Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate

Over the last few decades there has been increasing interest in oxometalate and polyoxometalate applications to medicine and pharmacology. This interest arose, at least in part, due to the properties of these classes of compounds as anti-cancer, anti-diabetic agents, and also for treatment of neurodegenerative diseases, among others. However, our understanding of the mechanism of action would be improved if biological models could be used to clarify potential toxicological effects in main cellular processes. Sarcoplasmic reticulum (SR) vesicles, containing a large amount of Ca^2 +-ATPase, an enzyme that accumulates calcium by active transport using ATP, have been suggested as a useful model to study the effects of oxometalates on calcium homeostasis. In the present article, it is shown that decavanadate, decaniobate, vanadate, tungstate and molybdate, all inhibited SR Ca²⁺-ATPase, with the following IC₅₀ values: 15, 35, 50, 400 μM and 45 mM, respectively. Decaniobate (Nb₁₀), is the strongest P-type enzyme inhibitor, after decavanadate (V₁₀). Atomic-absorption spectroscopy (AAS) analysis, indicates that decavanadate binds to the protein with a 1:1 decavanadate:Ca^2 +-ATPase stoichiometry. Furthermore, V₁₀ binds with similar extension to all the protein conformations, which occur during calcium translocation by active transport, namely E1, E1P, E2 and E2P, as analysed by AAS. In contrast, it was confirmed that the binding of monomeric vanadate (H₂VO₄^2 −; V₁₎ to the calcium pump is favoured only for the E2 and E2P conformations of the ATPase, whereas no significant amount of vanadate is bound to the E1 and E1P conformations. Scatchard plot analysis, confirmed a 1:1 ratio for decavanadate-Ca^2 +-ATPase, with a dissociation constant, k_d of 1 μM^− 1. The interaction of decavanadate V₁₀O₂₈^6 − (V₁₀) with Ca^2 +-ATPase is prevented by the isostructural and isoelectronic decaniobate Nb₁₀O₂₈^6 − (Nb₁₀), whereas no significant effects were detected with ATP or with heparin, a known competitive ATP binding molecule, suggesting that V₁₀ binds non-competitively, with respect to ATP, to the protein. Finally, it was shown that decaniobate inhibits SR Ca^2 +-ATPase activity in a non competitive type of inhibition, with respect to ATP. Taken together, these data demonstrate that decameric niobate and vanadate species are stronger inhibitors of the SR calcium ATPase than simple monomeric vanadate, tungstate and molybdate oxometalates, thus affecting calcium homeostasis, cell signalling and cell bioenergetics, as well many other cellular processes. The ability of these oxometalates to act either as phosphate analogues, as a transition-state analogue in enzyme-catalysed phosphoryl group transfer processes and as potentially nucleotide-dependent enzymes modulators or inhibitors, suggests that different oxometalates may reveal different mechanistic preferences in these classes of enzymes.     

Recent perspectives into biochemistry of decavanadate

The number of papers about decavanadate has doubled in the past decade. In the present review, new insights into decavanadate biochemistry, cell biology, and antidiabetic and antitumor activities are described. Decameric vanadate species (V10) clearly differs from monomeric vanadate (V1), and affects differently calcium pumps, and structure and function of myosin and actin. Only decavanadate inhibits calcium accumulation by calcium pump ATPase, and strongly inhibits actomyosin ATPase activity (IC50 = 1.4 μmol/L, V10), whereas no such ef- fects are detected with V1 up to 150 μmol/L; prevents actin polymerization (IC50 of 68 μmol/L, whereas no effects detected with up to 2 mmol/L V1); and interacts with actin in a way that induces cysteine oxidation and vanadate reduction to vanadyl. Moreover, in vivo decavanadate toxicity studies have revealed that acute exposure to polyoxovanadate induces different changes in antioxidant enzymes and oxidative stress parameters, in comparison with vanadate. In vitro studies have clearly demonstrated that mitochondrial oxygen consumption is strongly affected by decavanadate (IC50, 0.1 μmol/L); perhaps the most relevant biological effect. Finally, decavanadate (100 μmol/L) increases rat adipocyte glucose accumulation more potently than several vanadium complexes. Preliminary studies sug- gest that decavanadate does not have similar effects in human adipocytes. Although decavanadate can be a useful biochemical tool, further studies must be carried out before it can be conf irmed that decavanadate and its complexes can be used as anticancer or antidiabetic agents.     

Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadate

Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68+/-22 microM and 17+/-2 microM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2mM concentration of "metavanadate" solution that contains ortho and metavanadate species, as observed by combining kinetic with (51)V NMR spectroscopy studies. Although at 25 degrees C, decameric vanadate (10 microM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 microM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the "decavanadate" interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems.     

Behavior of vanadate and vanadyl ion in canine blood

Radiolabeled vanadium as either vanadyl ion or vanadate ion was injected intravenously into adult beagle dogs, and blood samples were collected at various times up to 48 hr post injection. For each sample, the distribution of vanadium between the cells and the plasma was determined, and the plasma was analyzed by electrophoresis to identify specific vanadium-binding proteins. Initially, vanadyl ion left the bloodstream more rapidly than vanadate, but the rates equalized after about 5 hr. A significant fraction of the vanadium in blood was associated with the cellular component following injection of both forms of vanadium. About 77% of the plasma vanadium was eventually bound by the serum iron transport protein transferrin, regardless of the vanadium species initially injected. For both vanadyl and vanadate, about 30 hr were required to reach the maximum degree of transferrin binding.     

Reaction of vanadate ion with chlorpromazine,formation of vanadyl ion and chlorpromazine free radical

The red colored product, which was identified as a chlorpromazine (CPZ) free radical, was observed in the reaction of CPZ with the vanadate ion (+5 oxidation state). The product and the mechanism for the reaction were characterized from optical and EPR spectrometries. Optimal conditions for generation of the free radical were determined as reaction time within one minute of pH 6 and free radical stabilizing time of 30 minutes by acidifying with HCl. Under these conditions, the stoichiometry for the reaction was found to be 1:1, indicating the involvement of one electron transfer from CPZ to the vanadate ion to form the free radical and vanadyl ion (+4 oxidation state). A possible reaction scheme was proposed:

The implications of this reaction were discussed with regard to the pharmacological action of the vanadate ion and CPZ.     

Interaction of porcine uterine fluid purple acid phosphatase with vanadate and vanadyl cation.

Uteroferrin, the purple acid phosphatase from porcine uterine fluid, is noncompetitively inhibited by vanadate in a time-dependent manner under both aerobic and anaerobic conditions. This time-dependent inhibition is observed only with the diiron enzyme and is absent when the FeZn enzyme is used. The observations are attributed to the sequential formation of two uteroferrin-vanadium complexes. The first complex forms rapidly and reversibly, while the second complex forms slowly and results in the production of catalytically inactive oxidized uteroferrin and V(IV), which is observed by EPR. The redox reaction can be reversed by treatment of the oxidized enzyme first with (V(IV)) and then EDTA to generate a catalytically active uteroferrin. Multiple inhibition kinetics suggests that vanadate is mutually exclusive with molybdate, tungstate, and vanadyl cation. The binding site for each of these anions is distinct from the site to which the competitive inhibitors phosphate and arsenate bind. The time-dependent inhibition by vanadate of uteroferrin containing the diiron core represents a new type of mechanism by which vanadium can interact with proteins and gives additional insight into the binding of anions to uteroferrin.     

人肠道病毒71型与宿主相互作用研究进展

自1969年首次从美国加利福尼亚患者分离出人肠道病毒71型(Human enterovirus 71,HEV71)后,该病毒已在世界范围内造成多次暴发流行,近年来又在中国大陆、中国台湾及其他亚太地区流行。该肠道病毒主要引起婴幼儿手足口病,但在某些患儿可表现为严重的神经系统感染和呼吸、循环功能     

Recent advances in ecosystem-atmosphere interactions: an ecological perspective

The atmosphere and terrestrial ecosystems are fundamentally coupled on a variety of time-scales. On short time-scales, this bi-directional interaction is dominated by the rapid exchange of CO(2), water and energy between the atmosphere and the land surface; on long time-scales, the interaction involves changes in ecosystem structure and composition in response to changes in climate that feed back through biophysical and biogeochemical mechanisms to influence climate over decades and centuries. After briefly describing some early pioneering work, I focus this review on recent advances in understanding long-term ecosystem-atmosphere interactions through a discussion of three case studies. I then examine how efforts to assess the stability and resilience of ecosystem-atmosphere interactions over these long time-scales using Dynamic Global Vegetation Models are hampered by the presence of important functional diversity and heterogeneity within plant communities. Recent work illustrates how this issue can be addressed through the use of Structured Ecosystem Models that more accurately scale between the short-term physiological responses of individual plants and the long-term, large-scale dynamics of heterogeneous, functionally diverse ecosystems.     

植物与内生真菌互作的生理与分子机制研究进展

在自然生态系统中,植物组织可作为许多微生物定居的生态位.内生真菌普遍存在于植物组织内,与宿主建立复杂的相互作用(互惠、拮抗和中性之间的相互转化),并且存在不同的传播方式(垂直和水平传播).内生真菌通过多样化途径来增强植物体的营养生理和抗性机能.但这种生理功能的实现有赖于双方精细的调控机制,表明宿主和真菌双方都进化形成特有的分子调控机制来维持这种互惠共生关系.环境因子(如气候、土壤性质等)、宿主种类和生理状态、真菌基因型的变化都将改变互作结果.此外,菌根真菌和真菌病毒等也可能普遍参与植物-内生真菌共生体,形成三重互作体系,最终影响宿主的表型.研究试图从形态、生理和分子水平阐述内生真菌与植物互作的基础.     

Recent insights into barley and Rhynchosporium commune interactions

Rhynchosporium commune is the causal pathogen of scald in barley (Hordeum vulgare), a foliar disease that can reduce yield by up to 40% in susceptible cultivars. R. commune is found worldwide in all temperate growing regions and is regarded as one of the most economically important barley pathogens. It is a polycyclic pathogen with the ability to rapidly evolve new virulent strains in response to resistance genes deployed in commercial cultivars. Hence, introgression and pyramiding of different loci for resistance (qualitative or quantitative) through marker-assisted selection is an effective way to improve scald resistance in barley. This review summarizes all 148 resistance quantitative trait loci reported at the date of submission of this review and projects them onto the barley physical map, where it is clear many loci co-locate on chromosomes 3H and 7H. We have summarized the major named resistance loci and reiterated the renaming of Rrs15 (CI8288) to Rrs17. This review provides a comprehensive resource for future discovery and breeding efforts of qualitative and quantitative scald resistance loci.     

Recent advances in bivalve-microbiota interactions for disease prevention in aquaculture

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Enhanced sensitivity of insulin-resistant adipocytes to vanadate is associated with oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4).

Vanadate (sodium orthovanadate), an inhibitor of phosphotyrosine phosphatases (PTPs), mimics many of the metabolic actions of insulin in vitro and in vivo. The potential of vanadate to stimulate glucose transport independent of the early steps in insulin signaling prompted us to test its effectiveness in an in vitro model of insulin resistance. In primary rat adipocytes cultured for 18 h in the presence of high glucose (15 mm) and insulin (10(-7) m), sensitivity to insulin-stimulated glucose transport was decreased. In contrast, there was a paradoxical enhanced sensitivity to vanadate of the insulin-resistant cells (EC(50) for control, 325 +/- 7.5 microm; EC(50) for insulin-resistant, 171 +/- 32 microm; p < 0.002). Enhanced sensitivity was also present for vanadate stimulation of insulin receptor kinase activity and autophosphorylation and Akt/protein kinase B Ser-473 phosphorylation consistent with more effective PTP inhibition in the resistant cells. Investigation of this phenomenon revealed that 1) depletion of GSH with buthionine sulfoximine reproduced the enhanced sensitivity to vanadate while preincubation of resistant cells with N-acetylcysteine (NAC) prevented it, 2) intracellular GSH was decreased in resistant cells and normalized by NAC, 3) exposure to high glucose and insulin induced an increase in reactive oxygen species, which was prevented by NAC, 4) EPR (electron paramagnetic resonance) spectroscopy showed a decreased amount of vanadyl (+4) in resistant and buthionine sulfoximine-treated cells, which correlated with decreased GSH and increased vanadate sensitivity, while total vanadium uptake was not altered, and 5) inhibition of recombinant PTP1B in vitro was more sensitive to vanadate (+5) than vanadyl (+4). In conclusion, the paradoxical increased sensitivity to vanadate in hyperglycemia-induced insulin resistant adipocytes is due to oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4). Thus, sensitivity of PTP inhibition and glucose transport to vanadate is regulated by cellular redox state.     

Tetrahymena actin: copolymerization with skeletal muscle actin and interactions with muscle actin-binding proteins

We have previously shown that actin from Tetrahymena pyriformis has a very divergent primary structure (Hirono, M., Endoh, H., Okada, N., Numata, O., & Watanabe, Y. (1987) J. Mol. Biol. 194, 181-192) and that though it shares essential properties with skeletal muscle actin, it does not interact at all with phalloidin or DNase I (Hirono, M., Kumagai, Y., Numata, O., & Watanabe, Y. (1989) Proc. Natl. Acad. Sci. U.S. 86, 75-79). In this study, we investigated the copolymerization of this actin with skeletal muscle actin by direct observation of the heteropolymers formed from the two actins by means of electron microscopy. We also examined the binding of actin-binding proteins from skeletal muscle or smooth muscle to Tetrahymena actin by means of a cosedimentation assay. The results show that (i) Tetrahymena actin copolymerizes with skeletal muscle actin and that (ii) muscle myosin subfragment 1 binds to it in the absence of ATP, like skeletal muscle actin. However, it was also shown that (iii) muscle alpha-actinin hardly binds to Tetrahymena actin and that (iv) muscle tropomyosin does not bind to it at all. The results show that Tetrahymena actin has both properties similar and dissimilar to those of skeletal muscle actin.     

The effects of orthovanadate,vanadyl and peroxides of vanadate on glucose metabolism in skeletal muscle preparations in vitro

The insulin-like effects of various vanadium compounds (orthovanadate, vanadyl and peroxides of vanadate) on rates of glucose oxidation, lactate formation and glycogen synthesis were measured in isolated incubated epitrochlearis (mainly type 11 fibres) and soleus (mainly type I fibres) muscle preparations. There was a small stimulation of the rate of glucose utilisation in soleus muscle preparations in vitro by orthovanadate (1 mM). Orthovanadate or vanadyl, at 1 mM, had little effect on the rates of lactate formation or glycogen synthesis in isolated incubated epitrochlearis muscle preparations. In contrast, peroxides of vanadate (peroxovanadates, at 1 mM) significantly stimulated glucose utilisation in both soleus and epitrochlearis muscle preparations in vitro. The stimulation of the rate of glycogen synthesis was associated with an increase in the percentage of glycogen synthase in the I (or a) form. Peroxovanadates were administered in the drinking water to rats made insulin deficient by streptozotocin treatment. There was no decrease in the elevated level of blood glucose over an 8 day administration period. (Mol Cell Biochem 109: 157–162, 1992)     

Recent advances in chromatographic and electrophoretic methods for the study of drug-protein interactions

Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines various chromatographic and electrophoretic methods that have been developed to study such interactions. An overview of each technique is presented along with a discussion of its strengths, weaknesses and potential applications. Formats that are discussed include the use of both soluble and immobilized drugs or proteins, and approaches based on zonal elution, frontal analysis or vacancy peak measurements. Furthermore, examples are provided that illustrate the use of these methods in determining the overall extent of drug-protein binding, in examining the displacement of a drug by other agents and in measuring the equilibrium or rate constants for drug-protein interactions. Examples are also given demonstrating how the same methods, particularly when used in high-performance liquid chromatography or capillary electrophoresis systems, can be employed as rapid screening tools for investigating the binding of different forms of a chiral drug to a protein or the binding of different proteins and peptides to a given pharmaceutical agent.     

Recent advances on conversion and co-production of acetone-butanol-ethanol into high value-added bioproducts

Butanol is an important bulk chemical and has been regarded as an advanced biofuel. Large-scale production of butanol has been applied for more than 100 years, but its production through acetone–butanol–ethanol (ABE) fermentation process by solventogenic Clostridium species is still not economically viable due to the low butanol titer and yield caused by the toxicity of butanol and a by-product, such as acetone. Renewed interest in biobutanol as a biofuel has spurred technological advances to strain modification and fermentation process design. Especially, with the development of interdisciplinary processes, the sole product or even the mixture of ABE produced through ABE fermentation process can be further used as platform chemicals for high value added product production through enzymatic or chemical catalysis. This review aims to comprehensively summarize the most recent advances on the conversion of acetone, butanol and ABE mixture into various products, such as isopropanol, butyl-butyrate and higher-molecular mass alkanes. Additionally, co-production of other value added products with ABE was also discussed.