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A simultaneous partition experiment of divalent metal ions was performed between sphalerite and 1 mol/L (Ni, Mg, Co, Fe, Mn)Cl2 aqueous solution under supercritical hydrothermal conditions of 500–800 °C and 100 MPa. The bulk partition coefficient that was defined by KPB(ZnS) = (xMeS/xZnS)/(mMeaq/mZnaq) followed the order of Zn ≑ Co ≑ Ni &gt, Fe &gt, Mn &gt, Mg at all temperatures. In the partition coefficient versus ionic radius (PC–IR) diagrams with the logarithmic value of the bulk partition coefficient (log KPB(ZnS)) on the vertical axis, and the ionic radius of the six-fold coordinated site on the horizontal axis, Ni shows a positive partition anomaly, and the other elements were almost on the PC–IR curve. Based on the PC–IR curve, the optimum ionic radius for sphalerite existed where the ionic radius was slightly larger than Zn (~0.76 Å). A Ni positive partition anomaly may result from its large electronegativity.

To clarify the element partitioning behavior between minerals and aqueous chloride solutions, we conducted experiments to investigate simultaneous partitioning of Ni2+, Mg2+, Co2+, Zn2+, Fe2+, and Mn2+ ions between alabandite (MnS) and 1 mol/L (Ni, Mg, Co, Zn, Fe)Cl2 aqueous solutions at 500&ndash, 800 &deg, C and 100 MPa. The bulk partition coefficients calculated using the following equation were in the order of Fe2+ &gt, Co2+ &gt, Ni2+ &asymp, Zn2+ &gt, Mn2+ &gt, &gt, Mg2+, KPN = (xMeS/mMeaq)/(xMnS/mMnaq). A partition coefficient-ionic radius (PC-IR) curve was plotted with the logarithmic value of the partition coefficient on the y-axis and the ionic radius at the six-fold coordinated site on the x-axis. The peak of this curve was located near the ionic radius of Fe2+ and not near the ionic radius of Mn2+. Zn2+ showed a slight negative partitioning anomaly, which increased in the order of sulfide minerals &lt, arsenic sulfide minerals &lt, arsenide minerals as the covalent bond became stronger. Ni2+ showed a positive partitioning anomaly, which indicated that it preferred an octahedral structure. The width of the PC-IR curve decreased in the order of sulfide minerals &gt, arsenic sulfide minerals &gt, arsenide minerals as the covalent bond became stronger, that is, the ion selectivity became stronger.

Surfactin, which is an anionic bio-surfactant, can be effective for enhanced oil recovery because it decreases interfacial tension between oil and water. However, it forms precipitation by binding with divalent cations. This study examined the countermeasure to prevent surfactin from forming precipitation for applying it to enhanced oil recovery. Alcohols, chelating agents, a cationic surfactant and an ion capturing substance were selected as the candidates for inhibiting surfactin from forming precipitation. Citric acid and trisodium citrate were selected as promising candidates through the measurements of turbidity of the mixture of the candidate, surfactin and calcium ions. Those chemicals also had a function as a co-surfactant for surfactin. However, the permeability of the Berea sandstone core into which the solution containing surfactin and trisodium citrate was injected was decreased significantly, whereas citric acid could be injected into the core without significant permeability reduction. Citric acid was therefore selected as the best inhibitor and subjected to the core flooding experiments. High enhancement of oil recovery of 9.4% (vs. original oil in place (OOIP)) was obtained and pressure drop was not increased during the injection of surfactin and citric acid. Those results suggest that citric acid has a dual role as the binding inhibitor and co-surfactant for surfactin.

Welan is a branched polymer in the gellan family of polysaccharides. It has good compatibility with divalent calcium ions with a potential use in cementing applications. It is also been utilized as an anti-washout material for underwater concrete placement. Herein, influence of divalent cations Ca2+, Ni2+, Mg2+, Zn2+ and Sr2+ on the gelling and thermal properties of welan have been investigated. The viscoelastic properties reveal strong gelation behavior that are stable up to 80 °C. Among all the five cations Ni2+, Ca2+, Zn2+ and Sr2+ are suited well, compared to Mg2+ ions, to promote favorable interactions among the welan chains leading to stronger junction zones and elastic moduli. The melting peak and enthalpy are dependent on the nature and size of the divalent cation. The outcome is deemed to aid to expand the welan utility in food and non-food applications.

The bacterial flagellar motor is a reversible rotary molecular nanomachine, which couples ion flux across the cytoplasmic membrane to torque generation. It comprises a rotor and multiple stator complexes, and each stator complex functions as an ion channel and determines the ion specificity of the motor. Although coupling ions for the motor rotation were presumed to be only monovalent cations, such as H+ and Na+, the stator complex MotA1/MotB1 of Paenibacillus sp. TCA20 (MotA1TCA/MotB1TCA) was reported to use divalent cations as coupling ions, such as Ca2+ and Mg2+. In this study, we initially aimed to measure the motor torque generated by MotA1TCA/MotB1TCA under the control of divalent cation motive force, however, we identified that the coupling ion of MotA1TCAMotB1TCA is very likely to be a monovalent ion. We engineered a series of functional chimeric stator proteins between MotB1TCA and Escherichia coli MotB. E. coli &Delta, motAB cells expressing MotA1TCA and the chimeric MotB presented significant motility in the absence of divalent cations. Moreover, we confirmed that MotA1TCA/MotB1TCA in Bacillus subtilis &Delta, motAB&Delta, motPS cells generates torque without divalent cations. Based on two independent experimental results, we conclude that the MotA1TCA/MotB1TCA complex directly converts the energy released from monovalent cation flux to motor rotation.

Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F1FO-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F1FO-ATPase either sustained by the natural cofactor Mg2+ or by Ca2+ by a similar uncompetitive inhibition mechanism, namely the tertiary complex (ESI) only forms when the ATP substrate is already bound to the enzyme, and with similar strength, as shown by the similar K'i values (0.82±0.07mM in presence of Mg2+ and 0.64±0.05mM in the presence of Ca2+). Multiple inhibitor analysis indicates that features of the F1 catalytic sites and/or the FO proton binding sites are apparently unaffected by PGO. However, PGO and F1 or FO inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg2+-activated F1FO-ATPase, whereas they do not mutually exclude to bind the Ca2+-activated F1FO-ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F1FO-ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation.

International audience; Two new octahedral molybdenum cyanide cluster compounds, namely [{Zn(H2O)(en)2}{Zn(en)2}-Mo6Br6Se2(CN)6].2H2O (1) and [{Cu(H2O)(en)2}2Mo6Br6Se2(CN)6].2H2O (2) (en = ethylenediamine), have been synthetized as single crystals. 1 was obtained by slow diffusion of a solution containing [Mo6Br6Se2(CN)6]4- cluster units into a solution of zinc acetate and ethylenediamine. 2 was obtained by slow diffusion of an ammonia solution of [Mo6Br6Se2(CN)6]4- cluster units into a solution of cupper chloride and ethylenediamine. Both compounds were structurally characterized by single-crystal X-ray diffraction analysis. 1 is a bimetallic one dimensional coordination compound and it crystallizes in the orthorhombic system (P212121; Z = 4; a = 10.675(1) Ŭ b = 15.073(2) Ŭ c = 25.812(4) Ŭ V = 4153.3(1) ų at T = 150 K). 2 is a bimetallic H bonded three dimensional compound and it crystallizes in the monoclinic system (P21/c; Z = 2; a = 9.4281(4) Ŭ b = 11.0220(5) Å, c = 21.1494(9) Ŭ β = 100.053(2)°, V = 2164.03(16) ų at T = 296 K).

Pyrazinamidase of Mycobacterium tuberculosis catalyzes the conversion of pyrazinamide to the active molecule pyrazinoic acid. Reduction of pyrazinamidase activity results in a level of pyrazinamide resistance. Previous studies have suggested that pyrazinamidase has a metal-binding site and that a divalent metal cofactor is required for activity. To determine the effect of divalent metals on the pyrazinamidase, the recombinant wild-type pyrazinamidase corresponding to the H37Rv pyrazinamide-susceptible reference strain was expressed in Escherichia coli with and without a carboxy terminal. His-tagged pyrazinamidase was inactivated by metal depletion and reactivated by titration with divalent metals. Although Co(2+), Mn(2+), and Zn(2+) restored pyrazinamidase activity, only Co(2+) enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu(2+), Fe(2+), Fe(3+), and Mg(2+) did not restore the activity under the conditions tested. Various recombinant mutated pyrazinamidases with appropriate folding but different enzymatic activities showed a differential pattern of recovered activity. X-ray fluorescence and atomic absorbance spectroscopy showed that recombinant wild-type pyrazinamidase expressed in E. coli most likely contained Zn. In conclusion, this study suggests that M. tuberculosis pyrazinamidase is a metalloenzyme that is able to coordinate several ions, but in vivo, it is more likely to coordinate Zn(2+). However, in vitro, the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn.

While Dicer alone has been shown to form stable complexes with double-stranded RNAs and short interfering RNAs, its interactions with single-stranded RNAs (ssRNAs) have not been characterized. Here, we show that recombinant human Dicer alone can bind 21-nt ssRNAs in vitro, independent of their sequence and structure. We also demonstrate that Dicer binds ssRNAs having a 5′-phosphate with greater affinity versus those with a 5′-hydroxyl. In addition, 3′-biotinylated ssRNAs are bound by Dicer with lower affinity than 3′-hydroxyl ssRNAs. The stability of ssRNA–Dicer complexes was found to depend on divalent cations. Together, our results suggest a role for the PAZ domain of Dicer in binding ssRNAs and may indicate roles for Dicer in cellular function beyond those currently known.

In vitro RNA-dependent RNA polymerase assays revealed that the JEV replication complex (RC) synthesized viral RNA utilizing a semiconservative and asymmetric mechanism. Peak viral replicase activity and levels of viral RNA observed 15-18 h postinfection (h p.i.) preceded maximum viral titers in the culture medium seen 21 h p.i. Among divalent cations, $Mg^{2+}$ was essential and exhibited cooperative binding for its two replicase-binding sites. $Mn^{2+}$, despite sixfold higher affinity for the replicase, elicited only 70% of the maximum $Mg^{2+}$-dependent activity, and deficit of either cation led to synthesis of incomplete RNA products. We also determined as a first instance for a flavivirus RC, kinetic parameters using cytoplasmic "virus-induced heavy membranes" after depleting endogenous nucleotides. Exhaustive trypsin treatment, which degraded the bulk of NS3 and NS5, had no effect on replicase activity, suggesting that the active flaviviral RC resides behind a membrane barrier and recruits minuscule proportions of the replicase proteins.

Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation,in vitroandin vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules’ basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others.

alpha7 nicotinic receptors are highly permeable to Ca(2+) as well as monovalent cations. We extended the characterization of the Ca(2+) permeation of non-desensitizing chick alpha7 receptors (S240T/L247T alpha7 nAChRs) expressed in Xenopus oocytes by (1) measuring the concentration dependence of conductance under conditions in which Ca(2+) or Ba(2+) were the only permeant cations in the extracellular solution, and (2) measuring the concentration dependence of Ca(2+) block of K(+) currents through the receptors. The first set of experiments yielded an apparent affinity of 0.96 mM Ca(2+) activity (2.4 mM concentration) for Ca(2+) permeation and an apparent affinity of 0.65 mM Ba(2+) activity (1.7 mM concentration) for Ba(2+) permeation. The apparent affinity of Ca(2+) inhibition of K(+) currents was 0.49 mM activity (1.5 mM concentration). The similarity of these apparent affinities in the millimolar range suggests that the pore of alpha7 receptors has one or more low-affinity Ca(2+) binding sites and no high-affinity sites.

The thermodynamic parameters of an antiparallel G-quartet formation of d(G4T4G4) with 1 mM divalent cation (Mg(2+), Ca(2+), Mn(2+), Co(2+), and Zn(2+)) were obtained. The thermodynamic parameters showed that the divalent cation destabilizes the antiparallel G-quartet of d(G4T4G4) in the following order: Zn(2+)>Co(2+)>Mn(2+)>Mg(2+)>Ca(2+). In addition, a higher concentration of a divalent cation induced a transition from an antiparallel to a parallel G-quartet structure. These results indicate that these divalent cations are a good tool for regulating the G-quartet structures.

Five sesquiterpene alcohol esters of the carotane series, from plants of the genus Ferula, were investigated with regard to their capacity to modify the ion permeability of both planar lipid bilayers and mitochondria. These compounds are subdivided into two structural groups that differ in their effects on membrane permeability. Complex esters of sesquiterpene alcohols with aliphatic acids, which constituted the first group (lapidin and lapiferin), do not possess ionophoric properties. The second group comprised complex esters of sesquiterpene alcohols with aromatic acids (ferutinin, tenuferidin and ferutidin), all of which increase cation permeability of lipid bilayers and mitochondria in a dose-dependent manner. A pronounced selectivity of the terpenoid-modified membranes for divalent cations versus monovalent cations was found. Evidence of a carrier mechanism for terpenoid-induced ion transport is demonstrated. A tentative complex composed of a divalent cation with two molecules of membrane-active terpenoid is proposed.

Membrane fusion between uncharged lipid vesicles can be triggered by the peptide sequence 'B18' from the fertilization protein 'bindin', but it only proceeds efficiently in the presence of Zn(2+) ions. We studied (i) the interaction of Zn(2+) with the fusogenic peptide B18, (ii) the binding of B18 to 1-palmitoyl-2-oleoylglycero-3-phosphocholine (POPC), and (iii) the ternary system POPC/B18/Zn(2+). The complex formation of Zn(2+) with the central histidine-rich motif of B18 appears to shift the secondary structure away from a beta-sheet towards an alpha-helical conformation. Here we observe for the first time an essentially alpha-helical structure of the peptide when immersed in POPC bilayers which appears to represent its functional fusogenic state. Infrared linear dichroism suggests a peripheral, oblique insertion mode of B18, mediated by the hydrophobic patches along one side of the amphipathic peptide. Furthermore, the hydration level of the peptide is reduced, suggesting that the hydrophobic region of the bilayer is involved in the lipid/peptide interactions. The hydration capacity of the POPC/B18/Zn(2+) system is distinctly smaller than that of POPC/Zn(2+) without peptide. The accompanying decrease in the number of tightly bound water molecules per lipid can be interpreted as a reduction in the repulsive 'hydration' forces, which usually prevent the spontaneous fusion of lipid vesicles. Binding of the B18 peptide in the presence of Zn(2+) effectively renders the membrane surface more hydrophobic, thus allowing fusion to proceed.

The interaction of aqueous phospholipid dispersions of negatively charged 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, sodium salt (DMPG) with the divalent cations Mg(2+), Ca(2+) and Sr(2+) at equimolar ratios in 100 mM NaCl at pH 7 was investigated by Fourier transform infrared spectroscopy. The binding of the three cations induces a crystalline-like gel phase with highly ordered and rigid all-trans acyl chains. These features are observed after storage below room temperature for 24 h. When the gel phase is heated after prolonged incubation at low temperature phase transitions into the liquid crystalline phase are observed at 58 degrees C for the DMPG:Sr(2+), 65 degrees C for the DMPG:Mg(2+), and 80 degrees C for the DMPG:Ca(2+) complex. By subsequent cooling from temperatures above T(m) these complexes retain the features of a liquid crystalline phase with disordered acyl chains until a metastable gel phase is formed at temperatures between 38 and 32 degrees C. This phase is characterized by predominantly all-trans acyl chains, arranged in a loosely packed hexagonal or distorted hexagonal subcell lattice. Reheating the DMPG:Sr(2+) samples after a storage time of 2 h at 4 degrees C results in the transition of the metastable gel to the liquid crystalline phase at 35 degrees C. This phase transition into the liquid crystalline state at 35 degrees C is also observed for the Mg(2+) complex. However, for DMPG:Mg(2+) at higher temperatures, a partial recrystallization of the acyl chains occurs and the high temperature phase transition at 65 degrees C is also detected. In contrast, DMPG:Ca(2+) exhibits only the phase transition at 80 degrees C from the crystalline gel into the fluid state upon reheating. Below 20 degrees C, the rate of conversion from the metastable gel to a thermodynamically stable, crystalline-like gel phase decreases in the order Ca(2+)z. Gt;Mg(2+)Sr(2+). This conversion into the crystalline gel phase is accompanied by a complete dehydration of the phosphate groups in DMPG:Mg(2+) and by a reorientation of the polar lipid head groups in DMPG:Ca(2+) and in DMPG:Sr(2+). The primary binding sites of the cations are the PO(2)(-) groups of the phosphodiester moiety. Our infrared spectroscopic results suggest a deep penetration of the divalent cations into the polar head group region of DMPG bilayers, whereby the ester carbonyl groups, located in the interfacial region of the bilayers, are indirectly affected by strong hydrogen bonding of immobilized water molecules. In the liquid crystalline phase, the interaction of all three cations with DMPG is weak, but still observable in the infrared spectra of the DMPG:Ca(2+) complex by a slight ordering effect induced in the acyl chains, when compared to pure DMPG liposomes.

International audience; Complexation of divalent cations (Mg2+, Co2+, Ni2+, Cu2+, Cd2+) by selenate ligand was studied by ACE (UV indirect detection) in 0.1 mol/L NaNO3 ionic strength solutions at various temperatures (15, 25, 35, 45 and 55°C). For each solution, a unique peak was observed as a result of a fast equilibrium between the free ion and the complex (labile systems). The migration time corresponding to this peak changed as a function of the solution composition, namely the free and complexed metal concentrations, according to the complexation reactions. The results confirmed the formation of a unique 1:1 complex for each cation. The thermodynamic parameters were fitted to the experimental data at 0.1 mol/L ionic strength: (25°C) = −(6.5 ± 0.3), −(7.5 ± 0.3), −(7.7 ± 0.3), −(7.7 ± 0.3), and –(8.1 ± 0.3) kJ/mol and = 2.5 ± 0.2, 4.7 ± 0.4, 4.5 ± 0.6, 8.4 ± 1.1, and 7.2 ± 0.6 kJ/mol for M2+ = Mg2+, Co2+, Ni2+, Cu2+, and Cd2+, respectively. Complexes with alkaline earth and transition metal cations could be distinguished by their relative stabilities. The effect of the ionic medium was treated using the specific ion interaction theory and the thermodynamic parameters at infinite dilution were compared to previously published data on metal–selenate, metal–sulfate, and metal–chromate complexes.

Concentrations of extracellular divalent cations (Ca2+ and Mg2+) fall substantially during intensive synaptic transmission as well as during some pathophysiological conditions such as epilepsy and brain ischemia. Here we report that a synthetic serine protease inhibitor, nafamostat mesylate (NM), and several of its analogues, block recombinant TRPM7 currents expressed in HEK293T cells in inverse relationship to the concentration of extracellular divalent cations. Lowering extracellular Ca2+ and Mg2+ also evokes a divalent-sensitive non-selective cation current that is mediated by TRPM7 expression in hippocampal neurons. In cultured hippocampal neurons, NM blocked these TRPM7-mediated currents with an apparent affinity of 27 μM, as well as the paradoxical Ca2+ influx associated with lowering extracellular Ca2+. Unexpectedly, pre-exposure to NM strongly potentiated TRPM7 currents. In the presence of physiological concentrations of extracellular divalent cations, NM activates TRPM7. The stimulating effects of NM on TRPM7 currents are also inversely related to extracellular Ca2+ and Mg2+. DAPI and HSB but not netropsin, blocked and stimulated TRPM7. In contrast, mono-cationic, the metabolites of NM, p-GBA and AN, as well as protease inhibitor leupeptin and gabexate failed to substantially modulate TRPM7. NM thus provides a molecular template for the design of putative modulators of TRPM7.

Serum albumin is an abundant protein in blood plasma, that is well-known for its ability to transport hydrophobic biomolecules and drugs. Recent hypotheses propose that serum albumin plays a role in the regulation of lipid metabolism in addition to its lipid transport properties. The present work explores the capacity of bovine serum albumin (BSA) to extract diacylglycerols (DAG) from phospholipid bilayers, and the inhibition of such interaction by divalent cations. Quantitative measurements using radioactive DAG and morphological evidence derived from giant unilamellar vesicles examined by confocal microscopy provide concurrent results. BSA extracts DAG from vesicles consisting of phosphatidylinositol/DAG. Long, saturated DAG species are incorporated more readily than the shorter-chain or unsaturated ones. Divalent cations hinder DAG uptake by BSA. For Ca(2+), the concentration causing half-maximal inhibition is approximately 10 muM; 90% inhibition is caused by 100 muM Ca(2+). Sr(2+) requires concentrations one order of magnitude higher, while Mg(2+) has virtually no effect. As an example on how DAG uptake by BSA, and its inhibition by Ca(2+), could play a regulating role in lipid metabolism, a PI-specific phospholipase C has been assayed in the presence of BSA and/or Ca(2+). BSA activates the enzyme by removing the end-product DAG, but the activation is reverted by Ca(2+) that inhibits DAG uptake.

Phase transitions Of L-carrageenan in a variety of monovalent (Li, Na, K) and divalent (Mg, Ca, Sr) cation solutions were studied during heating and cooling by using photon transmission technique. Photon transmission intensity (I-tr) was monitored against temperature to determine the transition temperatures and activation energies during the heating and cooling processes in the monovalent and divalent systems. Three distinct transition regions were observed during the heating and cooling cycles for the carrageenan-divalent salt system. At the first step of the heating process, dimer groups were transformed into dimers that presented (g-d) transitions, then these dimers were directly converted into a double helix by undergoing a (d-h) transition. In the higher temperature region, a double helix-to-coil (h-c) transition took place. During the cooling process, these transitions are arranged in the order of (c-h), (h-d), and (d-g). The carrageenan-monovalent salt system presented only coil-to-rod-like helix (c-r) and rod-like helix-to-coil (r-c) transitions during the cooling and heating processes, respectively. A hysteresis was observed between (r-c)-(c-r) and (g-d)-(d-g) transitions for the monovalent and divalent cations, respectively. Publisher's Verison Q3 WOS:000243487600004

Rehydration of red blood cells (RBC) in isotonic media after dehydration in hypertonic electrolyte or nonelectrolyte saline leads to their posthypertonic hemolysis (PH). Ca2+ ions at a concentration of more than 5 mM stimulated hemolysis of RBC treated by hypertonic sucrose but not NaCl if rehydration was carried out in the presence of cations. Zn2+ produced a more complex response of stimulation followed by inhibition as a concentration is increased. Mg2+, Ca2+, Zn2+, EDTA and sucrose exhibited only inhibition when added to isotonic NaCl media immediately after onset of rehydration or later on. At low ionic strength inhibition produced by divalent cations was markedly reduced and sucrose; was ineffective. An equimolar concentration of EDTA abolished the inhibition of PH by Zn2+ ions if they were introduced into the isotonic media after the cells, but activated hemolysis when rehydration was carried out in the presence of ions. The same divalent cations prevented shape transformation and hemolysis induced by melittin if they interacted with the plasma membrane prior to the addition of melittin. Subsequent chelation of cations by EDTA triggers the full sequence of events characteristic to the action of melittin alone and resulted in cell spherulation followed by hemolysis. Inhibition of melittin-induced hemolysis produced by all cations was reversible because EDTA abolished the action of divalent cations and even stimulated hemolysis in isotonic sucrose. Similarities in the mode of action of divalent cations and EDTA on posthypertonic hemolysis which is attributed to endogenous stimuli and melittin-induced hemolysis as far as the exogenous agent is concerned imply that in both cases common intrinsic mechanisms are involved in the process of cation-sensitive pore formation in erythrocyte membranes, while differences indicate that more complex pores are formed during posthypertonic injury.

The electrophysiological analysis of Ca 2+ -conducting ion channels in Xenopus oocytes is difficult due to secondary intracellular effects induced by Ca 2+ . In the presence of polyvinylpyrrolidone (PVP) membrane currents can be recorded in nominally divalent cation-free solutions. The Ca 2+ -permeable recombinant NMDA receptors of the NR1/NR2A subtype were used as assay system and the results show that PVP has no effect on NMDA receptor-induced currents. Ca 2+ and Ba 2+ depress NMDA-induced currents at submillimolar concentrations probably by interfering with the Na + /K + flux. This block is fully reversible as also observed for Mg 2+ but shows in contrast no pronounced voltage dependence. PVP-containing solutions may be useful for the analysis of divalent cation-dependent ion channels.

Using a reconstituted system for adenovirus DNA replication we tested the requirements for ATP and divalent cations. At the standard Mg2+ concentration ATP stimulated initiation 5 to 10-fold. However, this effect was caused by complexing Mg2+. At the optimal Mg2+ concentration ATP was not required for initiation or elongation. Besides Mg2+ also Mn2+, Ca2+ and Ba2+ were shown to support initiation whereas for elongation only Mg2+ was accepted. Since Mn2+could efficiently be used for DNA chain elongation on synthetic templates we hypothesize that Mg2+ is essential for the transition of initiation to elongation.

Calcineurin was dissociated into subunits A and B by SDS and the dissociated subunits were separated by Sephadex G-100 column chromatography in SDS. The phosphatase activity was associated with the A subunit and was detected only in the presence of MnCl2 of the various divalent cations tested. The Mn2+-dependent phosphatase of A subunit was stimulated (4-5-fold) by calmodulin. The subunit B increased only modestly Mn2+ stimulated phosphatase activity of subunit A but markedly increased it when assay also contained calmodulin. These results support the view that subunit B plays an important role in Mn2+/calmodulin regulation of subunit A phosphatase activity. They also lend further support to our earlier postulate ([1984] FEBS Lett. 169, 251-255) that Mn2+ is a powerful regulator of calcineurin phosphatase.

It was reported that the circulating blood cells, including neutrophils, were more likely adherent to the curved micro-vessels than the straight ones. Our previous work indicated that the vessel curvature, cell-cell interaction, and wall shear stress variation would greatly affect this preferential adhesion. It was also found that the affinity state of integrins would be influenced by different divalent cations, which provides an opportunity to explore the particular importance of integrins activation in neutrophils adhesion. In this study, we aim at numerically investigating the effects of divalent cations, i.e. Mn2+, Mg2+ plus EGTA, and Ca2+, on VLA-4/VCAM-1 adhesion under flow condition. The blood dynamics was carried out by the lattice Boltzmann method (LBM), the neutrophil dynamics was governed by the Newton's law of translation and rotation, and a refined neutrophil adhesive dynamics model that taken into account the effect of wall shear stress gradient on receptor-ligand bonds was applied to solve the VLA-4/VCAM-1 adhesion. The simulation results indicated that the divalent cations significantly influenced neutrophils adhesion under hydrodynamic condition. It was found that the capability of stimulating affinity state of VLA-4 to VCAM-1 was larger in Mg2+ plus EGTA than in Ca2+, and largest in the presence of Mn2+. Our results would be helpful to understand the β1-integrin-mediated neutrophils adhesion in real physiological situations.

We have investigated the regulation by divalent cations Mg 2+ , Ca 2+ and Mn 2+ of the functional activity of the human integrin VLA-1 expressed on neuroblastoma NB100 cells. VLA-1-mediated adhesion of NB100 cells to ligand collagen type I was supported by either mM concentrations of extracellular Mg 2+ or μM levels of Mn 2+ . In contrast, Ca 2+ alone did not induce activation of VLA-1 but exerted a potent inhibitory effect on the Mg 2+ -supported cell adhesion. We have also demonstrated that VLA-1 can be directly activated by the stimulatory monoclonal antibody TS2/16 specific for the integrin β1 subunit, resulting in effective adhesion of NB100 cells to type I collagen. This study has been possible by using a novel blocking VLA-α1 specific monoclonal antibody, 5E8D9.

A new method, based on the ion-translocating properties of the ionophores nigericin and A23187, is described for loading large unilamellar vesicles (LUVs) with the drugs vincristine and ciprofloxacin. LUVs composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (55:45 mol/mol) or sphingomyelin (SPM)/Chol (55:45 mol/mol) exhibiting a transmembrane salt gradient (for example, internal solution 300 mM MnSO4 or K2SO4; external solution 300 mM sucrose) are incubated in the presence of drug and, for experiments involving divalent cations, the chelator EDTA. The addition of ionophore couples the outward movement of the entrapped cation to the inward movement of protons, thus acidifying the vesicle interior. External drugs that are weak bases can be taken up in response to this induced transmembrane pH gradient. It is shown that both nigericin and A23187 facilitate the rapid uptake of vincristine and ciprofloxacin, with entrapment levels approaching 100% and excellent retention in vitro. Following drug loading, the ionophores can be removed by gel exclusion chromatography, dialysis, or treatment with biobeads. In vitro leakage assays (addition of 50% mouse serum) and in vivo pharmacokinetic studies (in mice) reveal that the A23187/Mn 2÷ system exhibits superior drug retention over the nigericin/K + system, and compares favorably with vesicles loaded by the standard ApH or amine methods. The unique features of this methodology and possible benefits are discussed. © 1998 Elsevier Science B.V. All rights reserved.

The divalent metal requirement of intrinsic phosphatase activity was investigated using native and trypsinized calcineruin. This was assessed by examining (1) the stimulation of the enzyme by various metals, (2) the inhibition of the enzyme activity by metal chelators (EDTA and EGTA), and (3) the restoration by various metals of the activity of the EDTA-inhibited calcineurin phosphatase. The results supported the view that a tightly bound trace metal is necessary for expression of the phosphatase activity of calcineurin and implicate Mn 2+ as the tighly bound metal.

Incubation of rat liver phosphorylase kinase in the presence of MgATP results in a time-dependent increase in activity, i.e., activation. Determination of the magnitude of activation depends, in large part, on the relative concentrations of Mg2+ and ATP used in the phosphorylase kinase activity assay, such that as the Mg2+ to ATP ratio increases less activation is detectable. Prior to activation, maximal activity of nonactivated phosphorylase kinase requires a 2–3 fold molar excess of Mg2+ (i.e., free Mg2+) over ATP. MgATP-dependent activation of the enzyme results in an alteration in the free Mg2+ requirement such that the activity of the activated enzyme is sharply inhibited by the free cation. Inhibition by free Mg2+ of the activated enzyme is rapidly reversed by removal of free Mg2+ but is not affected by addition of Ca2+. Both nonactivated and activated forms of enzyme appear to be inhibited by free ATP4–. The results show that the use of high concentrations of free Mg2+ in the phosphorylase kinase activity assay can blunt or completely obscure changes in enzyme activity following activation of the enzyme.