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Reaction of diastereomerically pure phosphinoselenoic acid salts with oxalyl chloride leads to enantiomerically pure P-chiral phosphinoselenoic chlorides with inversion of configuration at phosphorus; one of these chlorides is converted to a phosphinoselenothioic acid salt with a high degree of enantioselectivity.
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A Zn(2+)-glycerophosphocholine cholinephosphodiesterase (EC 3.1.4.38) purified from mouse brain was found to be reversibly inhibited by tellurium tetrachloride. This effect was characterized by a competitive pattern of inhibition, with apparent Ki values of 0.7 microM and 1.5 microM for the hydrolysis of p-nitrophenylphosphocholine and glycerophosphocholine respectively. Interestingly, the inhibitory effect of tellurium tetrachloride was found to be greatly potentiated by tetramethylammonium salt, indicative of a synergistic interaction between the two compounds. Additionally, it was observed that the effect of tellurium tetrachloride was not affected by a number of other metal ions, and was more pronounced at neutral pH, suggesting that the inhibitory role of the tellurium tetrachloride may be of importance under physiological conditions. Thus Zn(2+)-glycerophosphocholine cholinephosphodiesterase is proposed to be one of the target enzymes which is susceptible to the inhibitory effect of tellurium tetrachloride.
Insulin regulates the expression of genes involved in a variety of metabolic processes. In chick-embryo hepatocytes in culture, insulin amplifies the tri-iodothyronine (T3)-induced enzyme activity, and the level and rate of transcription of mRNA for both fatty acid synthase (FAS) and malic enzyme (ME). Insulin alone, however, has little or no effect on the expression of these genes. In chick-embryo hepatocytes, the mechanism by which insulin regulates the expression of these or other genes is not known. Several recent studies have compared the effects of zinc, vanadate and selenate on insulin-sensitive processes in an attempt to probe the mechanism of insulin action. Because zinc, vanadate and selenate mimic the effects of insulin on several processes, they have been termed insulin-mimetics. We have studied the effect of zinc, vanadate and selenate on the T3-induced expression of both FAS and ME. Like insulin, these agents had little or no effect on the basal activities for FAS and ME in chick-embryo hepatocytes in culture for 48 h. Unlike insulin, however, zinc, vanadate and selenate inhibited the T3-induced activities and mRNA levels of both FAS and ME. Maximal inhibition was achieved at concentrations of 50 microM zinc or vanadate, or 20 microM selenate. Zinc and vanadate also inhibited the T3-induced transcription of the FAS and ME genes. Although the mechanism of this inhibition is unknown, our results indicate that it is not mediated through inhibition of binding of T3 to its nuclear receptor nor through a general toxic effect. Thus zinc, vanadate and selenate are not insulin-mimetics under all conditions, and their effects on other insulin-sensitive processes may be fortuitous and unrelated to actions or components of the insulin signalling pathway.
A number of biochemically distinct systems have been characterized for the microbial reduction of the oxyanions, selenate (SeO(4)(2-)) and nitrate (NO(3)(-)). Two classes of molybdenum-dependent nitrate reductase catalyse the respiratory-linked reduction of nitrate (NO(3)(-)) to nitrite (NO(2)(-)). The main respiratory nitrate reductase (NAR) is membrane-anchored, with its active site facing the cytoplasmic compartment. The other enzyme (NAP) is water-soluble and located in the periplasm. In recent years, our understanding of each of these enzyme systems has increased significantly. The crystal structures of both NAR and NAP have now been solved and they provide new insight into the structure, function and evolution of these respiratory complexes. In contrast, our understanding of microbial selenate (SeO(4)(2-)) reduction and respiration is at an early stage; however, similarities to the nitrate reductase systems are emerging. This review will consider some of the common themes and variations between the different classes of nitrate and selenate reductases.
Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9 +/- 1.0 microm). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 microm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 microm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.
To investigate how plants acquire and assimilate sulfur from their environment, we isolated and characterized two mutants of Arabidopsis thaliana deficient in sulfate transport. The mutants are resistant to selenate, a toxic analogue of sulfate. They are allelic to each other and to the previously isolated sel1 (selenate-resistant) mutants, and have been designated sel1-8 and sel1-9. Root elongation in these mutants is less sensitive to selenate than in wild-type plants. Sulfate uptake into the roots is impaired in the mutants under both sulfur-sufficient and sulfur-deficient conditions, but transport of sulfate to the shoot is not affected. The sel1 mutants contain lesions in the sulfate transporter gene Sultr1;2 located on the lower arm of chromosome 1. The sel1-1, sel1-3 and sel1-8 mutants contain point mutations in the coding sequences of Sultr1;2, while the sel1-9 mutant has a T-DNA insertion in the Sultr1;2 promoter. The Sultr1;2 cDNA derived from wild-type plants is able to complement Saccharomyces cerevisiae mutants defective in sulfate transport, but the Sultr1;2 cDNA from sel1-8 is not. The Sultr1;2 gene is expressed mainly in roots, and accumulation of transcripts increases during sulfate deprivation. Examination of transgenic plants containing the Sultr1;2 promoter fused to the GUS-reporter gene indicates that Sultr1;2 is expressed mainly in the root cortex, the root tip and lateral roots. Weaker expression of the reporter gene was observed in hydathodes, guard cells and auxiliary buds of leaves, and in anthers and the basal parts of flowers. The results indicate that Sultr1;2 is primarily involved in importing sulfate from the environment into the root.
The possibility that microorganisms might use reduced humic substances (humics) as an electron donor for the reduction of electron acceptors with a more positive redox potential was investigated. All of the Fe(III)- and humics-reducing microorganisms evaluated were capable of oxidizing reduced humics and/or the reduced humics analogue anthrahydroquinone-2,6,-disulphonate (AHODS), with nitrate and/or fumarate as the electron acceptor. These included Geobacter metallireducens, Geobacter sulphurreducens, Geothrix fermentans, Shewanella alga, Wolinella succinogenes and 'S. barnesii'. Several of the humics-oxidizing microorganisms grew in medium with AHQDS as the sole electron donor and fumarate as the electron acceptor. Even though it does not reduce Fe(III) or humics, Paracoccus denitrificans could use AHQDS and reduced humics as electron donors for denitrification. However, another denitrifier, Pseudomonas denitrificans, could not. AHODS could also serve as an electron donor for selenate and arsenate reduction by W. succinogenes. Electron spin resonance studies demonstrated that humics oxidation was associated with the oxidation of hydroquinone moieties in the humics. Studies with G. metallireducens and W. succinogenes demonstrated that the anthraquinone-2,6-disulphonate (AQDS)/AHQDS redox couple mediated an interspecies electron transfer between the two organisms. These results suggest that, as microbially reduced humics enter less reduced zones of soils and sediments, the reduced humics may serve as electron donors for microbial reduction of several environmentally significant electron acceptors.
A Gram-negative bacterium, identified as Stenotrophomonas maltophilia by fatty acid analysis and 16S rRNA sequencing, was isolated from a seleniferous agricultural evaporation pond sediment collected in the Tulare Lake Drainage District, California. In cultures exposed to the atmosphere, the organism reduces selenate (SeO4(2-)) and selenite (SeO3(2-)) to red amorphous elemental selenium (Se degrees ) only upon reaching stationary phase, when O2 levels are less than 0.1 mg l(-1). In 48 h, S. maltophilia removed 81.2% and 99.8% of added SeO4(2-) and SeO3(2-) (initial concentration of 0.5 mM), respectively, from solution. Anaerobic growth experiments revealed that the organism was incapable of using SeO4(2-), SeO3(2-), SO4(2-) or NO3- as a terminal electron acceptor. Transmission electron microscopy of cultures spiked with either Se oxyanion were found to contain spherical extracellular deposits. Analysis of the deposits by energy-dispersive X-ray spectroscopy revealed that they consist of Se. Furthermore, S. maltophilia was active in producing volatile alkylselenides when in the presence of SeO4(2-) and SeO3(2-). The volatile products were positively identified as dimethyl selenide (DMSe), dimethyl selenenyl sulphide (DMSeS) and dimethyl diselenide (DMDSe) by gas chromatography-mass spectrometry. Our findings suggest that this bacterium may contribute to the biogeochemical cycling of Se in seleniferous evaporation pond sediments and waters. This organism may also be potentially useful in a bioremediation scheme designed to treat seleniferous agricultural wastewater.
Complementary and alternative therapies are used with increasing frequency in men with prostate cancer. However, little is known about the efficacy of such therapies for this cancer. While epidemiological data support the association between intake of certain micronutrients with development of prostate cancer, there exist limited prospective data that support the chemopreventative or therapeutic value of such nutritional agents in prostate cancer. To date, one of the most studied treatments has been PC-SPES, a combination of eight herbal therapies with activity against prostate cancer. Studies in cell lines of human prostate cancer demonstrate significant dose-dependent decreases in cellular viability after exposure to extracts of this agent. Clinical studies suggested that PC-SPES could reduce prostate specific antigen levels in patients with either androgen-dependent or androgen-independent prostate cancer. Toxicity was mild, although there was a low risk of thromboembolic events with such treatment. Manufacture of PC-SPES was recently halted, after revelations that the herbal combination was contaminated with warfarin, which led to a recall by the manufacturer. Subsequent analyses also revealed the presence of diethylstilbestrol (DES) and indomethacin in some lots of PC-SPES. Available data regarding other alternative therapies are reviewed as well.
Owing to the low pKa of its selenohydryl group, reduced monoselenolipoic acid (MSL) can be expected to be a very versatile antioxidant with direct thioredoxin-like activity. Since MSL supports the growth of lipoate-dependent bacteria, it can be anticipated that MSL will be susceptible to reversible reduction by one or more of the enzymes that reduces lipoic acid - thus greatly potentiating MSL's utility as an antioxidant. If it is not metabolized to release toxic free selenide, MSL may have interesting pharmaceutical potential.
Most MS patients use unconventional therapies, usually as complementary measures in addition to the conventional treatment. Only a few adequate clinical trials exist in this field. By definition, the efficacy of these therapies is unproven. Moreover, the possible risks are also largely unknown. Some therapies rely on rational pathophysiological considerations, other must be regarded as potentially harmful. The influence of diet on MS is unproven. Possibly, unsaturated fatty acids are beneficial. However, a few randomized trials yielded inconclusive results. Long-term supplementation of Vitamin D is associated with a decreased MS incidence. There is, however, insufficient evidence for an influence of Vitamin D on the course of the disease. Because of the high prevalence of osteoporosis in MS patients, prophylaxis with Vitamin D and Calcium is widely accepted. The effects of various minerals, selenium, antioxidant compounds, fish oil or vitamins remain speculative. Many patients use cannabis to alleviate spasticity and pain. Small series indicated positive effects, but randomized trials were negative for spasticity. However, many patients report subjective improvement under cannabis even if their objective parameters remain unchanged. Hyperbaric oxygenation was the subject of several small studies with heterogeneous results which, overall, do not support its use. Generally, physical therapies are perceived as an established therapy for MS. Short-term effects are probable, whereas the possible favourable long-term effects are unclear.
A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase. A divalent metal ion, Mg2+, and a monovalent cation, K+, NH4+, or Rb+, are required for selenophosphate synthetase activity [Veres, Z., Kim, I. Y., Scholz, T. D. & Stadtman, T. C. (1994) J. Biol. Chem. 269, 10597-10603]. Na+ and Li+ are ineffective as activators and in the presence of K+ are inhibitory. Mn-ATP, although not able to replace Mg-ATP for catalytic activity, binds to the enzyme provided an active monovalent cation is present. No Mn-ATP is bound when K+ is replaced with Na+. The requirement for K+, both for Mn-ATP binding and for catalytic activity of the synthetase, indicates a specific monovalent cation-induced conformational state of the enzyme. Previously we reported that activity of the enzyme is markedly inhibited by micromolar levels of Zn2+ in the presence of millimolar levels of Mg2+ [Kim, I. Y., Veres, Z. & Stadtman, T. C. (1993) J. Biol. Chem. 268, 27020-27025]. Binding of Mn-ATP also is decreased upon addition of Zn2+, indicating that the inhibitory effect of Zn2+ is exerted at the substrate-binding step of the overall selenophosphate synthetase reaction. When a cysteine residue at position 17 or 19 is replaced with serine, Mn-ATP binding to these mutant enzymes is unaffected by Zn2+ addition. Direct involvement of these cysteine residues in the zinc binding site was shown by use of 65ZnCl2. Radioactive Zn2+ bound to wild-type enzyme and was retained after gel filtration, but under the same conditions the catalytically inactive Cys-17 mutant protein and the catalytically active Cys-19 mutant enzyme were unlabeled.
The selD gene from Haemophilus influenzae has been overexpressed in Escherichia coli. The expressed protein was purified to homogeneity in a four-step procedure and then carboxymethylated by reaction with chloroacetate. N-terminal sequencing by Edman degradation identified residue 16 as carboxymethyl selenocysteine, which corresponded to the essential cysteine residue in the glycine-rich sequence of the E. coli selenophosphate synthetase. It would be expected that an ionized selenol of a selenocysteine in place of a catalytically essential cysteine residue would result in an enzyme with increased catalytic activity. To test this hypothesis we kinetically characterized the selenocysteine containing selenophosphate synthetase from H. influenzae and compared its catalytic activity to that of the cysteine containing selenophosphate synthetase from E. coli. Our characterization revealed the Km values for the two substrates, selenide and ATP, were similar for both enzymes. However, the selenocysteine-containing enzyme did not exhibit the expected higher catalytic activity. Based on these results we suggest a role of selenocysteine in H. influenzae that is not catalytic.
The NIFS protein from Azobacter vinelandii is a pyridoxal phosphate-containing homodimer that catalyzes the formation of equimolar amounts of elemental sulfur and L-alanine from the substrate L-cysteine (Zheng, L., White, R. H., Cash, V. L., Jack, R. F., and Dean, D. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2754-2758). A sulfur transfer role of NIFS in which the enzyme donates sulfur for iron sulfur center formation in nitrogenase was suggested. The fact that NIFS also can catalyze the decomposition of L-selenocysteine to elemental selenium and L-alanine suggested the possibility that this enzyme might serve as a selenide delivery protein for the in vitro biosynthesis of selenophosphate. In agreement with this hypothesis, we have shown that replacement of selenide with NIFS and L-selenocysteine in the in vitro selenophosphate synthetase assay results in an increased rate of formation of selenophosphate. These results thus support the view that a selenocysteine-specific enzyme similar to NIFS may be involved as an in vivo selenide delivery protein for selenophosphate biosynthesis. A kinetic characterization of the two NIFS catalyzed reactions carried out in the present study indicates that the enzyme favors L-cysteine as a substrate compared with its selenium analog. A specific activity for L-cysteine of 142 nmol/min/mg compared with 55 nmol/min/mg for L-selenocysteine was determined. This level of enzyme activity on the selenoamino acid substrate is adequate to deliver selenium to selenophosphate synthetase in the in vitro assay system described.
Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. Kinetic characterization revealed the K(m) value for selenide approached levels that are toxic to the cell. Our previous demonstration that a Se(0)-generating system consisting of l-selenocysteine and the Azotobacter vinelandii NifS protein can replace selenide for selenophosphate biosynthesis in vitro suggested a mechanism whereby cells can overcome selenide toxicity. Recently, three E. coli NifS-like proteins, CsdB, CSD, and IscS, have been overexpressed and characterized. All three enzymes act on selenocysteine and cysteine to produce Se(0) and S(0), respectively. In the present study, we demonstrate the ability of each E. coli NifS-like protein to function as a selenium delivery protein for the in vitro biosynthesis of selenophosphate by E. coli wild-type SPS. Significantly, the SPS (C17S) mutant, which is inactive in the standard in vitro assay with selenide as substrate, was found to exhibit detectable activity in the presence of CsdB, CSD, or IscS and l-selenocysteine. Taken together the ability of the NifS-like proteins to generate a selenium substrate for SPS and the activation of the SPS (C17S) mutant suggest a selenium delivery function for the proteins in vivo.
Selenium, an essential biological trace element, has been shown to reduce and prevent the incidence of cancer. Our previous studies have shown that selenite is involved in the chemoprevention of cancer and induction of apoptosis of cancer cells. In this study, we demonstrate that selenite also inhibits the invasion of tumor cells. Cancer cell invasion requires coordinated processes, such as changes in cell-cell and cell-matrix adhesion, degradation of the extracellular matrix, and cell migration. We found that selenite inhibited invasion of HT1080 human fibrosarcoma cells. Adhesion of HT1080 cells to the collagen matrix was also inhibited by treatment with selenite, but cell-cell interaction and cell motility were not affected by selenite. Moreover, selenite reduced expression of matrix metalloproteinase-2 and -9 and urokinase-type plasminogen activator, which are involved in matrix degradation, but increased a tissue inhibitor of metalloproteinase-1. This inhibitory effect of selenite on the protease expressions was mediated by the suppression of transcription factors, NF-kappaB and AP-1. However, selenate showed no remarkable effect on all the steps of cancer cell invasion.