It's hard to be green: Reverse green value chain.

Firms have recently discovered that it is not enough to optimize internal processes and relationships with partners along the value chain to create a sustainable competitive market position. A clear customer orientation, which acknowledges that consumer buying behavior is complex and includes many elements implied in the value chain, is required. As companies offering green products are no exception to this rule, this study analyzes consumer behavior in Europe from a reserve green supply chain management perspective, using descriptive analyses and a structural equation model, with data collected by Flash Barometer comprising 26,573 responses from 28 European countries. The results suggest that European consumers are conscious of the green concept, but are not willing to buy or pay more for these products since the value is unclear. Companies offering green products must therefore rethink their strategies, especially in terms of value proposition, communication strategies, and eco-labeling.

Early disruption of the actin cytoskeleton in cultured cerebellar granule neurons exposed to 3-morpholinosydnonimine-oxidative stress is linked to alterations of the cytosolic calcium concentration.

Cytoskeleton damage is a frequent feature in neuronal cell death and one of the early events in oxidant-induced cell injury. This work addresses whether actin cytoskeleton reorganization is an early event of SIN-1-induced extracellular nitrosative/oxidative stress in cultured cerebellar granule neurons (CGN). The actin polymerization state, i.e. the relative levels of G-/F-actin, was quantitatively assessed by the ratio of the fluorescence intensities of microscopy images obtained from CGN double-labelled with Alexa594-DNase-I (for actin monomers) and Bodipy-FL-phallacidin (for actin filaments). Exposure of CGN to a flux of peroxynitrite as low as 0.5-1µM/min during 30min (achieved with 0.1mM SIN-1) was found to promote alterations of the actin cytoskeleton dynamics as it increases the G-actin/F-actin ratio. Because L-type voltage-operated Ca(2+) channels (L-VOCC) are primary targets in CGN exposed to SIN-1, the possible role of Ca(2+) dynamics on the perturbation of the actin cytoskeleton was also assessed from the cytosolic Ca(2+) concentration response to the L-VOCC's agonist FPL-64176 and to the L-VOCC's blocker nifedipine. The results showed that SIN-1 induced changes in the actin polymerization state correlated with its ability to decrease Ca(2+) influx through L-VOCC. Combined analysis of cytosolic Ca(2+) concentration and G-actin/F-actin ratio alterations by SIN-1, cytochalasin D, latrunculin B and jasplakinolide support that disruption of the actin cytoskeleton is linked to cytosolic calcium concentration changes.

Peroxynitrite-mediated oxidative modifications of myosin and implications on structure and function.

Abstract The peroxynitrite-induced functional impairment of myosin was studied in different reaction conditions, known to alter the oxidative chemistry of peroxynitrite, to better understand the molecular mechanisms of this interaction. It is shown that peroxynitrite is able to enhance the basal MgATPase activity up to 2-fold while inhibiting the actin-stimulated ATPase activity of myosin and that the extent of these functional alterations is dependent on the reaction medium. The observed changes in the stimulation of the MgATPase activity correlate with the extent of carbonyl formation in myosin. The enzyme inhibition is more potent in conditions where the efficiency of tyrosine nitration and peroxynitrite reactivity towards sulphydryls are lower. Together with the observation that reversion of sulphydryl oxidation did not lead to the recovery of myosin functional and structural impairments, these results point out to the importance of protein carbonylation as a post-translational modification in the peroxynitrite-induced myosin functional impairment.

L-type calcium channels and cytochrome b5 reductase are components of protein complexes tightly associated with lipid rafts microdomains of the neuronal plasma membrane.

The presence of cytosolic calcium microcompartments in neurons is well established. L-type voltage calcium channels play a leading role in the rise of cytosolic calcium in the neuronal soma and are sensitive to redox modulation. In a recent work [Samhan-Arias, A.K., García-Bereguiaín, M.A., Martín-Romero, F.J. and Gutiérrez-Merino, C. (2009) Mol. and Cell. Neurosci. 40, 14-26], we have shown that cytochrome b(5) reductase, whose deregulation leads to an overshot of superoxide anion production at the neuronal plasma membrane that triggers apoptosis in primary cultures of cerebellar granule neurons in culture, forms a large mesh of redox centres associated with lipid rafts in these neurons. In this work, we have implemented the use of fluorescent antibodies as reagents for quantitative Förster resonance energy transfer measurements and analysis using fluorescence microscopy images of cerebellar granule neurons in culture. The results of this study show that L-type voltage-operated calcium channels are also enriched in lipid rafts associated protein microdomains at a distance between 10 and 100 nm from cytochrome b(5) reductase. The methodological improvements done in this work can be also valuable for the study of proteins compartmentalization within other subcellular microdomains in any cell type in culture.

Sarcoplasmic reticulum calcium ATPase is inhibited by organic vanadium coordination compounds: pyridine-2,6-dicarboxylatodioxovanadium(V), BMOV, and an amavadine analogue.

The general affinity of the sarcoplasmic reticulum (SR) Ca (2+)-ATPase was examined for three different classes of vanadium coordination complexes including a vanadium(V) compound, pyridine-2,6-dicarboxylatodioxovanadium(V) (PDC-V(V)), and two vanadium(IV) compounds, bis(maltolato)oxovanadium(IV) (BMOV), and an analogue of amavadine, bis( N-hydroxylamidoiminodiacetato)vanadium(IV) (HAIDA-V(IV)). The ability of vanadate to act either as a phosphate analogue or as a transition-state analogue with enzymes' catalysis phosphoryl group transfer suggests that vanadium coordination compounds may reveal mechanistic preferences in these classes of enzymes. Two of these compounds investigated, PDC-V(V) and BMOV, were hydrolytically and oxidatively reactive at neutral pH, and one, HAIDA-V(IV), does not hydrolyze, oxidize, or otherwise decompose to a measurable extent during the enzyme assay. The SR Ca (2+)-ATPase was inhibited by all three of these complexes. The relative order of inhibition was PDC-V(V) > BMOV > vanadate > HAIDA-V(IV), and the IC 50 values were 25, 40, 80, and 325 microM, respectively. Because the observed inhibition is more potent for PDC-V(V) and BMOV than that of oxovanadates, the inhibition cannot be explained by oxovanadate formation during enzyme assays. Furthermore, the hydrolytically and redox stable amavadine analogue HAIDA-V(IV) inhibited the Ca (2+)-ATPase less than oxovanadates. To gauge the importance of the lipid environment, studies of oxidized BMOV in microemulsions were performed and showed that this system remained in the aqueous pool even though PDC-V(V) is able to penetrate lipid interfaces. These findings suggest that the hydrolytic properties of these complexes may be important in the inhibition of the calcium pump. Our results show that two simple coordination complexes with known insulin enhancing effects can invoke a response in calcium homeostasis and the regulation of muscle contraction through the SR Ca (2+)-ATPase.

Binding modes of decavanadate to myosin and inhibition of the actomyosin ATPase activity.

Decavanadate, a vanadate oligomer, is known to interact with myosin and to inhibit the ATPase activity, but the putative binding sites and the mechanism of inhibition are still to be clarified. We have previously proposed that the decavanadate (V(10)O(28)(6-)) inhibition of the actin-stimulated myosin ATPase activity is non-competitive towards both actin and ATP. A likely explanation for these results is that V(10) binds to the so-called back-door at the end of the Pi-tube opposite to the nucleotide-binding site. In order to further investigate this possibility, we have carried out molecular docking simulations of the V(10) oligomer on three different structures of the myosin motor domain of Dictyostelium discoideum, representing distinct states of the ATPase cycle. The results indicate a clear preference of V(10) to bind at the back-door, but only on the "open" structures where there is access to the phosphate binding-loop. It is suggested that V(10) acts as a "back-door stop" blocking the closure of the 50-kDa cleft necessary to carry out ATP-gamma-phosphate hydrolysis. This provides a simple explanation to the non-competitive behavior of V(10) and spurs the use of the oligomer as a tool to elucidate myosin back-door conformational changes in the process of muscle contraction.

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.

Inhibition of skeletal muscle S1-myosin ATPase by peroxynitrite.

Exposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine (SIN-1) produced a time-dependent inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity, reaching 50% inhibition with 46.7 +/- 8.3 microM SIN-1 for 8.7 microM S1, that is, at a SIN-1/S1 molar ratio of approximately 5.5. The inhibition was due to the peroxynitrite produced by SIN-1 decomposition because (1) decomposed SIN-1 was found to have no effect on S1 ATPase activity, (2) addition of SIN-1 in the presence of superoxide dismutase and catalase fully prevented inhibition by SIN-1, and (3) micromolar pulses of chemically synthesized peroxynitrite produced inhibition of F-actin-stimulated S1 Mg(2+)-ATPase activity. In parallel, SIN-1 produced the inhibition of the nonphysiological Ca(2+)-dependent and K(+)/EDTA-dependent S1 ATPase activity of S1 and, therefore, suggested that the inhibition of F-actin-stimulated S1 Mg(2+)-ATPase activity is produced by the oxidation of highly reactive cysteines of S1 (Cys(707) and Cys(697)), located close to the catalytic center. This point was further confirmed by the titration of S1 cysteines with 5,5'-dithiobis(2-nitrobenzoic acid) and by the parallel decrease of Cys(707) labeling by 5-(iodoacetamido)fluorescein, and it was reinforced by the fact that other common protein modifications produced by peroxynitrite, for example, protein carbonyl and nitrotyrosine formation, were barely detected at the concentrations of SIN-1 that produced more than 50% inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity. Differential scanning calorimetry of S1 (untreated and treated with different SIN-1 concentrations) pointed out that SIN-1, at concentrations that generate micromolar peroxynitrite fluxes, impaired the ability of ADP.V(1) to induce the intermediate catalytic transition state and also produced the partial unfolding of S1 that leads to an enhanced susceptibility of S1 to trypsin digestion, which can be fully protected by 2 mM GSH.

Peroxynitrite induces F-actin depolymerization and blockade of myosin ATPase stimulation.

Treatment of F-actin with the peroxynitrite-releasing agent 3-morpholinosydnonimine (SIN-1) produced a dose-dependent F-actin depolymerization. This is due to released peroxynitrite because it is not produced by 'decomposed SIN-1', and it is prevented by superoxide dismutase concentrations efficiently preventing peroxynitrite formation. F-actin depolymerization has been found to be very sensitive to peroxynitrite, as exposure to fluxes as low as 50-100nM peroxynitrite leads to nearly 50% depolymerization in about 1h. G-actin polymerization is also impaired by peroxynitrite although with nearly 2-fold lower sensitivity. Exposure of F-actin to submicromolar fluxes of peroxynitrite produced cysteine oxidation and also a blockade of the ability of actin to stimulate myosin ATPase activity. Our results suggest that an imbalance of the F-actin/G-actin equilibrium can account for the observed structural and functional impairment of myofibrils under the peroxynitrite-mediated oxidative stress reported for some pathophysiological conditions.

Interactions of vanadium(V)-citrate complexes with the sarcoplasmic reticulum calcium pump.

Among the biotargets interacting with vanadium is the calcium pump from the sarcoplasmic reticulum (SR). To this end, initial research efforts were launched with two vanadium(V)-citrate complexes, namely (NH(4))(6)[V(2)O(4)(C(6)H(4)O(7))(2)].6H(2)O and (NH(4))(6)[V(2)O(2)(O(2))(2)(C(6)H(4)O(7))(2)].4H(2)O, potentially capable of interacting with the SR calcium pump by combining kinetic studies with (51)V NMR spectroscopy. Upon dissolution in the reaction medium (concentration range: 4-0.5mM), both vanadium(V):citrate (VC) and peroxovanadium(V):citrate (PVC) complexes are partially converted into vanadate oligomers. A 1mM solution of the PVC complex, containing 184microM of the PVC complex, 94microM oxoperoxovanadium(V) (PV) species, 222microM monomeric (V1), 43microM dimeric (V2) and 53microM tetrameric (V4) species, inhibits Ca(2+) accumulation by 75 %, whereas a solution of the VC complex of the same vanadium concentration, containing 98microM of the VC complex, 263microM monomeric (V1), 64microM dimeric (V2) and 92microM tetrameric (V4) species inhibits the calcium pump activity by 33 %. In contrast, a 1 mM metavanadate solution, containing 460microM monomeric (V1), 90.2microM dimeric (V2) and 80microM tetrameric (V4) species, has no effect on Ca(2+) accumulation. The NMR signals from the VC complex (-548.0ppm), PVC complex (-551.5ppm) and PV (-611.1ppm) are broadened upon SR vesicle addition (2.5mg/ml total protein). The relative order for the half width line broadening of the NMR signals, which reflect the interaction with the protein, was found to be V4>PVC>VC>PV>V2=V1=1, with no effect observed for the V1 and V2 signals. Putting it all together the effects of two vanadium(V)-citrate complexes on the modulation of calcium accumulation and ATP hydrolysis by the SR calcium pump reflected the observed variable reactivity into the nature of key species forming upon dissolution of the title complexes in the reaction media.

Decavanadate as a biochemical tool in the elucidation of muscle contraction regulation.

Recently reported decameric vanadate (V(10)) high affinity binding site in myosin S1, suggests that it can be used as a tool in the muscle contraction regulation. In the present article, it is shown that V(10) species induces myosin S1 cleavage, upon irradiation, at the 23 and 74 kDa sites, the latter being prevented by actin and the former blocked by the presence of ATP. Identical cleavage patterns were found for meta- and decavanadate solutions, indicating that V(10) and tetrameric vanadate (V(4)) have the same binding sites in myosin S1. Concentrations as low as 50 muM decavanadate (5 muM V(10) species) induces 30% of protein cleavage, whereas 500 muM metavanadate is needed to attain the same extent of cleavage. After irradiation, V(10) species is rapidly decomposed, upon protein addition, forming vanadyl (V(4+)) species during the process. It was also observed by NMR line broadening experiments that, V(10) competes with V(4) for the myosin S1 binding sites, having a higher affinity. In addition, V(4) interaction with myosin S1 is highly affected by the products release during ATP hydrolysis in the presence or absence of actin, whereas V(10) appears to be affected at a much lower extent. From these results it is proposed that the binding of vanadate oligomers to myosin S1 at the phosphate loop (23 kDa site) is probably the cause of the actin stimulated myosin ATPase inhibition by the prevention of ATP/ADP exchange, and that this interaction is favoured for higher vanadate anions, such as V(10).

Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity.

Decameric vanadate (V(10)) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K(i) = 0.27 +/- 0.05 microM) in myosin subfragment-1 (S1). The binding of V(10) to S1 can be monitored from titration with V(10) of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V(10) binding site per monomer with a dissociation constant of 0.16-0.7 microM, indicating that S1 labeling with these dyes produced only a small distortion of the V(10) binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V(10) indicated that the V(10) binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V(10) to S1 is not competitive either with actin or with ADP.V(1) or ADP.AlF(4); (ii) the affinity of V(10) for the complex S1/ADP.V(1) and S1/ADP.AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand P(1)P(5)-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V(10) is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V(10) to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.