Amphetamine-type stimulants (ATS), having one or more asymmetric carbons, are important targets in forensic science field. For chiral analysis, capillary electrophoresis (CE) has advantages over other chromatographic methods, especially, in fastness, resolution, cost performance and simplicity. This review summarizes chiral separation techniques of ATS using CE with UV detection or CE/mass spectrometry mainly by the present authors' research group.
Fibroblast growth factors (FGFs) stimulate the proliferation of a variety of cells and protect against stress-induced apoptosis. We have demonstrated that basic FGF (bFGF) significantly stimulates intestinal cell proliferation and protects against dexamethasone-induced apoptosis. In this study, we further investigate the effects of bFGF on radiation-induced inhibition of thymocyte and splenocyte proliferation and apoptotic cell death. Mice were subjected to whole-body X-rays irradiation by at a dose of 0.5 Gy or 1.0 Gy and 14 hr later thymocytes and splenocytes were collected for in vitro culture with or without bFGF. Radiation induced a significant decrease in the cell proliferation of both thymocytes and splenocytes, which was not significantly inhibited by incubation with bFGF. Radiation with 0.5 and 1.0 Gy also significantly increased apoptotic cell death in both thymocytes and splenocytes, and incubation with bFGF did not significantly affect the apoptotic effects of radiation. These results suggest that incubation of bFGF in vitro with thymocytes and splenocytes from irradiated mice did not significantly prevent radiation-induced inhibition of cell proliferation and apoptotic effects.
We established a method for determining capsaicin glucuronide in rat urine samples using liquid chromatography-mass spectrometry combined with enzymatic hydrolysis. Capsaicin was not detected in urine samples of rats administered capsaicin intraperitoneally (i.p., 2 and 4 mg/kg), but after hydrolysis with β-glucuronidase, extraction with methanol and solid-phase extraction, capsaicin was clearly detected by liquid chromatography-mass spectrometry with electrospray ionization. The limit of detection was obtained to be 0.1 ng/ml in urine sample matrix. The conditions for the enzymatic hydrolysis of the conjugate were optimized for β-glucuronidases from Ampullaria, Escherichia coli, bovine liver, Helix pomatia and Patella vulgata. The optimal conditions among those examined [pH (3.3-9.0), temperature (37-70°C) and enzyme amount (50-2500 U)] for 1 ml of the urine sample were as follows: β-glucuronidase from Ampullaria (pH 4.2, 45°C, 500 U), from Escherichia coli (pH 6.0-7.2, 37°C, 500 U), from bovine liver (pH 5.0, 45°C, 500 U), from Helix pomatia (pH 5.0, 60°C, 1250 U) and from Patella vulgata (pH 3.8, 45-60°C, 2500 U). Among the enzymes examined, β-glucuronidase from Ampullaria was found to be suitable for hydrolysis of the conjugate. Under the optimal conditions of Ampullariaβ-glucuronidase, the incubation of 1 ml of urine sample for 90 min was sufficient for hydrolysis of capsaicin glucuronide in the urine sample. The urinary recovery values of capsaicin collected for 0-48 hr after administration of 2 and 4 mg/kg capsaicin to rats were 1.4 and 1.1%, respectively.
The microbial water quality in canals of metropolitan Bangkok, Thailand, was assessed using the fluorescent bacteriophage assay (FBA) and fluorescence in situ hybridization (FISH). When FISH was used with oligonucleotide probes targeted to Escherichia coli (E. coli) and anaerobic Bacteroides group, < 0.1% to 7% and < 0.1% to 11%, respectively, of the total bacteria were identified. Estimates of viable E. coli determined by FBA with 4',6-diamidino-2-phenylindole (DAPI)-labeled T4 bacteriophage, which accounted for 1 to 8% of total cells, were significantly higher than those of fecal coliforms determined by the most probable number method. The FBA procedure can specifically detect viable E. coli in canal water samples that are grossly contaminated with fecal flora within 30 min, and it can quickly deliver accurate findings for risk assessment of environmental contamination in water samples.
Licorice flavonoid oil (LFO) is a new dietary ingredient for functional foods consisting of licorice hydrophobic polyphenols in medium-chain triglycerides (MCT). In an effective dose finding study conducted previously, LFO has exhibited a dose-dependent body fat-reducing effect. Here we report the weight-reducing effect of LFO in a placebo-controlled, double-blind, long-term (12 weeks) ingestion study at 300 mg/day, the minimal effective dose observed in the dose finding study. A total of 103 overweight subjects [body mass index (BMI): 24-30] completed this study and were analyzed. Body weight increased in the placebo group, but was maintained at close to pre-ingestion level in the LFO group, resulting in significant (p < 0.05) differences in the changes in body weight and BMI between the LFO group and the placebo group at each time-point. Dual-energy X-ray absorptiometry (DXA) measurement of body fat indicated that the weight-reducing effect was attributable to reduced body fat. No clinically significant adverse events occurred during the 12-week ingestion period. To confirm the safety of LFO for practical use we also conducted a placebo-controlled, double-blind safety study in 40 overweight subjects with a 4-week excessive ingestion at 1800 mg/day; 6 times the dose of the 300 mg/day study that exhibited a weight-reducing effect. No clinically significant adverse events occurred during the 4-week ingestion period. Based on these findings in both human studies it was shown that LFO is a safe ingredient for functional foods even for long-term or excessive ingestion, with a potential weight-reducing effect.
To determine the effect of concomitant oral ingestion of germanium-132 (Ge-132) and curcumin on the onsets of hepatitis and hepatic cancer in Long-Evans Cinnamon (LEC) rats, 40 rats were administered sterile water (NT), corn oil (Vehicle), Ge-132 (Ge), curcumin (Cur), or Ge-132 plus curcumin (GeCur) for 50 weeks. Plasma enzyme levels of asparate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyltrasferase (γGTP), and lactate dehydrogenase (LDH) were measured at Pre (6 weeks), Early (18-26 weeks), Late (30-38 weeks) and End (42-50 weeks) stages. Liver damage was assessed by the Histology Activity Index (HAI). In the group of Ge, LDH was significantly decreased at the End stage. In the group of Cur, LDH was remarkably decreased in the Early stage, whereas AST and LDH were significantly decreased in the End stage. In the GeCur group, AST, ALT, and γGTP were significantly increased in the Early stage, whereas LDH was significantly decreased in the End stage. The onset rate of icterus and the mortality rate were significantly increased in the GeCur groups (vs. Vehicle group, p < 0.01). There was no statistically significant difference in HAI among the groups. Thus, concomitant oral ingestion of Ge-132 and curcumin may aggravate hepatic dysfunction, thereby increasing the mortality rate in LEC rats.
Dynamics of redox relating biotrace elements, selenium (Se), iron (Fe), and zinc (Zn) in liver, kidney, and spleen of selenium deficient Wistar male rats in a series of feeding period (from 0 to 8 weeks) were studied using instrumental neutron activation analysis (INAA). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) for the plasma fraction of the rat bloods and the concentration of vitamin C and vitamin E in the liver homogenates were measured. The initial purpose of this study was to find Fe and Zn as sensitive indices of the tissue oxidative stress levels. However, the relationships among the biotrace elements and the oxidative stress/injury were much complicated. Control group, which was fed Se-deficient diet with Na2SeO4 (0.1 mg selenium/l) in drinking water, showed strange response of Se and Zn contents in the kidney and showed high BUN. Supplementation of inorganic Se by biased Se source may serve as another source of a stress especially in the kidney. The Fe and Zn contents in the liver and kidney look sensitive to the Se-deficiency and/or relating oxidative stresses. Short term exposure to the Se-deficiency appeared to consume Fe and Zn in the liver and kidney. In contrast, long term or chronic exposure to Se-deficiency appeared to accumulate Fe and Zn in liver and kidney.
The abundance and phylogenetic composition of antibiotic resistant bacteria in canals of metropolitan Bangkok, Thailand, were investigated using a microcolony method and fluorescence in situ hybridization (FISH). Cells were directly trapped from aquatic samples onto polycarbonate membranes and incubated for 24 hr on selective agar containing antibiotics. Individual antibiotic resistant bacterial microcolonies samples were classified on the filter using FISH with rRNA-targeted probes. The numbers of microcolony forming units (mCFU) on selective medium containing antibiotics were 0.5 to 8.1-fold (average, 3.4-fold) higher than those of colony forming units (CFU) in all samples, and mCFU and CFU closely correlated in all samples (r2 = 0.89). Estimates of Escherichia coli (E. coli) determined by FISH with rRNA-targeted probe accounted for approximately 1% of bacteria detectable by probe EUB338 among microcolony-forming bacteria on nonselective medium. However, they accounted for approximately 10% of bacteria detectable by probe EUB338 among microcolony-forming norfloxacin/tetracycline-resistant bacteria. Microcolony-FISH on selective medium containing antibiotics would be a valuable tool that could help in obtaining information about the numbers and phylogenetic affiliations of yet-to be-cultured antibiotic-resistant bacteria in aquatic environments.
The suspension and the boiling water extract of dried powder from the aerial parts of Bidens pilosa L. var. radiata SCHERFF (Tachiawayukisendangusa: MMBP) on the Japanese island of Miyako have antiinflammatory and antiallergic properties in experimental diseases. Oral administration of MMBP suspension in carboxy-methyl-cellulose sodium solution inhibited the production of IgE 10 days after immunization with DNP-ascaris in mice. The extract inhibited histamine release from rat peritoneal mast cells induced by compound 48/80 or antigen-antibody reaction. Oral administration of the suspension inhibited dye exudation in rat skin induced by passive cutaneous anaphylaxis. Oral administration of the suspension inhibited dye exudation in rat skin induced by chemical mediators (histamine, substance P, and serotonin). These findings suggest that MMBP may be clinically useful in the prevention of type I allergic disease.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is frequently used to analyze urinary proteins, but appropriate urine sample preparation is essential, because urinary proteins are present in small concentrations, and urine contains high concentrations of salts and metabolic wastes. The aim of this study was to determine the optimum method for urine sample preparation. The effects on urinary protein recovery of pretreating the ultrafiltration apparatus with various agents (Triton X-100, Tween20, PEG compound, and SDS) were studied; Triton X-100 was found to be the most effective agent. Ultrafiltration and acetone precipitation were compared as sample preparation methods for SDS-PAGE. Recovery of urinary protein using ultrafiltration (89.5 ± 8.1%) was better than that achieved by using acetone precipitation (75.1 ± 12.2%) (p < 0.01). Integrated densitometric values for five protein bands (62, 52, 39, 31, and 6 kDa) were higher for ultrafiltration samples than for acetone samples (42350 ± 2568 vs. 37010 ± 725, 34665 ± 1519 vs. 32355 ± 2278, 10755 ± 1683 vs. 8870 ± 941, 65920 ± 3354 vs. 60680 ± 3651, and 1355 ± 160 vs. 1130 ± 211, respectively) (p < 0.05), although differences for the other nine bands were not significant. In conclusion, ultrafiltration is a suitable method for preparing urine samples for SDS-PAGE.
A novel aseptic urine collection kit has been devised, and its effectiveness in collecting and storing bacteria-free urine specimens was evaluated. The kit, which is based on filtration sterilization, was proven effective for these purposes. For the evaluation of its efficiency, urine samples spiked with nitrazepam at 0.5 μg/ml were treated with the kit and monitored for 6 months at 4°C and 25°C. An automated column-switching liquid chromatography-mass spectrometry (LC-MS) procedure was utilized for the direct determination of the analyte in urine. In severely-contaminated urine with bacteria, there were significant losses of nitrazepam at 25°C. However, such degradation was successfully suppressed by the use of the kit even at a storage temperature of 25°C, while requiring no chemical additives.
Relation between blood uric acid levels and brain cell functions were examined using potassium oxonate-induced hyperuricemic mice and allopurinol-induced hypouricemic mice. In hyperuricemic mice and hypouricemic mice, erythrocyte catalase activity was significantly lower and higher, respectively, than that in the control group. No significant change of superoxide dismutase (SOD) activity or glutathione peroxidase activity was observed in either group. Cerebral lipid peroxide levels expressed as thiobarbituric acid reactive substances (TBARS) were significantly higher in hyperuricemic mice, and cerebral mitochondrial ATP synthesis and manganese-containing SOD (Mn-SOD) activity were significantly diminished in the same mice. In hypouricemic mice, no significant change was observed for TBARS levels, mitochondrial ATP synthesis and Mn-SOD activity. Then significant negative correlation between erythrocyte catalase activity and cerebral TBARS levels, and significant positive correlation between catalase activity and mitochondrial ATP synthesis were confirmed. Furthermore, when mouse erythrocytes were treated with uric acid, their catalase activities were particularly diminished. These results suggest that the reduction of erythrocyte catalase activity resulted from the elevation of blood uric acid levels is a major cause of cerebral oxidative stress and mitochondrial dysfunction in hyperuricemic mice. Therefore, it is possible that hyperuricemia and gout are risk factors of cerebral disorders.
Although anti-inflammatory effect of human placenta extract (HPE) was observed in rheumatoid arthritis and carrageenin-induced edema, effect of HPE on the arthritis of hyperuricemia and gout patients had never been examined. Excess uric acid is regarded to be a major risk factor in gout, renal diseases, cardiovascular diseases and cerebrovascular diseases. In our experiment, in order to investigate the effect of HPE on blood uric acid levels and xanthine oxidase (XO) activity, HPE 100 mg/kg was administered to the abdominal cavity of potassium oxonate-induced hyperuricemic mice. Blood uric acid levels and XO activity were measured 3 hr after administration. Furthermore, effect of HPE on in vitro superoxide anion generation in xanthine-XO system was also measured. In experimental mice, oxonate-induced significant elevation of blood uric acid levels was suppressed by the HPE administration similar to the allopurinol administration. Similarly, both in HPE-treated mice and in allopurinol-treated mice, blood XO activity was significantly reduced in comparison with vehicle-treated mice and oxonate-treated mice. Furthermore, there was significant positive correlation between blood uric acid levels and XO activity (p < 0.01). In in vitro experiments, xanthine concentration was increased by the addition of HPE while simultaneously reducing uric acid concentration. These results indicate that HPE contains some XO inhibitors which reduce blood uric acid levels with inhibiting uric acid generation in purine metabolism. Then, XO inhibitors of HPE could be used in the treatment of hyperuricemia and gout patients to lower blood uric acid levels.
Previously, we have reported that geniposide, a compound isolated from an extract of Gardenia fructus, has neuritogenic activity in PC12h cells, a subclone of the rat pheochromocytoma cell. In this study, we have examined the effects of seven geniposide-related compounds (S-1, 6α-hydroxygeniposide; S-2, 6β-hydroxygeniposide; S-3, 6α-methoxygeniposide; S-4, 6β-methoxygeniposide; S-5, loganin; S-6, 7-ketologanin; and S-7, syringopicroside) isolated from various medicinal herbs. The geniposide-type iridoids S-1, S-2, S-3, and S-4, and S-7 induced neurite outgrowth that was similar or more potent to that of geniposide. S-2 and S-4, which are optical isomers of S-1 and S-3, respectively, were particularly potent. The 2 loganin-type iridoids, S-5 and S-6, showed less activity than geniposide. The neuritogenic activity of geniposide-type iridoids appears to be not necessarily correlated directly to their hydrophobicity. These results suggest that geniposide-type iridoids have potent neuritogenic activity and that specific configurations for the interactions between iridoid compounds and the target molecule are necessary for neuritogenic function.
There has been a growing interest in studying the role played by lipid peroxidation and antioxidant status in breast cancer. Free radicals are found to be involved in both initiation and promotion of multistage carcinogenesis. In the present study, effects of Kalpaamruthaa (KA), a modified Siddha preparation on the levels of lipid peroxides (LPO) and status of antioxidants in several tissues were studied in mammary carcinoma rats. The levels of reactive oxygen species (ROS) were also measured in the control and experimental animals. A significant increase in the levels of LPO, ROS and a decreased levels of antioxidants observed in mammary carcinoma bearing rats were found to be reverted back to near normal levels on treatment with KA. Simultaneous treatment with KA showed more effect than post treatment with KA. Drug control animals showed no significant changes in the levels of ROS when compared with control animals. These results suggest that the free radical mediated damage during mammary carcinoma could have been controlled by KA by its free radical quenching and antioxidative potential. The above results also show that KA exert its anticancer effect on the development of breast cancer.
The effects of prolonged intake of juice prepared from Satsuma mandarin (Citrus unshiu MARC.) containing β-cryptoxanthin on circulating biochemical markers of bone metabolism in subjects, including menopausal woman, were investigated. Ninety volunteers, aged 27-65 years (19 men and 71 women), were enrolled in this study. The 71 females included 35 premenopausal women (ages, 27-50 years) and 36 postmenopausal women (ages, 46-65 years). Volunteers were divided into four groups; placebo juice without β-cyptoxanthin (5 men and 19 women), juice containing β-cyptoxanthin at 1.5 mg/200 ml of juice/day (4 men and 17 women), 3.0 mg/day (5 men and 17 women), and 6.0 mg/day (5 men and 18 women). Placebo or juice (200 ml) was ingested once a day for 28 or 56 days. Serum β-cryptoxanthin concentrations were significantly increased after the intake of juice containing β-cryptoxanthin (1.5, 3.0, or 6.0 mg/day) for 28 or 56 days, and the increases were dose-dependent. 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. Bone-specific alkaline phosphatase activity was significantly increased after the intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/day) for 56 days as compared with the value obtained before intake. γ-Carboxylated osteocalcin concentration was significantly increased after the intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/day) for 28 or 56 days as compared with the value obtained before intake or after the intake of placebo juice. Serum TRACP activity and type I collagen N-telopeptide concentration were significantly decreased after the intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/day) for 28 or 56 days as compared with the value obtained before intake or after intake of placebo juice, and significant decreases were also seen after the intake of 1.5 mg/day β-cryptoxanthin as compared with the value obtained before intake. In menopausal women, bone-specific alkaline phosphatase activity and γ-carboxylated osteocalcin concentration were significantly increased after the intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/day) for 56 days as compared with the value obtained after placebo intake. Also, this intake caused a significant decrease in bone TRACP activity. Meanwhile, serum calcium, inorganic phosphorous, and parathyroid hormone (intact) were not changed after the intake of β-cryptoxanthin-containing juice for 28 or 56 days. This study demonstrates that the prolonged intake of juice fortified with β-cryptoxanthin has stimulatory effects on bone formation and inhibitory effects on bone resorption in humans, and that the intake has an effect in menopausal women.
We have previously demonstrated that a natural iridoid compound, genipin, induces neurite outgrowth mediated by nitric oxide (NO) production in PC12h cells. However, genipin could not induce neurite outgrowth by PC12 cells, the parental cells of PC12h cells. The difference in neuritogenic response to genipin may be due to a lack of neuronal NO synthase (NOS) protein, most likely neuronal NOS, in PC12 cells. In this study, we have investigated whether neuronal NOS protein innately expressed in PC12h cells plays any functional role in neuritogenesis. L-Lysine and L-norvaline, inhibitors of arginase which uses the same substrate as NOS, significantly induced neurite outgrowth in PC12h cells but not in PC12 cells. In PC12h cells, L-lysine-induced neurite outgrowth was completely inhibited by a nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) but was not inhibited by its negative isomer D-NAME, suggesting that up-regulation of NOS activity by arginase inhibitors with increasing intracellular concentrations of substrate induces neuritogenesis in NOS-expressing cells. Thus, it is concluded that innately expressed neuronal NOS has a functional role in neuritogenesis in PC12h cells.
Cypermethrin, a synthetic pyrethroid, has broad-spectrum use in agriculture, domestic and veterinary applications due to its high bioefficacy, enhanced stability and considerably low mammalian toxicity. The objective of this study was to investigate the cypermetrin-induced alterations in the liver tissue of Wistar male rats, based on the histopatological, enzymological analyses and apoptotic changes. The animals of the experimental groups were orally fed with laboratory chow combined 60, 150, and 300 mg/kg Kral 250 EC during 28 consecutive days. At the end of the treatment, no significant change was found in relative liver weights, liver total proteins and cholinesterase enzyme activities of cypermethrin treated rats, when compared with control animals. Histopathological changes such as vacuolar degeneration, enlargement of the sinusoids, degeneration in hepatic cords and hepatocytes, vacuole formations in hepatocytes, pleomorphism in nucleus, and congestion were observed in liver tissues of only 150 and 300 mg/kg cypermethrin treated rats. Mononuclear cell infiltration and an increase in the Kupffer cells in liver parenchymatous tissue were also determined. In all cypermethrin treated groups, the apoptotic index in livers of rats was significantly increased compared to control group (p < 0.001). These results suggest that cypermethrin might cause hazardous effects in different levels to non-target organisms.
The effects of graded doses of verapamil on ethanol-induced stomach mucosal damage were studied in rats. Gastric lesions were induced in vivo by oral administration of 80% ethanol and evaluated with regard to ulcer index, gastric mucus content, free and total acidity, lipid peroxidation and nonprotein sulfhydryl groups. Orally administered ethanol markedly increased the ulcer index and lipid peroxidation. Pretreatment of rats with verapamil (1, 5, and 25 mg/kg i.p.) was carried out 1 h before the administration of ethanol. Verapamil showed a protective effect against ethanol-induced mucosal damage only at high dose (25 mg/kg). Verapamil dose dependently decreased the total acidity, lipid peroxidation, and nonprotein sulfhydryl content. Verapamil 25 mg/kg also increased significantly the gastric mucus secretion. L-arginine (100 mg/kg) or L-nitroarginine (100 mg/kg) with verapamil were also administered to the animals to determine the role of nitric oxide in the mechanism of the gastroprotective activity of verapamil (25 mg/kg). The results indicate that reduced acidity and lipid peroxidation and increased mucus secretion participate in the protective effects of verapamil against ethanol damage. On the other hand, a decrease in the nonprotein sulfhydryl content was observed with decreased gastroprotective effects of verapamil.
Blackfoot disease (BFD) is a local endemic disease that prevails in the southwest coastal areas of Taiwan. From a pathological point of view, BFD is a chronic peripheral vascular atherosclerosis obliterans disease affecting the lower limbs. This study compared the calcium, magnesium, potassium, and sodium concentrations found in the hair of hyper endemic village BFD patients (n = 123), and in the hair of inhabitants without BFD (n = 39) by using an atomic absorption spectrophotometer. Analysis revealed, when compared to the hyper endemic village inhabitants without BFD, BFD patients have a higher mean calcium concentration (p < 0.05), but a lower mean magnesium concentration (p < 0.05) and a lower mean potassium concentration (p < 0.01) in hair. In addition, we found BFD patients have a higher ratio of [Ca/Mg] (p < 0.05), but have a lower ratio of [K/Na] (p < 0.01) in hair. From this point of view, we hypothesize the lower mean concentrations of magnesium and potassium and the higher mean calcium concentration may have certain correlation with the BFD. Furthermore, this assumption may be used to explain why some of the hyper endemic village inhabitants can avoid the BFD. However, it needs further investigation.
Previous study on in vitro antiplasmodial activity of diaza phenanthrene analogs indicated that the 1,10-phenanthroline skeleton represents a potential antimalarial leader compound. Based on those skeletons, six derivatives of N-alkyl and N-benzyl-1,10-phenanthroline were synthesized and the in vitro antiplasmodial activities was evaluated. This paper reported the in vivo antiplasmodial activity study of the 1,10-phenanthroline derivatives performed by the classical 4-day suppressive test against Plasmodium berghei. Acute toxicity of each compound was determined after a single injection of the compound intraperitoneally in Swiss mice. The 50% effective dose (ED50) of the compound ranged from 2.08 to 50.93 mg/kg of body weight, and the therapeutic indices (TIs) ranged from 2.06 to 7.57 except (1)-N-benzyl-1,10-phenantrolinium iodide, which was 58.38. All of the 1,10-phenanthroline derivatives had in vivo antiplasmodial activity and (1)-N-benzyl-1,10-phenantrolinium iodide was the most potent.
The complete microbial degradation of Phthalic Acid (PA) is described. PA was thought to be an endocrine-disrupting chemical. A pure culture (strain No. A-1) from soil sample capable of utilizing PA as the sole source of carbon and energy was identified as Flavobacterium sp. Degradation patterns of PA were observed on the high-performance liquid chromatogram (HPLC) of the culture filtrates of this strain, and growth of bacteria was measured as protein by the Kennedy and Fewson method. The growth yield of this strain was about 6.1 g of protein per mole of carbon source of PA, and was similar to that in the case of glucose as a carbon source. Complete degradation of PA has been achieved (1660 mg/l) in less than 2 days using Flavobacterium sp. strain No. A-1. The transient intermediates of PA were not detectable on the high-performance liquid chromatogram of the culture filtrates of this strain. This strain could not degrade dimethyl, diethyl phthalate ester and phthalic anhydride.
N-methyl-1-(3,4-methylenedioxyphenyl)butan-2-amine (MDP-2-MB, MBDB) is a new homologue of N-methyl-1-(3,4-methylenedioxyphenyl)propan-2-amine (MDMA), which is strictly controlled as a narcotic. As part of our continuous survey on illegal designer drugs in the Japanese market, we found that N-methyl-4-(3,4-methylenedioxyphenyl)butan-2-amine (MDP-3-MB, HMDMA) was being sold as MBDB. As this is the first time that HMDMA has been revealed to be in market distribution, and its physico-chemical data is thus far unreported, we describe the structure elucidation of HMDMA and comparative analysis with related compounds.
Toxicities of antifouling chemicals and natural marine samples were evaluated by three assays, among which bioluminescence assay using freshly incubated Vibrio fischeri (V. fischeri) cells (NZ assay) and MicroTox were regarded as short-term assays, and growth inhibition assay was conducted as long-term assay. Short-term toxicity levels evaluated by NZ assay were in good agreement with those by MicroTox assay for all of the samples examined. Based on the EC50 values of each chemical by respective assay, NZ assay showed prior reproducibility and similar levels of sensitivity when compared with those of MicroTox assay. On the other hand, growth inhibition assay showed lower sensitivity and reproducibility than NZ and MicroTox assays. Four kinds of antifouling chemicals, Irgarol 1051, Diuron, thiabendazole (TBDZ), and N-dichlorofluoromethylthio-N',N'-dimethyl-N-phenylsulfamide (DCF), were detected to possess delayed toxicity from the judgments on the difference of short-term and long-term toxicities. Four out of 16 seawater samples collected in Japan showed remarkable toxicity in NZ assay, suggesting that they were contaminated by several types of antifouling chemicals. Considering time consumed, facility for operation, cost, and requirements, NZ assay was proved to be efficient for toxicity evaluations for artificial and natural samples.
The in vivo effects of drinking a water product, which had been confirmed to have antioxidant activities in vitro, were preliminarily studied by monitoring the blood concentrations of oxidative stress marker substances in the two group subjects who ingested the same quantity of the water product and a tap water solution during the same time. The results indicated that hydrogen gas and reductive vanadium ions as the components responsible for the antioxidant activities in vitro cannot enhance the scavenging ability for reactive oxygen species in vivo after being drunk and absorbed into the human body, although it was suggested that an ingestion of a greater quantity of water than usual gives a slight reduction, overall, in the oxidative stress.
To investigate the inhibitory effect of aza-polycyclic aromatic compounds on cytochrome P450 (CYP) 2C9 activity and analyze the fluorine-substitution effects on the CYP2C9 inhibition, benzo[h]quinoline (BhQ) and its fluorinated derivatives, 3-F-, 5-F-, 6-F-, 9-F-, 10-F-, 3,6-diF-, 5,6-diF-, 7,10-diF-BhQ, were subjected to analysis of their inhibitory effects on recombinant human CYP2C9.1-catalyzed tolbutamide hydroxylation. Although the inhibitory activity of BhQ itself on tolbutamide hydroxylation was not very strong (IC50 = 157 μ M), the inhibitory activities of BhQ derivatives on tolbutamide hydroxylation varied with the substituted fluorine positions. Their inhibitory activities decreased in the following order; (BhQ) > 7,10-diF ≥ 3,6-diF, 9-F ≥ 10-F > 6-F ≥ 5-F, 3-F ≥ 5,6-diF-BhQ. Moreover, the fluorine-substitution effect on the inhibition of tolbutamide hydroxylation catalyzed by wild-type CYP2C9 (CYP2C9.1) was different from the effects on the hydroxylation catalyzed by its polymorphic isozymes CYP2C9.2 and CYP2C9.3. The inhibitory activities of BhQs on tolbutamide hydroxylation by CYP2C9.2 decreased in the following order; 5-F ≥ 10-F, 9-F ≥ (BhQ) ≥ 7,10-diF > 6-F ≥ 5,6-diF, 3,6-diF > 3-F-BhQ, and those on the hydroxylation by CYP2C9.3 decreased in the follwing order; 7,10-diF, 10-F, (BhQ) ≥ 9-F, 5-F > 3-F ≥ 5,6-diF, 6-F, 3,6-diF-BhQ. The results thus showed that the position-specific substitution by fluorine atom(s) altered the CYP2C9 inhibition by BhQ derivatives in different manners depending on the polymorphic isozymes involved. These results suggest that inhibitory profiles obtained with fluorine-substituted analogs of the key inhibitor molecule may be useful as a new tool for phenotyping the polymorphic CYP isoforms.
The effects of menaquinone-4 (MK-4; vitamin K2) supplemented egg shell calcium (Cal K2) on bone components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues of rats was investigated. Cal K2 contained calcium carbonate (Ca 100 mg/g) and MK-4 (593 μg/g). Rats were orally administered a solution of Cal K2 (10, 25, or 50 mg/ml/100 g body weight) or placebo (without MK-4, 50 mg/ml/100 g body weight) once daily for 7 days. The serum r-carboxylated osteocalcin concentration, which is produced in osteoblastic cells, was significantly increased after the administration of Cal K2 (50 mg/100 g body weight) as compared with that in the control group or placebo-control group without MK-4. Calcium content and alkaline phosphatase activity in the femoral-diaphyseal tissues were significantly increased after Cal K2 (25 or 50 mg/100 g) as compared with that in the placebo-control group. Femoral-metaphyseal calcium content was significantly increased after the administration of Cal K2 (25 or 50 mg/100 g) as compared with that in the control group. DNA content in the femoral-diaphyseal and -metaphyseal tissues was significantly increased after the administration of Cal K2 (50 mg/100 g) as compared with that in the control or placebo-controlled group. This study demonstrates that the oral administration of Cal K2 containing MK-4 has anabolic effects on bone components in rats. Supplemental Cal K2 may have a role in the prevention of bone loss with aging.
Effects of estrogenic compounds (bisphenol A, alkyl phenols, phthalate esters, and genistein) on T lymphocyte apoptosis were investigated in vitro. The assays were performed in the absence or presence of low concentrations of apoptosis-inducing agents etoposide or dexamethasone to detect apoptosis-inducing, -enhancing, and -suppressing activities of the test compounds. When T lymphatic Jurkat cells were exposed to 10 μM bisphenol A for 20 hr, apoptosis was not induced, but apoptosis induced by 1 μM etoposide was significantly enhanced. 4-n-Nonylphenol (10 μM) showed an apoptosis-inducing activity significantly. 4-tert-Octylphenol (10 μM) exhibited an apoptosis-enhancing activity. In contrast, phthalate esters including di-n-butyl phthalate and di-2-ethylhexyl phthalate showed neither activity at 10 μM. Genistein (10 μM) significantly exhibited apoptosis-inducing and enhancing activities. On the other hand, 17β-estradiol did not show any of these activities at 10 nM, the concentration exerting the estrogenic activity comparable to or much higher than that of 10 μM of the test compounds. Effects of these estrogenic compounds on apoptosis were also investigated using mouse primary thymocytes. Mouse thymocytes similarly exposed to the estrogenic compounds in the absence or the presence of dexamethasone for 6 hr were characterized. In agreement with Jurkat cells, apoptosis-inducing or/and -enhancing activities were observed for the cells co-incubated with bisphenol A, alkyl phenols, and genistein, but not those with di-2-ethylhexyl phthalate or 17β-estradiol. The apoptosis-inducing or/and -enhancing effects of the estrogenic compounds observed here appear to be due to their unidentified properties other than estrogenic activity.
We examined whether or not changes occur in the quantity of carbon monoxide (CO) production as a consequence of the intake of ordinary meals. The study was performed by following diurnal changes in the concentration of CO in the breath over approximately 12 hr after each meal on 17 healthy subjects. CO is a product of the reaction of the enzyme heme oxygenase-1 (HO-1) induced through oxidative stress. The average value for diurnal variations in the concentration of CO in the breath was 1.9 ± 0.5 (0.8 to 3.3) ppm, and the maximum range of variation in individual cases came within the narrow range of 0.4 to 1.3 ppm. There were no significant differences in average values before and after meals. The value for the total quantity of CO in the breath 12 hr sought on the basis of the area under the diurnal variation curve was 22.8 ppm (0.39 mmol). This figure was very close to the heme breakdown quantity of 0.35 mmol (theoretical value, corresponding to the CO production quantity) every 12 hr.
Stress is one of the basic factors in the etiology of number of diseases. The present study was aimed to investigate the antioxidant properties of Triphala (Terminalia chebula, Terminalia bellerica and Emblica officinalis) during cold-stress. Four groups of albino rats were employed namely control, Triphala, cold-stress and Triphala with cold-stress. The oxidative stress was assessed by measuring the lipid peroxidation (LPO), enzymatic superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and non-enzymatic (Vitamin C) antioxidant status in adrenal tissue and plasma corticosterone level. Following cold-exposure (8°C for 16 hr/d/15 days), enzymatic and non-enzymatic antioxidants were significantly reduced with concomitant increase in LPO and corticosterone levels were observed. Administration of Triphala (1 g/kg/body weight/48 days) significantly prevents the cold-stress-induced oxidative stress and elevation in LPO and corticosterone levels. This study concludes that Triphala supplementation significantly prevents the cold-stress-induced oxidative stress may due to its antioxidant properties.
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