The biological functions of selenium (Se) are mediated by the expression of selenoproteins such as glutathione peroxidase. Recent progress in biochemical characterization of selenoproteins has elucidated new functions of newly identified and classical selenoproteins. Most selenoproteins are involved in protection against oxidative injury and in redox regulation of cellular events. On the other hand, mechanisms of anticarcinogenic effects of Se compounds appear to include not only the expression of selenoproteins but also pharmacological actions of Se metabolites.
Selenocysteine (Sec) is the 21st amino acid, because Sec has a specific tRNA and codon UGA, and shares a major stop codon UGA. The present article surveys the structure of mammalian tRNASec, the identity elements, the enzymes to syntheize Sec, and selenophosphate as a Se donor. Sec-tRNA is converted from Ser-tRNA by Sec synthase. tRNASec has a long 9bp AA-stem, 6bp D-stem and 4bp T-stem, differing from Ser tRNAs. The 9bp AA-stem and 6bp D-stem were identity elements necessary for selenylation, however, the 4bp T-stem is not essential. This finding was supported by the active mutants derived from major tRNASer by changing the AA-stem to 9bp and the D-stem to 6bp. There are many enzymes and factors that produce and bring Sec-tRNA to ribosomes. The reaction proceeds as follows. The first step is Ser-tRNA synthetase which recognizes the discriminaition base G73 and the long extra arm. Bovine Sec synthase, composed of two protein species, recognizes Ser-tRNASec among three Ser-tRNAs and changes Ser-tRNASec to Sec-tRNASec, by the addition of Se from selenophosphate (SeP). The stability of SeP and some properties of the synthetase are discussed. Sec-tRNA is recognized by an elongation factor specific to Sec-tRNA, and is brought to Se-protein mRNA. The discrimination mechanism of the Sec UGA codon from a major stop codon UGA remains to be resolved.
Selenocysteine is incorporated into selenoproteins by an in-frame UGA codon whose readthrough requires the selenocysteine insertion sequence (SECIS), a conserved hairpin in the 3' untranslated region (3'UTR) of eukaryotic selenoprotein mRNAs. To identify new selenoproteins, we developed a strategy that obviates the need for prior amino acid sequence information. A computational screen was used to scan nucleotide sequence databases for sequences presenting a potential SECIS secondary structure. The computer-selected hairpins were then assayed in vivo for their functional capacities and the cDNAs corresponding to the SECIS winners were identified. Four of them encoded novel selenoproteins as confirmed by in vivo experiments. Among these, SelZf1 and SelZf2 share a common domain with the mitochondrial thioredoxin reductase TrxR2. The three proteins, however, possess distinct N-terminal domains. We found that another protein, SelX, displays sequence similarity to a protein involved in bacterial pili formation. For the first time, four novel selenoproteins were discovered based on a computational screen for the RNA hairpin directing selenocysteine incorporation.
Selenoprotein P (SeP : the "P" denotes its presence in plasma) is the major selenoprotein in plasma. All selenoproteins so far reported contain one atom of selenium (Se) as selenocysteine per molecule except SeP, which is thought to contain ten atoms of Se as selenocysteine per molecule. SeP contains 50% of the total Se in plasma. The function of Sep is currently unknown, although several indirect lines of evidence suggest that SeP is a free radical scavenger or Se transporter. Recently, we reported that SeP is capable of reducing phospholipid hydroperoxide in the presence of glutathione, and characterized the enzymatic nature of SeP. SeP is also reported to function as a peroxynitrite scavenger or cell survival factor in primary culture of neurons. In this minireview, we discuss the relationship between the structure and function of SeP.
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) which is a selenoprotein and a key enzyme in the protection of biomembranes exposed to oxidative stress. Involvement of mitochondrial PHGPx in apoptosis was studied using RBL2H3 cells that overexpressed mitochondrial PHGPx. The elevation of hydroperoxides in mitochondria, release of cytochrome c, the activation of caspase and the fragmentatoin of DNA associated with apoptosis sequentially occurred in control cells upon exposure of cells to hypoglycemia with 2-deoxyglucose (2DG). Overexpression of mitochondrial PHGPx prevented the rise of hydroperoxide in the earliest event of apoptosis and then blocked apoptosis. Mitochondrial PHGPx overexpressing cells were also resistant to apoptosis induced by etoposide, staurosporine and UV irradiation, which mediate apoptosis through the mitochondrial pathway. These results indicate that mitochondrial PHGPx might play a critical role as an anti-apoptotic factor in the mitochondrial-death pathways. PHGPx was exclusively expressed in the mitochondria of human spermatozoa. We found dramatic decrease in the expression of mitochondrial PHGPx in the spermatozoa of 10% of infertile males. Mitochondrial PHGPx-deficient spermatozoa lost the mitochondrial membrane potential and showed an abnormal mitochondrial morphology. Failure of the expression of mitochondrial PHGPx might cause a dysfuction of the mitochondria in spermatozoa and a defect in spermatogenesis in which apoptosis occurs.
Selenium (Se) is an essential trace element and a toxicant for animals. Selenocystine (CySeSeCy) and selenomethionine (SeMet), selenoamino acids, are one of the chemical forms in which selenium exists in foods. This review summarized recent studies on the relation of toxicity and metabolism of selenite, CySeSeCy and SeMet in experimental animals. Hepatotoxicity is caused by repeated oral administration of CySeSeCy. CySeSeCy is metabolized by reduced glutathione (GSH) and/or glutathione reductase to hydrogen selenide (H2Se) via selenocysteine-glutathione selenenyl sulfide (CySeSG). The H2Se is a key intermediate in the methylation process of inorganic and organic selenium compounds. Accumulation of H2Se resulting from inhibition of the Se-methylation metabolism, the detoxification pathway of selenium, is found in animals following repeated oral administration of a toxic dose of CySeSeCy. The Se-methylation inhibition is caused by a reduction in the Sadenosylmethionine (SAM) level due to the repression of methionine adenosyltransferase activity. The excess of H2Se produced by inhibition of methionine adenosyltransferase contributes to the hepatotoxicity caused by CySeSeCy. Moreover, SeMet is now known to be directly metabolized to monomethylselenol (MMSe) as selenide by γ-elimination enzyme in mouse liver. The disturbances in detoxification pathway of Se compounds such as methylation process may be involved in the development of selenosis.
The modifying effects of novel synthesized oragnoseleniums on carcinogenesis have been examined in several organs. p-Methoxybenzeneselenol (MBS), benzylselenocyanate (BSC) and 1, 4-phenylenebis(methylene)-selenocyanate (p-XSC) have been synthesized, respectively. MBS reduced benzo[a]pyrene (B[a]P)-induced forestomach tumors in female CD-1 mice and azoxymethane (AOM)-induced colon, liver and kidney neoplasms in female F344 rats. BSC has been effective on AOM-induced colon neoplasms and liver preneoplastic lesions in male F344 rats, and dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in female SD rats. p-XSC reduced AOM induced colon neoplasms, 4-(methlynitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung neoplasms in female A/J mice, DMBA induced mammary neoplasms in female SD rats, and 4-nitroquinoline oxide(4-NQO)-induced tongue carcinogenesis in male F344 rats. BSC increased selenium-dependent glutathione peroxidase in the kidney, colon and small intestine. An increase in total liver cytochrome P-450 was also found in BSC-treated rats. Following AOM treatment, signyficantly less O6-methylguanine and 7-methylguanine was present in the colon DNA from rats consuming the BSC diet than in the rats fed control diets. Those results indicate that dietary BSC induces the enzymes to hydroxylate or oxidate the carcinogens and decrease DNA alkylation. p-XSC inhibited NNK-induced oxidative damage in the lung of A/J mice or F344 rats, DMBA-DNA adducts in rat mammary tissue, and induced apoptosis. These mechanisms may account for their chemopreventive activities.
The impact of synchrotron radiation as a new X-ray source with its polychromatic nature and associated high intensity and fine collimation has brought important advances in the field of macromolecular crystallography. It has extended structure determinations of proteins to higher resolution, allowed use of smaller crystals with larger unit cells. In particular, selenoprotein is a suitable material for X-ray crystal structure analysis with synchrotron radiation, since its polychromatic nature and anomalous diffraction from the selenium atom(s) in the protein allow the multiple-wavelength anomalous-diffraction (MAD) method to be used for phase determination. RIKEN beam line I (BL45XU), installed in the SPring-8 synchrotron radiation facility, has been designed and developed to optimize MAD data collection based on a 'trichromatic concept'. This concept facilitates simultaneous data collection by use of a'trichromator', of three intensity data-sets at three different wavelengths from a single protein crystal, and thus results in the minimization of systematic errors in the measurement of anomalous diffraction by the MAD method. The X-ray crystallographic analysis of selenoprotein with SPring-8 synchrotron radiation, therefore, results in very fast data collection and high resolution structural analysis using a single protein crystal, which should lead to elucidation of the structure-function relationship of selenoprotein.
Peptides are known to be widely distributed in the body, particularly in the gut and the nervous system. In 1982, the presence of a novel brain peptide, neuropeptide Y (NPY) was reported. Subsequently, it was established that NPY is colocalized and co-released with norepinephrine and is distributed in both the central and the peripheral nervous systems. Other reports have indicated the possible role of NPY as a neuromodulator for the catecholaminergic neurons. Since altered sympathetic activity plays a crucial role in the pathophysiology of congestive heart failure, we examined the role of NPY in the modulation of sympathetic activity in various cardiovascular diseases including congestive heart failure. This review article will provide an insight into the status of the NPY receptor the possible therapeutic targets in congestive heart failure.
A number of different methods have been used for modeling the adsorption and biotransformation of volatile organic chlorinated compounds such as tetrachloroethylene perchloroethylene (PCE). In this study, PCE was degraded by granular biological activated carbon (GBAC) in an anaerobic Erlenmeyer flask reactor. In order to study GBAC, we performed experiments in which the efficiencies of GBAC, granular activated carbon (GAC) and anaerobic sludge for the removal of PCE were compared. In this paper, we describe how PCE was microbially biotransformed and adsorbed on GBAC. The biotransformational products were trichloroethylene (TCE) and cis-1, 2-dichloroethylene (cis-1, 2-DCE), which were adsorbed by GBAC (at solid phase). Thus, the GBAC acted not only as a microbiological carrier, but was also instrumental in adsorbing PCE and the biotransformation products. Since the adsorbed PCE was biotransformed on the GBAC, we concluded that GBAC might be effective in the treatment of PCE in synthetic wastewater. The model confirmed that the GBAC was involved in the degradation of PCE under our conditions. The model confirmed that the GBAC was instrumental in treating PCE.
The exact mechanism by which troglitazone improves insulin sensitivity is not well understood. Eight 35-week-old male diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats were treated with troglitazone (30mg/kg body weight/d) for 20d (OLETF-T). Body composition, glucose tolerance, serum lipid profile and expression of glucose transporter 4 (Glut 4) in OLETF-T were compared with those in 8 male control OLETF rats and in 18 normal Long Evans Tokushima Otsuka (LETO) rats. Body weight, visceral fat weight, and pancreas weight in OLETF-T rats were significantly lower than those in OLETF rats (p<0.05). Furthermore, troglitazone treatment attenuated atrophy and fibrosis of the pancreas. Serum concentrations of glucose, triglyceride, total cholesterol and immunoreactive insulin (IRI) were also significantly lower in OLETF-T rats. Expression of Glut 4 in plasma membrane fractions of skeletal muscle and viscera fat was detected by Western blot. The amount of Glut 4 protein in skeletal muscle in OLETF rats was 52% of that in LETO rats, and 75% for OLETF-T rats. In visceral fat, Glut 4 expressions in OLETF and OLETF-T rats were 38% and 83%, respectively, of that in LETO rats. Thus, treatment with troglitazone prevented the decrease of Glut 4 expression seen in OLETF rats. Glucose tolerance was improved significantly by the treatment, and the amount of secreted IRI in response to oral glucose tolerance test was 1798pM, 702.2pM, and 1103.5pM, in OLETF, OLETF-T and LETO rats, respectively. The data presented suggest that treatment with troglitazone increased the Glut 4 expression in both skeletal and visceral fat tissues of OLETF rats, which may result in the improvement of insulin sensitivity and preservation of pancreas function.
To reduce formaldehyde in indoor environments, non-formalin products for wallpaper adhesives have been increased in number. In this study, an analysis method using gas chromatography (GC) and gas chromatography mass spectrometry (GC/MS) specific to each of several antimicrobial agents (2-methyl-4-isothiazolin-3-one (MIT), 5-chloro-2-methyl-4-isothiazolin-3-one (Cl-MIT), 1, 2-benzisothiazolin-3-one (BIT), 2-n-octyl-4-isothiazolin-3-one (OIT) and 2, 4, 5, 6-tetrachloro-isophthalonitrile (TPN)) was developed aiming to clarify the actual states of utilization of these agents. These antimicrobials have been reported to induce health impairments such as contact dermatitis. Furthermore, seven non-formalin adhesives for wallpaper sold as building materials were analyzed according to the established method. The present results demonstrated that these adhesives contained a combination of antimicrobials (MIT, Cl-MIT, OIT and TPN), and one of them contained TPN at a high concentration (2, 480μg/g), which is highly contact-allergenic.
In order to determine the safety or otherwise of amlodipine, we evaluated lipid and lipoprotein indices as well as fibrinolytic parameters in Japanese hypertensive patients undergoing amlodipine treatment. Lipids, lipoproteins, tissue plasminogen activator (t-PA), total and free plasminogen activator inhibitor-1 (PAI-1) and t-PA-PAI-1 complexes were determined in twenty-seven patients with essential hypertension before and after 3 months of amlodipine treatment. Plasma renin and noradrenaline levels were also determined. The mean systolic and diastolic blood pressures and heart rate were reduced, while the plasma renin and noradrenaline levels remained unchanged after amlodipine therapy. Triglycerides, very low density lipoprotein-cholesterol (VLDLC), total and free PAI-1 levels were significantly reduced, while the levels of total cholesterol (TC), high density lipoprotein-cholesterol (HDLC), low density lipoprotein-cholesterol (LDLC), HDLC/TC ratio, t-PA and t-PA-PAI-1 complex did not change from their pretreatment values after amlodipine treatment. There was a significant positive correlation between HDLC and t-PA-PAI-1 complex levels after amlodipine treatment. The findings from the present study show that amlodipine is effective for the treatment of hypertension and dose not cause reflex tachycardia in Japanese patients. We also found that amlodipine treatment is a safe antihypertensive agent characterized by beneficial lipid changes and enhanced fibrinolysis in Japanese hypertensive patients. The direct relationship between lipid levels and fibrinolytic function seen in this study has added to our understanding and knowledge of the pathogenesis of atherosclerosis during antihypertensive pharmacotherapy.
In order to grasp the actual conditions of use of three kinds of organoiodine compounds, 3-iodo-2-propynylbutylcarbamate (IPBC), 1-bromo-3-ethoxycarbonyloxy-1, 2-diiodo-1-propene (BECDIP), and 4-chlorophenyl-3-iodopropargylformal (CPIP), which are used as household antimicrobial/antifungal agents and wood preservatives, an analytical method using a gas chromatograph equipped with a flame ionization detector (FID-GC) and gas chromatography mass spectrometry (GC/MS) was employed.The calibration curve (peak area) by FID-GC showed good linearity in the range from 20 μg/ml to 100μg/ml (correlation coefficient R=0.99 or more). A purification method using a liquid-liquid partition by n-hexane/water and the cartridge column (florisil or alumina) was established, and the addition recovery exceeded 90% by this method. By employing this method, the analysis was carried out for 18 commercially available samples. As a result, IPBC was detected from 2 samples of aerosol type fungicides, BECDIP from 4 samples of wood preservatives, and CPIP from one sample of an aerosol type antifungal/insecticidal paint.
Althouh p-phenylenediamine (PPD) and related compounds have been used as antioxidants in rubber products, they commonly display sensitizing properties and have been associated with contact dermatitis. N-Substitution of PPD influences the sensitization potential, so it is important to examine which PPD antioxidants are used in commercially available rubber products, to prevent the occurrence of contact dermatitis in sensitized patients. In this study we developed a method for the determination of PPD derivatives, such as N-(1-methylheptyl)-N'-phenyl-p-phenylenediamine (MHPPD), N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD) and N-1, 3-dimethylbutyl-N'-phenyl-p-phenylenediamine (DMBPPD), using high performance liquid chromatography (HPLC), and investigated the PPD antioxidant content of rubber boots used by farmers. The PPD derivatives were extracted from rubber boots with acetone : chloroform (1 : 1). The extract was loaded then on to a silica-gel column, and eluted with 50ml of diethylether : hexane mixtures (5 : 95, 10 : 90, 20 : 80 and 50 : 50) in that order. MHPPD and DMBPPD were eluted in the diethylether : hexane (10 : 90) fraction. The recovery of MHPPD by fractionation was 95±8% (n=5). IPPD was detected in the diethylether : hexane (20 : 80) fraction. Each fraction was evaporated, and the residue was dissolved in dichloromethane and subjected to HPLC with an ODS coulmn and a UV detector (detection wavelength 290nm). The mobile phase was methanol : water (85 : 15). After fractionation, the retention times of these PPD derivatives were found not to overlap with other rubber additives. The linear calibration curves for MHPPD, DMBPPD and IPPD were obtained over the range of 0.1-400μg/ml. Using this method, eight types of rubber boot used by farmers were analyzed. MHPPD was not found in any of the rubber boots, but DMBPPD and IPPD were detected.
The effect of genistein and casein phosphopeptides (CPP) on bone components in the femoral tissue of young and elderly rats was investigated. Genistein has been shown to directly stimulate bone formation, and CPP has been reported to increase intestinal calcium absorption. Genistein (10 or 50μg/100g body weight) or CPP (40mg/100g) was orally administered to young (5 weeks old) or elderly (50 weeks old) rats for 14d. The administration of genistein (50μg/100g) resulted in a significant increase in the femoral dry weight, calcium content, alkaline phosphatase activity, and deoxyribonucleic acid (DNA) content in the femoral-diaphyseal and metaphyseal tissue of young and elderly rats. The administration of CPP (40mg/100g) caused a significant increase in the femoral dry weight of young and elderly rats. CPP administration increased significantly the diaphyseal calcium content in young rats, but it did not have an effect on the diaphyseal and metaphyseal alkaline phosphatase activity and DNA content. The genistein (50μg/100g)-increased femoral dry weight, calcium content, alkaline phosphatase activity, and DNA content in the diaphyseal and metaphyseal tissue of young rats was significantly enhanced by the simultaneous administration of CPP (40mg/100g). In elderly rats, this enhancement was resulted in the metaphyseal tissue. This study demonstrates that the combination of genistein and CPP administration has a synergistic-anabolic effect on bone components in rats with increasing age, suggesting that it may have a role in the prevention of osteoporosis.
Mechanism of Zn2+-induced stimulation and Cd2+-induced inhibition of bone nodule (BN) formation was studied in calvarial osteoblastic cells isolated from rats of various ages. Zn2+ dose-dependently increased BN formation at 10-8м or higher concentrations in the cells from 10-week-old (young) and 90-week-old (aged) rats and the degree of stimulation was much smaller in the aged rat cells. Conversely, Cd2+ dose-depedently inhibited BN formation in the young and aged rat cells, with the effect being more prominent in the aged rat cells. The lowest concentrations required to reduce the area of BN in the young and aged rat cells were 10-8 and 10-9м, respectively. Insulin-like growth factor (IGF)-I (10-6м) increased the area of BN by 350% and 50% in the young and aged rat cells, respectively. Zn2+ (3×10-6м) stimulated IGF-I production by 580% and 100% in the young and aged rat cells, respectively. Anti-IGF-I antibody almost completely inhibited the stimulatory effect of Zn2+ on BN formation in the young and aged rat cells. These results indicate that the effect of Zn2+ is mediated through the production of IGF-I. The stimulation of IGF-I production by Zn2+ (3×10-6м) was blocked by Cd2+ (10-7м) in the young and aged rat cells. Cd2+ (10-7м) also inhibited the increase in BN formation by IGF-I in the aged, but not in the young rat cells. These results indicate that Cd2+ inhibits the induction of IGF-I production by Zn2+ and the subsequent action of IGF-I on osteoblasts in the aged rat cells, while only the IGF-I production is affected in the young rat cells.
Alpha-linolenic acid (LL) was tested for antidiabetic activity in KK-Ay mice, an animal model of type 2 diabetes. Repeated administration of LL (300mg/kg) to KK-Ay mice significantly suppressed the increment of blood glucose at 21d (p<0.05). However, no affect on the blood glucose level in normal mice was seen, indicating that LL is useful in treating type 2 diabetes. In addition, LL improved hyperinsulinemia in KK-Ay mice (p<0.01). LL also significantly decreased the blood glucose at 120min in the insulin tolerance test (p<0.05). The muscle content of facilitative glucose transpoter isoform 4 (GLUT4) protein content in the total membrane fraction from KK-Ay mouse muscle significantly increased in the LL-treated mice when compared to that in the controls (p<0.01). From these findings, it seems likely that the hypoglycemic effect of LL is derived, at least in part, from the decrease in insulin resistance, due presumably to the increase of GLUT4 protein content in total membrane of the muscle.
A rapid and sensitive bioassay for detecting cytotoxicity was developed in this study to be used in evaluating many kind of chemicals. This assay, based on the LDL (low density lipoprotein)-uptake activity of human hepatoblastoma cells, Hep G2, can evaluate cytotoxicity for 48h with high sensitivity and selectivity using a 96 well plate and a fluorescent plate reader. We evaluate the toxicity of 230 kinds of chemicals and formulate the dose response data by a simple mathematical equation. The toxicity parameters derived by the formulation had some correlations in terms of chemical groups, which were classified as aromatics, organics, metals, and so on.
Subacute vanadium toxicity was investigated in rats. Female Wistar strain rats, weighing 180g (approximately 6 weeks old), were divided into three groups of seven each. They were fed a base diet with addition of various concentrations of vanadium (0, 50 or 100ppm) for 10 weeks. The amounts of vanadium found in kidney and liver increased in response to vanadium exposure. A significant increase in metallothionein (MT) was observed in kidney of both the 100ppm and 50ppm vanadium-fed groups. Significant decreases in hemoglobin, hematocrit and leukocyte counts were also observed in the former, as well as decreased lymphocyte counts, B cells and immunoglobulin G and M levels.
We evaluated the efficacy of flavonoid quercetin as an antioxidant in hepatic lysosomal fractions of mice using the hydrophilic radical generator 2, 2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and the lipophilic radical generator 2, 2'-azobis(2, 4-dimethylvaleronitrile) (AMVN). Quercetin inhibited lipid peroxidation in lysosomal fractions, measured as thiobarbituric acid reactive substances (TBARS), and inhibited the release of lysosomal enzymes more obviously against AAPH than against AMVN. Whereas inhibitory effects of quercetin on lipid peroxidation were weaker than those of the synthetic lipophilic antioxidant 2, 6-di-tert-butyl-p-cresol (BHT), lysosomal fractions preloaded with quercetin effectively quenched 1, 1-diphenly-2-picrylhydrazyl radicals, similar to BHT. Rutin, a glycoside of quercetin, was less potent than quercetin in these experiments. These findings suggest that quercetin could have potent antioxidative activity in the spaces between an aqueous phase and a lipid phase in biological systems owing to the localization within membranes as well as evident antioxidative activity.