An increase of mitochondrial membrane permeability is one of the key events in apoptosis, since it leads to the release of mitochondrial apoptogenic factors, such as cytochrome c, into the cytoplasm that activate downstream target of apoptotic cell death. Bcl-2 family is one of the best-characterized proteins that directly regulate mitochondrial functions. A major role of the Bcl-2 family of proteins is to alter mitochondrial membrane permeability, thus controlling the release of caspase-activating cytochrome c. Recent reports describe about involvement of interesting apoptogenic regulators other than Bcl-2 family in regulation of mitochondrial function. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a member of the heat-shock family of mitochondrial proteins, and substantially homologous to members of the 90-kDa families of heat-shock proteins (HSP90). TRAP1 seems to have specific functions differ from those of other members of the HSP90 family. Downregulation of TRAP1 expression enhances the release of cytochrome c from mitochondria. Moreover, reactive oxygen species (ROS) are involved in the regulation of the TRAP1 expression, indicating that TRAP1 is a sensor that involved in ROS mediated regulation of apoptosis. Here, we describe the mechanisms underlying the regulation of mitochondrial functions during apoptosis by TRAP1.
This review describes a comprehensive investigation in our laboratory focusing on development of sensitive analytical methods by utilizing luminescence detections, and leading to practical applications for quantification of biologically related compounds, especially drugs of abuse. The developed methods mainly used a high-performance liquid chromatographic separation technique with fluorescence and chemiluminescence detections. The main targets of our investigation are ultra-small amounts of biologically active compounds, especially centered on abused drugs such as stimulants, narcotics and anorectics in various matrices. Furthermore, drug-drug interaction of these compounds was also examined. The results obtained in our study might contribute to the prediction of and the protection of human health from the risks of abused drugs. The developed methods might be useful for pharmaceutical, clinical and forensic studies on drugs of abuse as well as other important biologically active compounds.
The cytochrome P450 (P450, CYP) enzyme superfamily is associated with the metabolism of drugs, environmental pollutants and endogenous substrates with broad overlapping substrate specificities. In addition, species differences between experimental animals and humans in the roles of P450 enzymes in drug metabolism are determinant factors in the drug discovery process. The induction and inhibition of P450-dependent metabolism, especially in the case of P450 3A enzyems, can cause serious problems in clinical practice and in the attrition of drug candidates because of adverse toxicology or pharmacokinetics. Recently, chimeric mice whose livers have been replaced to a level of 80% or more with human hepatocytes were established using urokinase-type plasminogen activator transgenic/severe combined immunodeficiency (uPA/SCID) mice. In the livers of these chimeric mice, hepatic cords and sinusoid-like structures were observed. Moreover, bile canaliculi associated with human hepatocytes were also detected. The mRNA expressions derived from humans of 52 phase I and 26 phase II enzymes (including 20 P450 s) and 21 transporters were ascertained in the chimeric mice liver. Taken together, these chimeric mice exhibited the glutathione conjugate form of a possible quinine-imine metabolite and responded to treatment with rifampicin or rifabutin by induction of P450 3A enzyems. This animal model should prove to be a good in vivo tool to assess the safety of drug candidates in terms of toxicity and drug-drug interactions caused by P450 induction.
To investigate the effect of dichloroacetate (DCA) on the mean pulmonary artery pressure (mPAP), pulmonary artery (PA) remodeling and voltage-gate K+ (Kv) channel expression in pulmonary arterial smooth muscle cells (PASMCs) in high altitude-induced pulmonary artery hypertension (HA-PAH) rats. Sprague-Dawley rats were randomly assigned to normal control (N), high altitude (HA), and HA+DCA (70 mg/kg DCA administration daily) groups (n=8 each). Rats were housed in a hypobaric, hypoxic chamber to mimic an altitude of 5000m for 21 days; then the mPAP and the wall thickness (WT) of the PA smooth muscle were measured. PASMCs apoptosis was examined using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stain. Real-time PCR, immunohistochemistry and western blot analyses were carried out to detect Kv1.5 and Kv2.1 expression in PASMCs. The expression of Kv1.5 and Kv2.1 was decreased in HA rats. With DCA treatment, the expression of Kv1.5 and Kv2.1 was restored, and the established HA-PAH was ameliorated. Compared with the HA-PAH rats, the DCA-treated rats displayed a decreased mPAP, WT of the PAs, right ventricular hypertrophy and ([Ca2+]i), and more PASMCs were apoptotic. DCA partially reversed the downregulation of Kv1.5 and Kv2.1 in the PASMCs of HA-PAH rats. DCA can reverse the remodeling of the PA and upregulate Kv1.5 and Kv2.1 expression in the PASMCs of HA-PAH rats. This result suggests that DCA may be an effective drug for treating HA-PAH and that restoring Kv1.5 and Kv2.1 can partially decrease mPAP.
In this study, effect of an ethanolic extract from rhizome of Costus igneus were investigated on activity of following enzyme in liver, kidney, pancreas of streptozotocin (STZ) induced diabetic rats: carbohydrate metabolic enzymes such as glucokinase glucose-6-phosphatase, and fructose-1,6-bisphosphatase in the liver; hepatoproductive enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in plasma and liver; and antioxidative enzymes such as superoxide dimutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione content (GSH), total sulfhydryl groups (TSH), lipid peroxides (LPO) in liver, kidney and pancreas. An ethanol extract from Costus igneus rhizome was administrated orally at a single dose of 100 or 200 mg/kg per day to diabetes induced rats for 30 days. This study demonstrated that glucose-6-phosphatase fructose-1,6-bisphosphatase, AST, ALT, ALP and LPO levels were significantly increased (p<0.05), whereas glycolytic enzyme glucokinase, SOD, CAT, GPx, GSH and TSH levels were significantly decreased (p<0.05) in STZ induced diabetic rats. Oral administration of Costus igneus rhizome ethanol extract [CiREE, 100 or 200 mg/kg per body weight (bw)] and glibenclamide to diabetic rats for 30 days significantly (p<0.05) reversed levels of these enzymes to normal. Bioactive compound quercetin and diosgenin were isolated from Costus igneus rhizome by high performance thin layer chromatography (HPTLC). These results suggest that, components of CiREE quercetin and diosgenin exhibit antioxidant activity, which was sufficient to reverse oxidative stress in the liver, pancreas and kidney of diabetic rats as well as to stimulate glycolytic enzymes and control gluconeogenesis in diabetic animals.
A new method to identify liothyronine (T3, 3,3',5-triiodo-L-thyronine) and levothyroxine (T4, L-thyroxine), which are thyroid hormones, in dietary supplements by high-performance liquid chromatography was developed using a pre-column derivative with 4-fluoro-7-nitrobenzofurazan (NBD-F), without using liquid chromatography (LC)/MS. T3 and T4 in a dietary supplement were extracted with 10% ammonia water. The extract was treated with a polyvinylpolypyrrolidone (PVPP) column. The T3 and T4 adsorbed on the PVPP were eluted with 60% acetonitrile. T3 and T4 present in the eluate from the PVPP column were then derivatized with NBD-F. The reaction of T3 and T4 with NBD-F was complete after 30 min at 60°C. These derivatives were measured by HPLC using an octadecylsilane (ODS) column with a fluorescent detector. 0.1% Phosphoric acid was added to 630 ml of acetonitrile to a final volume of 1000 ml, and the mixture was used as the mobile phase. The excitation wavelength on the fluorescent detector was 470 nm, and the emission wavelength was 530 nm. The stability of the peak area of T3-NBD and T4-NBD was maintained for 12 hr. The linearity of T3 and T4 as a coefficient of the correlation value was 0.9999, and the quantitation limit of T3 and T4 was 0.002 μg/ml. The results confirmed that T3 and T4 were added to eight dietary supplements. The recovery rate was in the range of 80.4-103.7% for T3 and 79.8-101.5% for T4, and the precisions, as measured by the standard deviation, were within 4.8% and 4.3%, respectively.
3,6-Dinitrobenzo[e]pyrene (DNBeP) is an extremely strong mutagen in Salmonella Typhimurium TA98 without a mammalian metabolic system (S9 mix). 3,6-DNBeP shows genotoxicity in vitro to mammalian cells, and produces DNA damage in the cells of several organs in mice in the comet assay. In a previous study, we developed an analytical method and clarified that 3,6-DNBeP widely exists in the environment, i.e., surface soil and airborne particles, and that diesel engines and municipal incinerators are probable sources of 3,6-DNBeP. In this study, we improved the method of analyzing 3,6-DNBeP by combining one step of clean-up and fluorescence detection utilizing a two-dimensional HPLC system, and analyzed 3,6-DNBeP in tea leaves (n=6), which is a possible exposure source of 3,6-DNBeP in our daily life, and in human hair samples (n=8), as a possible biomarker of 3,6-DNBeP. 3,6-DNBeP was detected in all examined tea leaves and human hair as single peaks on the chromatograms, and was well purified by the HPLC system. 3,6-DNBeP was detected in the range of 8-1823 pg/g of tea leaves and the amount of 3,6-DNBeP in tea leaves differed depending on the growth site of the tea leaves. 3,6-DNBeP in human hair was detected in the range of 11-121 pg/g of hair and 86-1576 pg/mg of eumelanin. These results suggested that tea leaf is a possible source of exposure to 3,6-DNBeP and that 3,6-DNBeP detected in hair might reflect human exposure to 3,6-DNBeP.
Synthetic amorphous silica nanoparticles (SiNPs) are seeing increased and widespread application in diagnosis, imaging, and drug delivery. In the present study, the inflammatory responses and histopathological changes caused by inflowing of SiNPs into the lung were studied in mice after a single intratracheal instillation. Changes in gene expression were also investigated in lung tissue using microarray analysis. As results, weight gain was significantly decreased in SiNP-treated mice on day 1 and 7 after instillation. The secretion of pro-inflammatory cytokines [interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α] and transforming growth factor (TGF)-β were also increased, and the distribution of cytotoxic T cells, natural killer (NK) cells, and natural killer T (NKT) cells with G1 arrest were increased. Microgranulomatous changes were observed on day 7 and 14. Furthermore, the gene expression was significantly affected on day 7. A total of 331 genes were up-regulated more than 2 fold, and 128 genes were down-regulated more than 2 fold. The secretion of inflammatory related cytokines, histopathological abnormalities, and the severity of gene expression changes decreased in a time-dependent manner. Based on these results, we suggest that SiNPs induce subchronic inflammatory responses and tissue damage in mice after a single intratracheal instillation.
We previously reported that novel polyselenodithiols are produced along with selenotrisulfide when a thiol (penicillamine) is reacted with selenite (H2SeO3). Here, we report the production of oligoselenodiglutathiones and their suppressive effects on oxidative DNA damage. Oligoselenodiglutathiones were produced by exceeding the conventional reaction ratio of [H2SeO3]/[glutathione (GSH)]=1/4. In liquid chromatography-mass spectrometry (LC/MS) analysis, the observed isotope patterns showed good agreement with the calculated isotope patterns assuming that two, three, or four selenium (Se) atoms were incorporated in the molecules. Based on 1H NMR and MS data, the structures of oligoselenodiglutathiones were assumed to have a common symmetrical structure that was centered by linearly bound Se atoms “wedged” in the disulfide bond of two GSH molecules. At 8 μM, selenodiglutathione (GSSeSG) and diselenodiglutathione (GSSe2SG) showed 80% suppression of the formation of 8-oxo-7,8-dihydroxy-2'-deoxyguanosine (in calf thymus DNA) induced by carcinogenic catechol and copper. The suppressive effects of GSSeSG and GSSe2SG were around threefold higher than that of glutathione disulfide at the same concentration, and the suppressive effect was not observed for H2SeO3 or GSH. Thus, formation of oligoselenodiglutathiones is important for Se or GSH to exert its protective effects on biomolecules from oxidative damage.
Mutanase, α-1,3-glucanase, catalyzes the hydrolysis of α-1,3-glucans, and is expected for preventive medicine, since the enzyme has an ability for hydrolysis of insoluble glucans that are synthesized by cariogenic streptococci. We previously isolated the mutanase of Paenibacillus humicus from fermented soybeans. In the present study, Paenibacillus mutanase was characterized with respect to its hydrolysis efficiency of insoluble glucans, and mode of action on α-1,3-glucan oligosaccharides. Recombinant mutanase hydrolyzed insoluble glucans of cariogenic streptococci efficiently. Enzymatic reaction on hydrolysis of mutan, we assumed that the enzyme cleaved the substrate in an endo-catalytic manner. The hydrolysis of α-1,3-glucan oligosaccharides gave α-1,3-glucan tetrasaccharide as the primary final product but α-1,3-glucan pentasaccharide was the minimal size of substrate on which the enzyme catalyzed. Mutanase hydrolyzed borohydride-treated α-1,3-glucan hexasaccharide into the tetrasaccharide and the disaccharide-alditol. Thus, the enzyme cleaved the fourth α-1,3-glucosidic linkage from the non-reducing end of the oligosaccharides. Mutanase in fermented food should be capable of removing streptococcal insoluble glucans that can induce dental caries.
In our previous paper, we reported that spontaneous hypothermia (HT) during ischemia protects against delayed neuronal death in the hippocampus but not against acute brain edema following transient forebrain ischemia, which is induced by occlusion of the bilateral common carotid arteries (BCCA) in C57BL/6J mice. We here demonstrate that artificial HT after reperfusion (rHT) suppresses the aggravation of acute brain edema in the BCCA occlusion C57BL/6J mouse model. Our results suggest that mechanisms regulated by rHT are involved in the attenuation of acute brain edema after reperfusion in this model of cerebral ischemia.
One of the principal regulators of mitogenesis in vascular smooth muscle cells (VSMCs) is platelet-derived growth factor-BB (PDGF-BB). An increase of PDGF-BB expression has been observed in atherosclerotic lesions. The aim of this study was to elucidate the effects and molecular mechanism of (2E)-3-(4-hydroxy-3-methoxyphenyl) phenylpro-2-en-1-one (KTJ2242), a newly synthesized benzylideneacetophenone derivative, on PDGF-BB-stimulated rat aortic VSMCs. KTJ2242 induced accumulation of cells in the G1 phase of the cell cycle of VSMCs. We observed that KTJ2242 inhibited PDGF-BB-stimulated [3H]-thymidine incorporation into the DNA of VSMCs, and the cell number was significantly reduced in a concentration-dependent manner. Also, we observed that KTJ2242 decreased PDGF-BB-stimulated extracellular-regulated kinase 1 and 2 (ERK1/2) and Akt phosphorylation. These results suggest the possibility that KTJ2242 may be a potential agent with which to control vascular disorders and its antiproliferative mechanism may be mediated through partial Akt and ERK1/2-dependent signaling pathways.
Previously, we showed that obesity affects the gene expression of acetoacetyl-CoA synthetase (AACS), the enzyme responsible for the utilization of ketone bodies for lipid synthesis, in adipocytes. Therefore, we examined whether obesity also changes the AACS level in skeletal muscle. AACS mRNA expression was decreased in the skeletal muscle of leptin-deficiency-induced obese rats. In undifferentiated C2C12 myoblast cells, a high concentration of glucose induced the expression of AACS mRNA but decreased the AACS level in differentiated C2C12 cells. Moreover, leptin directly increased the AACS mRNA level in C2C12 myocytes. The expression level of succinyl-CoA: 3-oxoacid CoA-transferase (SCOT), another enzyme that induces ketone body consumption, was not changed in C2C12 cells. Our results suggest that AACS gene expression is differently affected by leptin and glucose during muscle differentiation and leptin plays a regulatory role not only in lipid consumption but also in ketone body utilization via AACS in skeletal muscle.
Many lipid-binding proteins such as pleurotolysin and ostreolysin have been isolated from the edible mushroom Pleurotus ostreatus. In this study, we detected a novel lipid-binding protein with a molecular weight of 62 kDa by measuring via centrifugation the association of aqueous extracts of the mushroom with lipid vesicles composed of various phospholipids. The 62-kDa protein (p62) was purified by sedimentation of the mixture of protein extracts and acidic phospholipid-containing lipid vesicles. The purified p62 bound to phosphatidylglycerol (PG)/phosphatidylcholine/cholesterol (5:45:50) vesicles but not to vesicles composed of other phospholipids including phosphatidylserine (PS), phosphatidylinositol, phosphatidic acid, lysoPS, and lysophosphatidylinositol. The p62 protein specifically associated with the PG-containing vesicles but not with other polyglycerophospholipid vesicles consisting of cardiolipin, bis(monoacylglycero)phosphate, monolysocardiolipin, or dilysocardiolipin, suggesting that p62 recognized a precise molecular structure of PG. Intrinsic tryptophan fluorescence of p62 was changed by incubation of p62 with PG-containing vesicles. Staining of giant unilamellar vesicles with fluorescence-labeled p62 showed that p62 bound to PG-containing vesicles but not PS-containing vesicles. These observations signify the potential usefulness of p62 as a tool for studying the functions of PG molecules in biological membranes.
DNA cleavage by quinones contained in diesel exhaust particles (DEP) was examined in a cell-free system using supercoiled FX174 DNA as the target DNA. In the presence of Cu(II) and NADPH, 9,10-phenanthrenequinone (PQ) caused the transformation of the supercoiled FX174 DNA into open circular and then linear forms in a concentration-dependent manner. This DNA damage by PQ was decreased by catalase, a superoxide anion scavenger and a Cu(I)-specific chelator, but not by superoxide dismutase and a hydroxyl radical scavenger, suggesting that the ultimate reactive product responsible for the DNA scission may be Cu(I)-OOH generated from hydrogen peroxide and Cu(I) rather than hydroxyl radicals. In addition, 1,2-naphthoquinone (1,2-NQ) damaged DNA more severely than PQ, while 1,4-NQ and 9,10-anthraquinone (AQ) did not induce significant DNA damage. When a purified aldo-keto reductase (AKR) 1C isozyme, which catalyzes the two-electron reduction of PQ, was included in the reaction mixture, the PQ-induced DNA damage became more extensive. Addition of the AKR1C isozyme also increased the 1,2-NQ-induced DNA damage and conferred the ability to cause DNA damage on 1,4-NQ, but had no effect on AQ. The severity of the DNA damage induced by DEP quinones was solely related to both NADPH consumption and reactive oxygen species (ROS) generation. These findings indicate that the generation of ROS via redox cycling of DEP quinones is a causative event in DNA scission and that the AKR1C isozyme accelerates the redox cycling of DEP quinones that are utilized as substrates, thereby resulting in the promotion of oxidative stress and DNA damage.
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