl ether, luteolin 4'-glucoside, luteolin 3'-7-diglucoside and orientin, significantly inhibited cataracts induced in ovine lenses incubated in 45% hypotonic HBS for 24 hr. Different degrees of opacification were produced by hypotonic stress in ovine lenses. The significant inhibition of cataracts caused by four derivatives of luteolin in vitro may be considered as a preliminary evidence for the putative anticataract properties of stingless bee honeys.

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The hypoglycemic and hypolipidemic effects of Tangzhiqing (abbreviated TZ) a mixture of Mulberry Leaf, Lotus Leaf, Danshen Root, and Hawthorn Leaf, were investigated in KK-Ay mice, an animal model of type 2 diabetes. TZ reduced blood glucose, total cholesterol, and triglyceride levels of KK-Ay mice at 4 weeks after oral administration. TZ also improved sucrose or maltose tolerance. Maltase activity in small intestine significantly decreased in the TZ-treated KK-Ay mice. These results support the hypothesis that TZ improves glucose metabolism by reducing α-glycosidase activity. Therefore TZ might have beneficial effects on hyperglycemia and hyperlipidemia in type 2 diabetes.

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Influence of dietary protein levels on mercury (Hg) fate and on tissue metallothionein (MT) levels was investigated in mice. Twenty-four hr after single administration of mercuric chloride (2.5 mg Hg/kg, subcutaneous), the hepatic Hg concentration was enhanced by dietary protein deficiency, whereas the levels in other tissues and excrements were not affected. At that time, MT inductions by mercuric chloride in liver and kidney were suppressed by dietary protein deficiency, despite no observable differences in basal levels. Thus, Hg levels in the liver and kidney showed little correlation with MT levels. A further experiment demonstrated an enhancement of Hg concentration in the liver by dietary protein deficiency at 3 and 12hr but not at 1hr, and the Hg concentration in the kidney was transiently enhanced at 3 hr. Accordingly, the differences in Hg fate would arise considerably earlier, probably before MT induction. The present results suggest that dietary protein status modifies the fate of inorganic Hg, especially in the liver, probably independent of the differences in dietary protein level-dependent varying levels of MT.

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The quantity of formate was determined using apparatus comprised of a reactor with immobilized formate dehydrogenase (FDH) in the flow line. NADH formed by an enzymatic reaction was fluorometrically detected. The optimal concentration of NAD⁺ in the carrier was determined. The maximum peak area due to NADH was observed at pH 7.0 when the pH of the carrier consisting of piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES) buffer ranged from 6.0 to 8.0. Various buffer types were also examined as carrier media at pH 7.0 and PIPES buffer showed the maximum peak area. When the carrier composed of PIPES buffer (0.1 M, pH 7.0) was used, the calibration curve for formate was linear in the range of 0.5-50 μM (r=1.000). Relative standard deviations of the peak area at 1 μM and 10 μM were 2.6% (n=7) and 1.6% (n=7), respectively. This method was applied to the analysis of formate in foodstuffs, and formate content determined by this method agreed with that determined by a commercially available test-kit.

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Recently, it has been questioned whether mevalonate pyrophosphate decarboxylase (MPD) is predominantly located in the peroxisomes or cytosol. We previously reported that a small amount of MPD in the liver of rats fed a CP diet (5% cholestyramine and 0.1% pravastatin) existed in the peroxisomes, although MPD is predominantly located in the cytosol in the liver of rats fed normal chow and a CP diet for 12 days. In the present study, we examined the subcellular distribution of MPD in mouse melanoma cells (B16 and B16F10) treated with or without lovastatin, using digitonin permeabilization and immunoblotting. In permeabilized B16 by digitonin after treatment with or without lovastatin, 95% and 5%, or 98% and 2% of MPD existed in the cytosol and membrane/organelle (M/O) fraction, respectively. Using B16F10 under the same conditions, 80% and 20%, or 91% and 9% of MPD existed in the cytosol and M/O fraction, respectively. These results indicated that MPD was predominantly located in the cytosol in both mouse melanoma cells treated with or without lovastatin.

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An analytical method for the quantitative determination of monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT) and triphenyltin (TPhT) compounds in human breast milk is described. After the addition of surrogates (deuterium derivatives), milk samples were extracted with hexane-diethyl ether (4:6) in the presence of HCl and NaCl. Each extract was purified by cation exchange chromatography and treated with Grignard reagent to yield ethyl derivatives, which were determined by gas chromatography (GC) with flame photometric detection operated in the tin mode (610 nm). These organotinchlorides, spiked to milk at 12.5, 25, and 50 ng/ml (ppb), were recovered within a range of 85 to 105%. Detection limits were 1.3 ng/ml for DBT, TBT, and TPhT, and 2.5 ng/ml for MBT. This analytical method was used to determine organotins in about 70 breast milk samples obtained from mothers who had given birth within the previous week. DBT dichloride levels varied from undetectable to 9.5 ng/ml in human milk from mothers who habitually ate fish, however, the other organotins were not detectable. No significant difference was observed in DBT contents between mothers who ate fish more than twice a week and those who ate fish less than once a week. Thus, since the levels of organotin even in the milk of mothers who liked to eat fish were very low, human breast milk should be considered safe for feeding infants, at least concerning with regard to the possible transmission of organotin compounds.

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The rapid identification of bacteria in many kinds of samples, i.e., clinical, food, water, and material, is important from a hygienic standpoint. In this paper, we describe the development of a convenient bacterial identification system using bacterial cells by a plastic DNA array. The small plastic base, which was cut from an S-BIO^® PrimeSurface^® plastic base developed for the covalent immobilization of amino-modified DNA, was used as a substitute for a glass base. The species-specific primers were immobilized on the small plastic base and the spots specific to each bacterial species were observed after 2 types of thermal cycles in a single PCR tube using bacterial culture broth as a sample. The results obtained using the culture broth were the same as those obtained with total DNA extracted from bacterial cells. The detection limits of Staphlococcus aureus (S. aureus) ATCC25923 and Escherichia coli (E. coli) ATCC25922 were 8.7×10³ cells/μl and 2.1×10² cells/μl, respectively. This system is useful and convenient for the identification of bacteria in many types of samples. Moreover, further improvements in conditions, such as the ingredients in the reaction mixture, thermal cycles, and the steps of visualization would result in a more efficient system of identification.

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Chronological changes of bisphenol A (BPA) concentration were investigated for a week in mature medaka (Oryzias latipes) and spawned eggs (embryo) after exposing the fish to BPA at a concentration of 100 μg/l in water. The BPA concentrations in mature fish and spawned eggs increased beginning at the day after initial exposure, and reached an approximately constant level on the second day. On the other hand, the decrease in BPA concentration in the parent body was rapid after being placed back into pure water (average decrease was 87% on the first day, and 98% on the second day), while approximately 24% of the BPA in the spawned eggs from the parents remained after the fourth day in pure water. Thus, it was assumed that there is no system for BPA excretion from eggs. Another experiment was conducted, in which eggs spawned from a parent on the fourth day of exposure were raised in pure water. The BPA concentration in the eggs on the sixth day was approximately 29% lower when compared to that at spawning. Therefore, it was assumed that the embryo cannot conjugate BPA incorporated into the egg due to an underdeveloped metabolic or excretion mechanism.

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Seasonal and diurnal fluctuations in pharmaceuticals and personal care products (PPCPs) concentrations in residential sewage water were ascertained in an area with no businesses industry (e.g., plants or offices) upstream. PPCPs with high detection rates included ibuprofen, acetaminophen and indomethacin (antipyretic analgesics), atenolol and disopyramide (antiarrhythmics), clarithromycin (antibiotic), levofloxacin (synthetic antimicrobial agent) and triclosan (disinfectant). In summer, the concentration of triclosan was the highest, while in winter, the concentrations of ibuprofen and acetaminophen were higher than the others. Moreover, three types of diurnal fluctuations were observed: no marked diurnal changes (triclosan), high daytime concentrations (disopyramide) and high nighttime concentrations (acetaminophen).

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Recent studies have provided evidence that exposure to manganese (Mn) induces Parkinson's disease (PD)-like symptoms. We investigated the mechanism of Mn neurotoxicity in the SN4741 dopaminergic (DA) neuronal cell line. The results indicated that the p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases were activated during DA cell death by MnCl₂. Moreover, p38 inhibition with SB203580, a specific inhibitor of p38, induced more toxic effects in MnCl₂-treated cells. Among the p38 subfamily members, p38α attenuated the caspase-3 activation and cell death induced by MnCl₂. However, the expression of JNK stimulated the activity of caspase-3 and mediated the Mn-induced cell death. These results suggest that there are multiple pathways in MnCl₂-induced DA neuronal cell death.

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