Endothelium forms an innermost lining of blood vessel and it regulates the vascular tone and permeability. A healthy vascular endothelium is antiatherogenic in nature because of its properties such as inhibition of platelet aggregation, adhesion cascades, smooth muscle cell proliferation and leukocyte adhesion. Vascular endothelial dysfunction (VED) is associated with reduced synthesis and release of nitric oxide, proinflammatory and prothrombotic properties followed by diminished vasodilation. VED has been implicated in the pathogenesis of artherosclerosis, hypertension, myocardial infarction, heart failure, renal failure and stroke. Various pharmacological interventions such as angiotensin converting enzyme (ACE) inhibitors, statins, insulin sensitizers, L-arginine as well as agents that target endothelial nitric oxide synthase (eNOS) “coupling” such as folates or tetrahydrobiopterin (BH4) have been noted to improve the function of vascular endothelium. In this review, we discussed various recently developed pharmacological interventions to improve the function of endothelium. Moreover, the novel targets sites involved in the pathogenesis of vascular endothelial dysfunction have been delineated.

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The reaction of 4,4'-methylenedianiline (MDA) with chlorine in aqueous solution was investigated by gas chromatography/mass spectrometry (GC/MS) in order to determine its chemical changes in water. MDA was shown to react readily with chlorine under conditions likely to be found during water treatment disinfection. The reaction of MDA with chlorine in water was dependent on the solution pH and the molar ratios of chlorine to MDA. The disinfection products of p-benzoquinone, p-chloroaniline, 2,4-dichloroaniline, 2,4,6-trichloroaniline, and p-aminophenol were identified and them of p-aminobenzyl chloride, p-aminobenzaldehyde and p-methoxyaniline were estimated in the chlorination MDA solution. The mutagenic activity of the chlorination byproducts extracted from the chlorination MDA solution was evaluated using umu-test.

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The current situation of chiral drug development in Japan was investigated. The trend in the Japanese pharmaceutical development is increasingly moving towards the development of single isomers rather than racemates. The development of single-enantiomer drugs was made possible by the current technologies of asymmetric synthesis and chiral separation, and encouraged by the guidelines on the development of chiral drugs worldwide. Japan has not issued specific guidelines on the development of chiral drugs, however, the chiral drug development approached in Japan were essentially consistent with the approaches recommended by the U.S.A. and EU guidelines.

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To examine the association between poor mental health state and having headache in the Japanese general population, and the association between demographic characteristics and use of medical resources among subjects with headache. The subjects aged 18 through 75 years were chosen from the Japanese general population. We administered a questionnaire to obtain baseline data, and asked the participants to keep a personal health-diary during October 1-31, 2003. The state of mental health (MH) was examined using the SF-8 Health Survey in the baseline questionnaire. Of the 2371 subjects, 716 had a headache on at least one day during the study period. Of the 716, 52 consulted a physician, 475 used other medical resources, and 189 did nothing about the headache. There was an association between MH score and headache [adjusted odds ratio (OR)=1.166 (95% confidence interval (CI): 1.010-1.346) for 10-point increment of MH score]. There was also an association between use of medical resources and having a family physician. Poor mental health was associated with headache in the Japanese general population, and one of the predictors of consulting a physician among the headache sufferers was having a family physician whom the subject greatly trusts.

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Neurological diseases such as ischemia and dementia increase with aging, while whether seizure susceptibility increases with aging remains debatable. Seizure activity frequently originates in the hippocampus and is linked to excess of extracellular glutamate. To analyze seizure susceptibility in aged rats, the response of hippocampus excited excessively was evaluated based on extracellular concentrations of neurotransmitters and epileptic behavior. In 8-week-old and 90-100-week-old rats, which exhibited normal passive avoidance behavior, the hippocampus was stimulated with 50-100 mM KCl using in vivo microdialysis. The basal concentrations of extracellular glutamate and γ-amino butyric acid (GABA) were almost the same between young and aged rats. The changes in their concentrations after stimulation with 50-100 mM KCl were not also appreciably different between them. However, seizures, i.e., myoclonic jerks, were observed only in aged rats during the stimulation. These results suggest that aged rats are vulnerable to seizures induced by dyshomeostasis of potassium and chloride ions in the hippocampal extracellular fluid. Homeostasis of electrolytes in the hippocampal extracellular fluid seems to be important in preventing seizure activity in the elderly.

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The aim of this study was to investigate the role of ascorbic acid (AsA) in neural differentiation induced by nerve growth factor (NGF) in PC12 cells, a neural precursor cell line. Cells were cultivated in the presence of L-ascorbic acid-2-O-α-D-glucopyranoside (AA-2G) and evaluated for potentiation of neural differentiation induced by a small amount of NGF. AA-2G enhanced the proportion of neurite-bearing cells in a concentration-dependent manner (<1 mM). When cultivated with relatively high concentrations of AA-2G (1-5 mM), the cells tended to extend neurites further and to form a neuron-like network. The resultant number of neurite-bearing cells was apparently increased by the addition of AA-2G, with a concurrent decrease in the number of total cells in the same culture dish. These results suggest that AsA in the brain not only promotes NGF-induced differentiation of neural precursor cells but also participates in the development of neural network-forming cells.

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The extract of Sargassum horneri (S. horneri) has been shown to have an anabolic effect on bone components due to stimulating bone formation and to inhibiting bone resorption in rat femoral tissues in vitro and in vivo. This study was undertaken to determine the effect of supplemental intake of the water-solubilized S. horneri extract on circulating bone metabolic markers in healthy humans. Thirty-six volunteers, aged 20-60 years (16 men and 20 women), were enrolled in this study. Volunteers were divided into three groups; placebo tablet without S. horneri extract (5 men and 7 women), tablet containing S. horneri extract at 300 mg/day (6 men and 7 women) or 900 mg/day (5 men and 6 women). Placebo or S. horneri extract tablet was ingested once a day for 4 or 8 weeks. Bone-specific alkaline phosphatase and γ-carboxylated osteocalcin are serum bone markers of bone formation, and bone tartrate-resistant acid phosphatase (TRACP) and N-telopeptides of type I collagen are markers of bone resorption. Serum bone-specific alkaline phosphatase or γ-carboxylated osteocalcin concentration was not significantly changed after the intake of S. horneri extract (300 or 900 mg/day) for 4 or 8 weeks. Serum bone TRACP activity was significantly decreased after the intake of S. horneri extract (300 or 900 mg/day) for 8 weeks. Serum N-telopeptides of type I collagen concentration was significantly decreased after the intake of S. horneri extract (900 mg/day) for 8 weeks. Meanwhile, serum calcium, inorganic phosphorus, and other biochemical findings were not changed after the intake of S. horneri extract (300 or 900 mg/day) for 4 or 8 weeks. This study demonstrates that the prolonged intake of S. horneri extract has inhibitory effects on bone resorption in humans.

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Homocysteine is a risk factor for vascular diseases such as atherosclerosis, and proteoglycans (PGs) derived from arterial smooth muscle cells are the key molecules in atherosclerosis progression. We investigated the effect of homocysteine on the synthesis of PGs in vascular smooth muscle cells in vitro. Homocysteine significantly decreased the accumulation of PGs, particularly in the conditioned medium, independent of cell density without nonspecific cell damage. DEAE-Sephacel ion exchange chromatography of PGs showed that homocysteine selectively decreases chondroitin/dermatan sulfate PGs (CS/DSPGs) with low charge density, i.e., the small CS/DSPG biglycan and decorin, in the conditioned medium. Fluorophore-assisted carbohydrate electrophoresis analysis demonstrated that homocysteine selectively increases the disaccharide unit of iduronic acid-4-O-sulfated N-acetylgalactosamine in chondroitin/dermatan sulfate chains. However, no change was observed in heparan sulfate chains. Therefore, it is suggested that homocysteine inhibits the synthesis of small CS/DSPGs with low charge density in vascular smooth muscle cells. Furthermore, homocysteine affects the microstructure of the chondroitin/dermatan sulfate chains. These alterations may influence the progression of atherosclerosis.

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The genotoxicity of furfuryl alcohol and 2-furyl methyl ketone was assessed in human lymphocytes in vitro using standard chromosome aberration analysis and sister chromatid exchange (SCE) analysis. Three doses ranging from 240 to 960 ppm which corresponded to in vivo doses were selected. Lymphocytes were cultured using TC-199 medium plus required supplements and initiated using blood drawn from healthy human donors who had not been exposed to any drugs or chemicals for a substantial period of time. After 24, 48, and 72 hr of compound exposure, slides were prepared and analyzed for variations in the mitotic index (MI) and chromosome aberrations. For SCE analysis, 10 μg of 5-bromodeoxy uridine was added 24 hr after culture initiation and slides were prepared and stained using the standard technique. Two hundred metaphase spreads were scored per dose per period to analyze chromosome aberrations, and 5000 cells were randomly scored to calculate the MI. Seventy-five metaphase spreads in second mitotic divisions were scored for each dose and period for SCE analysis. All data were subjected to stastical analysis. There was an insignificant induction of chromosome aberrations and mitodepression (MD), while SCEs showed an increased incidence with both compounds which was dose and period dependent.

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The quantity of D-malate was determined using apparatus comprised of a reactor with immobilized D-malate dehydrogenase (D-MDH) in a flow line. NADH formed by an enzymatic reaction was fluorometrically detected. The optimal concentration of NAD⁺ in the carrier was determined. The maximum peak areas due to NADH were observed at pH 8.0 when the pH of the carrier consisting of piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES) buffer ranged from 7.0 to 8.5. Various buffer types were also examined as carrier media at pH 8.0, and PIPES buffer showed the maximum peak area. When the carrier composed of PIPES buffer (0.1 M, pH 8.0) containing 1 mM NAD⁺ and 5 mM MgCl₂ was used, the calibration curve for D-malate was linear in the range of 0.02-50 μM (r=1.000). The detection limit (S/N=3) was 0.01 μM. Relative standard deviations of the peak area at 1 μM and 10 μM were 1.6% (n=7) and 0.48% (n=7), respectively. This method was applied to the analysis of D-malate in several beverages, and the recovery test of the added D-malate to samples was also carried out to afford good results.

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We investigated the active components responsible for the anti-allergic effect of Hypsizigus marmoreus on an oxazolone-induced type IV allergy in male ICR mice. Following both an oral administration at a dose of 250 mg/kg body weight and percutaneous administration at 0.10 mg/ear, significant anti-allergic activities were found in the 80% ethanolsoluble fraction (Fr. 1), and the low molecular weight fraction (Fr. 2, below a molecular weight of ca. 12000; Fr. 4, below ca. 3000) prepared from Fr. 1. These fractions included ergosterol, mannitol, and trehalose as antioxidants and/or anti-inflammatory components. The anti-allergic activities of these compounds and methionine were equal to or weaker than those of the fractions. Therefore, the molecular weights of the major active components in Hypsizigus marmoreus are thought to be less than ca. 3000, though ergosterol, mannitol, trehalose, and methionine would not be the main active components.

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Multiple antibiotic-resistant Helicobacter pylori (H. pylori), one of the major causes of gastric cancer, is now increasingly reported. The aim of this study was to screen medicinal plants widely used in Thailand as possible sources of medicines that can be used to treat H. pylori infection. Twenty-four extracts from 13 kinds of Thai herbs were tested for their antibacterial activity against 20 strains of antibiotic-resistant H. pylori. Inhibition of growth was tested by the paper disc agar diffusion method. Most strains of H. pylori examined were proved to be susceptible to seven medicinal plants; i.e., Peltophorum pterocarpum, Piper betle, Punica granatum (P. granatum), Quercus infectoria (Q. infectoria), Tamarindus indica, Uncaria gambir, and Walsura robusta. Among these extracts, P. granatum and Q. infectoria exhibited the greatest inhibitory potencies. Minimal inhibitory concentrations (MIC) were determined by the agar dilution method in Petri dishes with a Millipore filter membrane, and minimal bactericidal concentrations (MBC) were assessed with the extract that gave a significant MIC value against each bacterial strain by placing the Millipore filter membrane onto a fresh Isosensitest agar plate. Ethanolic extracts of P. granatum and Q. infectoria significantly reduced the growth of all strains of H. pylori, with the best MIC values at 0.8 and 3.1 mg/ml, and the best MBC values at 3.1 and 6.2 mg/ml, respectively. Effective fractions partially purified from both plant species yielded MICs and MBCs that were at least 10-fold less compared with the crude extracts. From the data obtained, it is hoped that P. granatum and Q. infectoria will become useful sources with which to develop new therapeutic agents for H. pylori infection.

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We developed a method for determining the following ten psychedelic phenethylamines and tryptamines by gas chromatography-mass spectrometry (GC-MS). The phenethylamines examined were 3,4,5-trimethoxyamphetamine (TMA), 2,4,5-trimethoxyamphetamine (TMA-2), 4-bromo-2,5-dimethoxyphenethylamine (2C-B), 4-iodo-2,5-dimethoxyphenethylamine (2C-I), 2,5-dimethoxy-4-ethylthiophenethylamine (2CT-2), and 2,5-dimethoxy-4-propylthiophenethylamine (2CT-7). The tryptamines examined were 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), α-methyltryptamine (AMT), N-isopropyl-5-methoxy-N-methyltryptamine (5-MeO-MIPT), and N,N-diisopropyl-5-methoxytryptamine (5-MeO-DIPT). As the reference standards, five compounds were newly synthesized, and five were purified from the products. These were all structurally ascertained by GC-MS and nuclear magnetic resonance (NMR) spectroscopy. Between April 2005 and March 2007, 8 compounds analyzed in this study were found in 100 out of the 178 products which were examined.

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Levels of serum lipids such as total cholesterol (TCHO), free cholesterol (F-CHO), low density lipoprotein cholesterol (LDL-C), triglycerides (TG), and phospholipids (PL) were examined in both sexes of Meishan pigs. In young adult pigs (4- to 5-monthold), levels of all above lipids mentioned were significantly higher in females than in males, while no such sex difference was observed until the age of 3 months. In young adult male pigs castrated at the age of 1 month, levels of these lipids were almost the same as levels in corresponding female pigs. In addition, no sex differences in the levels of high density lipoprotein cholesterol and non-esterified free fatty acids were observed. The present findings indicated that androgen might act as a down-regulator for the levels of serum lipids, such as T-CHO, F-CHO, LDLC, TG and PL, in pigs.

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Surfactin C is a biosurfactant produced by Bacillus subtilis from Korean soybean paste. Surfactin C is known to have several therapeutic effects including anti-inflammatory, fibrinolytic, and thrombolytic activities. However, there is little information concerning its safety. In this study, we evaluated the genetic and developmental toxicity of surfactin C. Bacterial reverse mutation and rodent micronucleus assays were performed to determine its genotoxic potentials. Surfactin C at 0, 125, 250, and 500 mg/kg of body weight/day was administered orally to pregnant ICR mice during the period of major organogenesis. There was no genetic toxicity related to surfactin C treatment in in vitro and in vivo systems. In the developmental study, surfactin C did not demonstrate maternal toxicity, fetotoxicity, and teratogenicity, and hence the no observed effect level was concluded 500 mg/kg per day in ICR mice.

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The stochastic properties of the daily variations in drug sales at a community pharmacy in the season of 2004/2005 are studied by a factor analysis. The four factors which can closely be related to chronic diseases, common cold, influenza and allergies are extracted from the data of 31 drugs. For most of the drugs, the sales time series can approximately be represented by the four factors alone. Similar results are obtained from the same pharmacy in the 2003/2004 season and also from two other pharmacies. Interestingly, the classification of drugs according to the factor analysis corresponds to their medicinal actions.

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Molecules with bi-functional activities are preferred targets in drug discovery research today. In this study, a possible dual role (anti-inflammatory and anti-bacterial activity) of a plant extract has been discussed. As an initiative, the extract is found to suppress inflammation caused by endotoxin release from an enteric pathogen. Enteropathogenic Escherichia coli (E. coli) (EPEC O127:H6) causes persistent diarrhea and continues to be a major health problem among infants in developing countries. Host inflammatory response during EPEC infection is weak when compared to infections caused by other enteric pathogens. This is due to the suppression of intestinal inflammatory response by type III secreted proteins of EPEC during infection process. The weak inflammatory response is thus possibly due to disruption of tight junctions, which in turn facilitate recruitment of macrophages from the underlying lamina propria or shedding of bacterial products like lipopolysaccharide (LPS) during antibiotic mediated bacterial lysis. Since intestinal cells are non-responsive to LPS, the inflammatory response would be elicited by the intestinal macrophages present at the site of infection. Apart from commercial LPS from E. coli O111:B4, LPS was isolated from EPEC and used for inducing inflammatory markers like TNFα, IL-1β, IL-8 and TLR4 in J774 A.1 murine macrophage like cells to make this study clinically relevant. Alpinia officinarum is a traditional, perennial herb used widely in China and India for treating arthritis and gastrointestinal disorders. The ethyl acetate extract of Alpinia officinarum effectively suppressed EPEC LPS induced inflammatory response thereby exhibiting potential in a clinical scenario, wherein diarrhea induced by EPEC or products of its lysis is either a consequence of macrophage induced inflammation or antibiotic treatment or due to a potential, yet to be analyzed, anti-bacterial activity of Alpinia officinarum extract against EPEC.

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Endocrine disrupting activities of three isomers of monohydroxylated 1-nitropyrene (1-NP) [3-, 6-, and 8-hydroxy-1-nitropyrenes (OHNPs)] were evaluated for the first time by yeast two-hybrid assay. OHNPs, which are not only metabolites of 1-NP but are also found in airborne particles, did not exhibit androgenic activity but exhibited estrogenic, antiestrogenic, and antiandrogenic activities. 6-OHNP showed the strongest estrogenic activity among the three OHNP isomers examined in this study. Concentrations of the OHNP isomers that gave 10% of activity of 1.0×10^-6M 17β-estradiol (E₂) were as follows: 3-OHNP, 6.0×10^-7M; 6-OHNP, 6.0×10^-8M; 8-OHNP, 9.0×10^-7M. On the contrary, 8-OHNP exhibited the strongest antiestrogenic and antiandrogenic activities of the three isomers. 1.0×10^-6M of 8-OHNP inhibited 32 and 90% of β-galactosidase activity induced by 1.0×10^-9M of E₂ and 1.0×10^-8M of 5α-dihydrotestosterone (DHT), respectively. These findings point out the necessity for detailed investigation of environmental sources and distributions of OHNPs as well as the parent 1-NP.

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We applied transcription in vitro following multiplex PCR in order to improve the sensitivity and rapidity of microbial detection in aquatic environment with an oligonucleotide microarray. Transcripts of 16S rRNA gene were fluorescently labeled and hybridized on fabricated oligonucleotide chip. The assay sensitivity was evaluated by detecting cultured bacteria inoculated into natural river water. By using our procedure, the assay was completed more rapidly (6 hr) than conventional oligonucleotide microarray assays (>12 hr), and its sensitivity was improved: detection limit was decreased by one order of magnitude. This method might be useful for monitoring pathogenic bacteria in aquatic environments.

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