Zinc is an extremely most abundant trace element in the brain. Substantial amounts of zinc exist in the presynaptic vesicles, and are released with glutamate during the neuronal excitation. Synaptically-released zinc is believed to play crucial roles in normal brain functions. Therefore, zinc deficiency impairs brain development and capabilities of learning and memory. Notwithstanding, recent studies have indicated that excess zinc is linked with several neurodegenerative diseases and has a causative role in delayed neuronal death after transient global ischemia. We have developed the sensitive assay system for zinc neurotoxicity in vitro using GT1-7 cells (immortalized hypothalamic neurons) to elucidate the functions of zinc in neurodegenerative diseases. Pharmacological experiments have exhibited the involvement of energy failure, metal-metal interaction, and disruption of calcium homeostasis in zinc-induced neurotoxicity. It is inferred that zinc might play its part in brain functions as Janus, an ancient Roman god with two faces, and that zinc homeostasis is essential for the neuronal survival. Our assay system provides a good method for screening the protective substances of zinc neurotoxicity as a therapeutic target of the global ischemia.
Analytical conditions for evaluating the chemical toxicity were examined using luminescent bacterium Vibrio fischeri (V. fischeri) deutsche sammluing von microorganismen (DSM) 7151, taking the medium composition into consideration. The manipulation of the cells for optimal assay was obtained as follows; the cells were grown in luminescence (LM) medium containing 3 g/l of glycerol for 18 hr. They were washed twice with artificial seawater (ASW) and then diluted about 128 folds by ASW, corresponding to 1.0-1.5 × 106 cfu/ml. Using 4,5-dichloro-2-(n-octyl)-4-isothiazolin-3-one (SeaNine 211) as a representative toxic chemical, we evaluated the extent of its toxicity in terms of present method, where 50% effective concentration (EC50) resulted in 0.51 and 0.35 mg/l at 15 and 30 min of incubations, respectively. Sensitivity of present assay was higher than those of traditional ones by over 2 folds from the comparison of EC50 for Cu2+. The results show that the present assay using freshly cultured V. fischeri has not only high sensitivity and reproducibility but also simplicity to estimate the contamination by chemicals.
We investigated the effects of BRAND'S Essence of Chicken (BEC) on the basic metabolism of plasma lipids in mice loaded with restraint stress. When a lipid emulsion was intravenously injected into mice, 20 hr restraint stress prolonged the elimination of plasma triglyceride (TG). The results indicated that lipid metabolism was definitely disrupted by stress, and that the use of TG as an energy source decreased. The plasma TG level was 320 ± 27 mg/dl 35 min after Intralipid® administration in restrained mice, while it was 253 ± 23 mg/dl in the restrained mice that were given 5 ml/kg of BEC. The improved plasma lipid metabolism was well explained by the finding that lipoprotein lipase in omental adipose tissue was remarkably improved by BEC. Our study shows that BEC improves metabolic dysfunction possibly by utilizing plasma lipids as energy sources and elevating lipoprotein lipase activity reduced by stress. The anti-stress effect of BEC may partly be related to improved plasma lipid metabolism for energy utilization.
Three hereditary human disorders, Werner's syndrome, Bloom's syndrome and a subset of Rothmund-Thomson syndrome, are associated with the loss of function of the respective RecQ homologues BLM, WRN, and RTS. These RecQ homologues are composed of a conserved helicase domain flanking C- and N-terminal domains. In contrast to BLM, WRN, and RTS, another RecQ homologue, RECQ5, possesses only short N-terminal region preceding the helicase domain and a long unique C-terminal domain. Although no disease has yet been genetically linked to a mutation in RECQ5, the prominent roles of RecQ helicase in the maintenance of genome stability suggest that RECQ5 helicase is likely to be important in vivo. To acquire a better understanding of RECQ5 function, we investigated protein interaction with the C-terminal domain of Drosophila melanogaster RECQ5/QE. A portion of Drosophila melanogaster retrotransposon mdg3, which corresponds to a nucleocapsid protein (gag-NC), was identified by use of the yeast two-hybrid system as interacting specifically with it. Glutathione S-transferase (GST) pull down experiments indicated that the mdg3 gag-NC bound mainly to an acidic region in the C-terminal domain of RECQ5/QE, which is adjacent to the RecQ helicase domain. The helicase activity of RECQ5/QE was stimulated by mdg3 gag-NC protein in vitro. These data suggest that RECQ5/QE helicase interacts physically and functionally with mdg3 gag-NC through the acidic region and that RecQ homologue might be involved in retrotransposition and genomic stability.
The effect of the carbenoxolone (CBX) on the subacute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in C57BL/6J mice. The loss of body weight due to TCDD was enhanced by simultaneous treatment with CBX. In agreement with the change in body weight, CBX failed to improve TCDD-induced hepatic hypertrophy and thymic atrophy. Co-treatment of CBX with TCDD tended to cause hepatic hypertrophy more extensively than did TCDD alone, and combined treatment induced significant renal hypertrophy and splenic atrophy which were not seen in mice treated with TCDD or CBX alone. CBX had no effect on the TCDD-mediated induction of hepatic ethoxyresorufin O-deethylase activity, a marker for aryl hydrocarbon receptor (AhR)-linked gene expression. These results suggest that CBX enhances TCDD toxicity by a mechanism(s) distinct from activation of the AhR.
A DNA microarray is a very useful tool for detecting multiple bacteria simultaneously, because it can be used to analyze the characteristics of various bacterial genes on one glass slide. This study was mainly performed to establish an assay protocol using a DNA microarray for detecting three food-borne bacteria (Salmonella enterica serovar Enteritidis, Yersinia enterocolitica, and Bacillus cereus) in fresh vegetables. To create the DNA microarray for detecting these three species of food-borne bacteria, four previously designed oligonucleotide probes corresponding to the 16S rRNA sequences of each food-borne bacterium were spotted onto a single glass slide. Bean sprouts and lettuce were used as representative fresh vegetables. Diluted cultures of the three bacteria were inoculated into the juices obtained from the fresh vegetables, and the inoculum was cultured with Soybean-Casein Digest broth to amplify the targeted bacterial 16S rRNA. RNAs extracted from the cultures were fluorescently labeled and hybridized to the DNA microarray. All three bacteria could be specifically detected in the fresh vegetable samples using this assay protocol. This DNA microarray provides a convenient approach for the simultaneous detection of food-borne bacteria in fresh vegetables in combination with conventional culture methods.
The effects of bee pollen extract on bone components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues of rats in vitro and in vivo were investigated. Bone tissues were cultured for 48 hr in serum-free Dulbecco's modified Eagle's medium containing either vehicle or water- or ethanol-solubilized extracts (10, 100, or 1000 μg/ml of medium) obtained from the bee pollen of Cistus ladaniferus. Calcium content in the femoral-diaphyseal or -metaphyseal tissues was significantly increased in the presence of water-solubilized extract (100 or 1000 μg/ml) and ethanol-solubilized extract (1000 μg/ml). An increase was also observed in the presence of water-solubilized extract (100 μg/ml) obtained from Fagopyrum esculentum, Camellia sinesis, or Brassica napus L. Alkaline phosphatase activity and DNA content in the femoral-diaphyseal or -metaphyseal tissues in vitro were significantly increased in the presence of water-solubilized extract (100 or 1000 μg/ml) obtained from the bee pollen. The effects of the bee pollen extract (100 μg/ml) in increasing bone components were completely inhibited in the presence of cycloheximide (10-6 M), an inhibitor of protein synthesis, in vitro. Moreover, the calcium content and alkaline phosphatase activity in the femoral-diaphyseal or -metaphyseal tissues were significantly increased by the oral administration of water-solubilized extracts (5 or 10 mg/100 g body weight) obtained from the bee pollen of Cistus ladaniferus once daily for 7 days. The DNA content in the diaphyseal or metaphyseal tissues was significantly increased by the oral administration of water-solubilized extract (10 mg/100 g) of bee pollen cistus. The dose of 1.0 mg/100 g caused a significant increase in the diaphyseal and metaphyseal alkaline phosphatase activity or the metaphyseal DNA content in vivo. This study demonstrates that the extract of bee pollen has an anabolic effect on bone components in rats in vitro and in vivo.
Air samples were collected at suburban Kanazawa and concentrations of dioxins in both gaseous and particulate phases were determined separately. The concentrations of the gaseous phase of dioxins increased with increasing temperature. Co-planar polychlorinated biphenyls (co-PCBs), whose vapor pressures are higher than those of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), were mainly in the gaseous phase samples in all the seasons. The gaseous phase ratios [gaseous phase/(gaseous phase + particulate phase)] of tetrachloro dibenzo-p-dioxins and dibenzofurans (TeCDD/DFs) were high regardless of the temperature. However, the gaseous phase ratios of penta-hepta CDD/DFs varied widely depending on the temperature. Gas/particle partitioning of atmospheric dioxins depended on not only the number of chlorine-substitutions but also the positions of the chlorine-substitutions. The position of chlorine-substitution in an isomer affects the isomer's molecular polarity. Dioxin isomers with higher molecular polarity, which have shorter retention times on the selected ion monitoring (SIM) chromatograms of their homologues, tended to be distributed unevenly in the gaseous phase. In addition, the differences in the gaseous phase ratios between the isomers with higher molecular polarity and those with lower molecular polarity increased with decreasing temperature.
To clarify the degradation pathway of chlorhexidine by a microbe, Pseudomonas sp. Strain No. A-3, the isolation and identification of microbial chlorhexidine degradation products were attempted. Two aromatic degradation products of chlorhexidine, named chlorhexidine aromatic degradation product (CHADP)-4 and CHADP-6, were isolated by column chromatography using Diaion HP-10, and purified by column chromatography using Diaion HP-20SS and Sephadex LH-20. The chemical structures of both compounds were examined by infrared, 1H NMR, 13C NMR and fast atom bombardment (FAB) mass spectra studies. Based on the spectroscopic data, CHADP-4 (molecular weight 335) and CHADP-6 (molecular weight 377) were found to be direct degradation products of chlorhexidine and were thought to be cleavage partners of p-chlorophenylurea (CHADP-5) and p-chloraniline (p-CA), respectively. Antimicrobial activity of CHADP-6 are similar to that of chlorhexidine, but antimicrobial activity of CHADP-4 decreased to 1/5-1/10 that of chlorhexidine.
To determine whether alterations in the L-methionine metabolism depend on nutritional conditions such as dietary protein levels, the effects of an L-methionine supplement for a protein-deficient diet on the urinary excretion of low molecular weight thiol compounds were compared to those of the supplement for an adequate protein diet. Although urinary concentrations of L-cysteine and L-homocysteine were increased by the L-methionine supplement irrespective of the dietary protein levels, the levels of increases were higher by the supplement to the protein-deficient diet than to the adequate protein diet. In addition, the urinary concentration of glutathione was increased only by the L-methionine supplement to the protein-deficient diet. However, the L-cysteinylglycine (CysGly) concentration was not affected by the L-methionine supplement to either diet. Thus, increases by the L-methionine supplement in the concentrations of these thiol compounds, except for CysGly, were more remarkable when the dietary protein level was deficient. These results suggest that the metabolism of excess L-methionine could be markedly affected by nutritional conditions, and that the alterations in the metabolism, at least partly, depend on the dietary protein levels.
Wavelength-dispersive X-ray fluorescence spectrometry following a simple pretreatment was developed to determine levels of dissolved tin in canned foods. Sample syrup or a homogenate solution of fruit (meat) was freeze-dried and diluted with the same weight of cellulose powder. The mixed powder was then quickly formed into a pellet for X-ray measurements. This analytical method (detection limit, 5 ppm) was used to determine levels of tin in several kinds of canned foods from the present markets. The analytical results indicated that high concentrations (100-300 ppm) of tin were present in cans of many kinds of fruit, and a relationship was observed between the concentration and the length of time after manufacture. After a can was opened, the amount of dissolved tin rapidly increased. These results are consistent with those of Horio et al., which suggests that the issue of tin dissolving from cans has not been adequately addressed, despite their previous warning.
A novel type of encephalopathy occurred in patients with chronic kidney diseases, which was associated with the ingestion of the Sugihiratake mushroom during the fall of 2004 in Japan. We attempted to investigate whether cyanide and thiocyanate are present in the Sugihiratake samples and determined the cyanide and thiocyanate levels in fifteen samples collected from different Japanese districts using HPLC with fluorometric detection. The cyanide ions and thiocyanate ions were detected in the ranges of N.D.-114.0 and N.D.-17.0 μg/g in the samples, respectively. This is the first study to quantitatively detect cyanide and thiocyanate in the Sugihiratake mushrooms. This result demonstrated that cyanide exposure could occur from the intake of Sugihiratake mushrooms in one's diet. Furthermore, we discussed the possible association between cyanide and the onset of encephalopathy.
We performed identification tests of aristolochic acid in Xixin, Mutong, Muxiang, and Fangji by reversed-phase TLC/scanning densitometry using aristolochic acid I as a marker compound. The developing solvents used were: I) acetonitrile/methanol/water mixture solution (3 : 1 : 1) and II) 2-butanone/methanol/sodium sulfate(1→20) mixture solution (2 : 1 : 1). The Rf value of aristolochic acid I was 0.54 using the I system and 0.57 using the II system. The maximum absorption wavelengths of aristolochic acid I observed by scanning densitometry were 254 and 325 nm.
To examine pathologically the influence of diesel exhaust (DE) exposure on fetal nervous system development, brain tissue (cerebral cortex and hippocampus) was collected from newborn mice whose mothers were exposed to DE during pregnancy. After DE exposure, these brain tissues showed evidence of numerous caspase 3-positive cells (a common enzymatic biomarker of apoptosis). Some cells were found to contain crescent-shaped spaces, which are suggestive of apoptotic processes. Granular perithelial (GP) cells, scavenger cells surrounding cerebral vessels of the blood-brain barrier, showed signs of apoptosis; furthermore, the GP cytoplasmic granules had degenerated and showed evidence of what appeared to be ultrafine, DE particles. Additionally, the swelling of astrocyte endfoot that surround capillaries showed degenerative changes similar to myelin figures. Furthermore, the apoptosis of endothelial cells and stenosis of some capillaries were observed. These findings varied in severity, depended on DE concentration, and were not observed in the control group. These observations suggest that exposure of pregnant mice to DE might carry a risk of cellular atrophy and might affect fetal brain development. Our findings also reveal that inhalation of DE might be hazardous to the general health of fetuses.
Heparin is known to stimulate the release of hepatic lipase (HTGL) activity from hepatocytes, but the action mechanisms have not been fully confirmed. Here, we investigated the involvement of the arachidonate pathway in the heparin-stimulated release of HGTL activity from rat hepatocytes. Heparin increased phospholipase (PL) A2 activity in the hepatocytes in a time- and dose-dependent manner. The stimulatory effect of heparin on PLA2 activity was markedly decreased by incubation with protein tyrosine kinase (TK) inhibitors. It was also observed that heparin rapidly increased leukotriene (LT) B4 and LTC4/D4/E4 contents in the hepatocytes. In addition, the stimulatory release of HTGL activity by heparin was suppressed by cytosolic PLA2, 5-lipoxygenase and LTA4 hydrolase inhibitors, but not by cyclooxygenase and thromboxane (TX) synthetase inhibitors or a TXA2 receptor antagonist. These findings suggest that the heparin-stimulated release of HTGL activity from hepatocytes is partly due to an action involving increases in cytosolic PLA2 activity and LTs production with associated of TK activity.
Nonylphenol (NP) is widely distributed in the environment as a microbial metabolite of nonylphenol polyethoxylates, which are commercially important surfactants. Although there are concerns regarding the estrogenic activity of NP and its effects on wildlife, this paper reports a new detrimental effect for NP; oxidative DNA damage. After UV or sunlight irradiation, several NP photoproducts were detected by high performance liquid chromatography (HPLC). Two HPLC fractions induced formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in calf thymus DNA. Based on gas chromatography/mass spectrometry and UV/visible spectrometry, one fraction was confirmed to contain 4-nonylcatechol. The other active compound remains unidentified. Non-irradiated NP did not exhibit any detectable 8-oxodG formation. These results suggest that NP induces oxidative DNA damage following environmental light exposure. This DNA damaging activity is a new adverse effect of NP and, in addition to estrogenic activity, should be investigated further and taken into consideration during NP risk assessment.
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