Effects Of Different Divalent Cations Research Articles

Effects of Axonal Demyelination, Inflammatory Cytokines and Divalent Cation Chelators on Thalamic HCN Channels and Oscillatory Bursting.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the progressive loss of oligodendrocytes and myelin and is associated with thalamic dysfunction. Cuprizone (CPZ)-induced general demyelination in rodents is a valuable model for studying different aspects of MS pathology. CPZ feeding is associated with the altered distribution and expression of different ion channels along neuronal somata and axons. However, it is largely unknown whether the copper chelator CPZ directly influences ion channels. Therefore, we assessed the effects of different divalent cations (copper; zinc) and trace metal chelators (EDTA; Tricine; the water-soluble derivative of CPZ, BiMPi) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that are major mediators of thalamic function and pathology. In addition, alterations of HCN channels induced by CPZ treatment and MS-related proinflammatory cytokines (IL-1β; IL-6; INF-α; INF-β) were characterized in C57Bl/6J mice. Thus, the hyperpolarization-activated inward current (Ih) was recorded in thalamocortical (TC) neurons and heterologous expression systems (mHCN2 expressing HEK cells; hHCN4 expressing oocytes). A number of electrophysiological characteristics of Ih (potential of half-maximal activation (V0.5); current density; activation kinetics) were unchanged following the extracellular application of trace metals and divalent cation chelators to native neurons, cell cultures or oocytes. Mice were fed a diet containing 0.2% CPZ for 35 days, resulting in general demyelination in the brain. Withdrawal of CPZ from the diet resulted in rapid remyelination, the effects of which were assessed at three time points after stopping CPZ feeding (Day1, Day7, Day25). In TC neurons, Ih was decreased on Day1 and Day25 and revealed a transient increased availability on Day7. In addition, we challenged naive TC neurons with INF-α and IL-1β. It was found that Ih parameters were differentially altered by the application of the two cytokines to thalamic cells, while IL-1β increased the availability of HCN channels (depolarized V0.5; increased current density) and the excitability of TC neurons (depolarized resting membrane potential (RMP); increased the number of action potentials (APs); produced a larger voltage sag; promoted higher input resistance; increased the number of burst spikes; hyperpolarized the AP threshold), INF-α mediated contrary effects. The effect of cytokine modulation on thalamic bursting was further assessed in horizontal slices and a computational model of slow thalamic oscillations. Here, IL-1β and INF-α increased and reduced oscillatory bursting, respectively. We conclude that HCN channels are not directly modulated by trace metals and divalent cation chelators but are subject to modulation by different MS-related cytokines.

Effects of different divalent cations in mTi-based codopants (m = Mg or Zn) on the piezoelectric properties of AlN thin films

The understanding of the effects of different divalent cations in codopants for AlN is important for the design of high-performance AlN-based piezoelectric thin films. The addition of Mg-based codopants (e.g., MgTi) into AlN has been one of the most effective methods for the promotion of a higher piezoelectric response (d33). In this study, Mg was replaced by Zn as the divalent cation in a single dopant or codopant and the effects on the piezoelectric responses were investigated. The substitution of Mg with Zn in a single dopant or codopant affected the crystallinity, composition, and eventually d33 of the thin film.

Effect of Different Divalent Cations on the Kinetics and Fidelity of DNA Polymerases

Effect of Different Divalent Cations on the Kinetics and Fidelity of DNA Polymerases

Effect of Different Divalent Cations on the Kinetics and Fidelity of RB69 DNA Polymerase.

Although Mg(2+) is the cation that functions as the cofactor for the nucleotidyl transfer reaction for almost all DNA polymerases, Mn(2+) can also serve, but when it does, the degree of base discrimination exhibited by most DNA polymerases (pols) is diminished. Metal ions other than Mg(2+) or Mn(2+) can also act as cofactors depending on the specific DNA polymerase. Here, we tested the ability of several divalent metal ions to substitute for Mg(2+) or Mn(2+) with RB69 DNA polymerase (RB69pol), a model B-family pol. Our choice of metal ions was based on previous studies with other DNA pols. Co(2+), and to a lesser extent Ni(2+), were the only cations among those tested besides Mg(2+) and Mn(2+) that could serve as cofactors with RB69pol. The incorporation efficiency of correct dNMPs increased by 5-fold with Co(2+), relative to that of Mg(2+). The incorporation efficiencies of incorrect dNMPs increased by 2-17-fold with Co(2+), relative to that with Mg(2+) depending on the incoming dNTP. Base selectivity was reduced even further with Mn(2+) compared to that observed with Co(2+). Substitution of Mn(2+), Co(2+), or Ni(2+) for Mg(2+) reduced the exonuclease activity of RB69pol by 2-, 6-, and 33-fold, respectively, contributing to the frequency of misincorporation. In addition, Co(2+) and Mn(2+) were better able to extend a primer past a mismatch than Mg(2+). Finally, Co(2+) and Mn(2+) enhanced ground-state binding of both correct and incorrect dNTPs to RB69pol:dideoxy-terminated primer-template complexes.

Divalent Cations are Antagonists of the Sodium-Dependent Potassium Channel Slo2.2

We studied the effect of different divalent cations on the Na+-dependent potassium channel Slo2.2 (Slack) using inside-out patches from Xenopus oocytes and a Slack-HEK cell line heterologously expressing Slack channels. In the presence of intracellular sodium, the addition of divalent cations reduces significantly Slack currents recorded in macropatches. Among the divalent cations studied, the most effective in reducing channel activity was Cd++ followed by Ni++, Ca++ and Mg++. Several results suggest that this effect is not caused by blocking the pore of the channel. First, the decrease in currents is not voltage dependent as expected for a cation blocking the channel pore. Second, single channel recordings show a decrease in open probability but not a significant reduction in single channel conductance. Third, some of these cations activate rather than block the Ca++-dependent channel Slo1, which is likely to have a pore structure similar to Slo2.2. Outside-out patches have been used to show that divalent cations do not have an inhibitory effect when applied to the extracellular side of the channel. We propose that the divalent cations may be competing with Na+ for the Na+ binding site. To test this hypothesis, we will examine the effect of divalent cations on several channel mutants and chimeras. These experiments also may help to reveal the structure and localization of the Na+ binding site.

Evaluation of Effects of Bivalent Cations on the Formation of Purine-rich Triple-Helix DNA by ESI-FT-MS

The GGA triplet repeats are widely dispersed throughout eukaryotic genomes. (GGA)n or (GGT)n oligonucleotides can interact with double-stranded DNA containing (GGA:CCT)n to form triple-stranded DNA. The effects of 8 divalent metal ions (3 alkaline-earth metals and 5 transition metals) on formation of these purine-rich triple-helix DNA were investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-MS). In the absence of metal ions, no triplex but single-strand, duplex, and purine homodimer ions were observed in mass spectra. The triple-helix DNA complexes were observed only in the presence of certain divalent ions. The effects of different divalent cations on the formation of purine-rich triplexes were compared. Transition-metal ions, especially Co(2+) and Ni(2+), significantly boost the formation of triple-helix DNA, whereas alkaline-earth metal ions have no positive effects on triplex formation. In addition, Ba(2+) is notably beneficial to the formation of homodimer instead of triplex.

The effect of different divalent cations on the reduction of hexavalent chromium by zerovalent iron

The effects of ferrous cation (Fe2+), copper cation (Cu2+) and calcium cation (Ca2+) on the reduction of hexavalent chromium Cr(VI) by zerovalent iron (Fe0) were investigated. The pseudo-first-order kinetic model was used for describing the reduction of Cr(VI). The experimental results showed that Cu2+ and Fe2+ could improve the reduction of Cr(VI) but Ca2+ might inhibit the reduction of Cr(VI) by Fe0. The improvement of the reduction of Cr(VI) by Cu2+ on Fe0 might be ascribed to Cu2+ acting as a catalyst or the media of charge transfer during the reaction. Different concentration of Cu2+ might lead to the different reduction kinetics of Cr(VI) by Fe0. The X-ray diffraction (XRD) results showed that the reacted iron powder was coated by a small amount of β-FeOOH. The results of scanning electron microscope (SEM), energy dispersive X-ray spectrometer (EDS) and Fourier-transform infra-red (FTIR) showed that the Fe–Cr coprecipitate or Fe–Cr–Cu coprecipitate might be formed during the reaction.

Nanoscale DNA - Zwıtterionıc Vesicle Formulation Compacted For Gene Delivery: Adiabatic Differential Scanning Calorimetric Study

Nucleic acids-membrane associations comprise the least functionally studied macromolecular assembly, yet attract the attention of researchers due to their potential in the field of gene therapy (Leckband & Israelashvili, 2001). The design of novel nucleic acid delivery formulations proceeds mainly as searches of alternatives to highly efficient but risky viral based vehicles (Templeton, 2001; Liu & Huang, 2002; Miller, 2003). The main objective is to achieve compaction of genetic material within highly restricted compartments, while decreasing its cytotoxicity. In the light of well-established potential of liposomes as gene carriers (Templeton, 2001), the current work concerns mainly the stability and physical properties of DNA within the lipid surrounding. Such particles, referred to as lipoplexes are composed of positively charged lipid species and a helper neutral lipid, used for the stabilization of the liposome complex. Despite the considerable efforts that had been made to characterize the structure of these complexes, the origin of molecular forces responsible for self-assembly formation, determination of their charge, colloidal properties, stability against dissociations, cytotoxicity issues, and unravelling characteristics related to efficient intracellular delivery and gene expression remain unclear. A possible alternative to the toxic cationic lipids is the employment of zwitterionic lipid species, which are much safer for target cells (Kharakoz, 1999). Neutral liposomeDNA self-organization is mediated by various inorganic cations, acting as condensing agents. In the light of recent strong evidence that divalent cations enhance the efficacy of plasmid DNA-cationic lipid formulations (Lam & Cullis, 2000), it is of particular interest to study the effect of different divalent cations on the transfection potency of lipid-DNA complexes. In this context, a preliminary results of promissimg ternary DNA-DPPC-Mg 2+ complex preparation and its thermodynamic properties are presented herein.

Induction of apoptosis in purified animal and plant nuclei by Xenopus egg extracts.

We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspension-cultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments. The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed. The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.

Binding properties of neuroligin 1 and neurexin 1beta reveal function as heterophilic cell adhesion molecules.

beta-Neurexins and neuroligins are plasma membrane proteins that are displayed on the neuronal cell surface. We have now investigated the interaction of neurexin 1beta with neuroligin 1 to evaluate their potential to function as heterophilic cell adhesion molecules. Using detergent-solubilized neuroligins and secreted neurexin 1beta-IgG fusion protein, we observed binding of these proteins to each other only in the presence of Ca2+ and in no other divalent cation tested. Only neurexin 1beta lacking an insert in splice site 4 bound neuroligins, whereas neurexin 1beta containing an insert was inactive. Half-maximal binding required 1-3 microM free Ca2+, which probably acts by binding to neuroligin 1 but not to neurexin 1beta. To determine if neurexin 1beta and neuroligin 1 can also interact with each other when present in a native membrane environment on the cell surface, we generated transfected cell lines expressing neuroligin 1 and neurexin 1beta. Upon mixing different cell populations, we found that cells aggregate only if cells expressing neurexin 1beta are mixed with cells expressing neuroligin 1. Aggregation was dependent on Ca2+ and was inhibited by the addition of soluble neurexin 1beta lacking an insert in splice site 4 but not by the addition of neurexin 1beta containing an insert in splice site 4. We conclude that neurexin 1beta and neuroligin 1 (and, by extension, other beta-neurexins and neuroligins) function as heterophilic cell adhesion molecules in a Ca2+-dependent reaction that is regulated by alternative splicing of beta-neurexins.

Enzymes of Δ1-Pyrroline-5-Carboxylate Metabolism in the Camel Tick Hyalomma dromedarii During Embryogenesis. Purification and Characterization of Δ1-Pyrroline-5-Carboxylate Dehydrogenases

The activity of P5C metabolizing enzymes: OAT, P5CR, PO, and P5CD, in the camel tick Hyalomma dromedarii has been followed throughout embryogenesis. The profiles of enzymatic activity showed clear differences in the four enzymes as the embryos grew older. During purification of P5CD to homogeneity the ion exchange chromatography steps lead to two separate forms (termed A and B) with different molecular weights (60,000–59,000 and 50,000–52,000 for the native and denatured enzymes, respectively), amino acid composition, Km for P5C and coenzymes, varying dehydrogenase activities with different substrate specificity when supplied with various aldehyde substrates. Both P5CD A and B exhibited sharp optima at pH 7.5. The effect of different divalent cations and competitive and noncompetitive inhibitors was examined. The changes in P5C metabolizing enzymes during embryogenesis suggest that H. dromedarii has the metabolic potential to convert ornithine into proline and glutamate.

Regulation of lipid polymorphism is essential for the viability of phosphatidylethanolamine-deficient Escherichia coli cells.

Escherichia coli strain AD93 is unable to synthesize the nonbilayer lipid phosphatidylethanolamine and requires high concentrations of specific divalent cations for growth. Previous studies suggested that in this strain, cardiolipin in combination with divalent cations functionally replaces phosphatidylethanolamine, reflecting polymorphic regulation of membrane lipid composition. However, it is also possible that divalent cations are required for regulation of lipid packing or membrane surface potential. 2H NMR was employed to measure the effect of different divalent cations on lipid packing in aqueous dispersions of lipid extracts isolated from AD93 and the wild type parental strain W3899, which were grown with [11,11-2H2]oleic acid. The results indicate that a range of acyl chain order is compatible with growth and that Ba2+, which cannot support growth of AD93, can increase chain packing to the wild type level. By means of microelectrophoresis, it was shown that the growth-promoting cations and Ba2+ have a strong and comparable ability to screen the surface charge of large unilamellar vesicles prepared from AD93 lipid extracts. Therefore, it is unlikely that the growth-promoting capacity of divalent cations is primarily due to their effect on lipid packing or their potency to decrease the surface potential. Furthermore, the addition of small amounts of Ba2+ to a AD93 lipid dispersion with excess Mg2+ diminished HII phase formation. This observation can explain the growth arrest in AD93 cultures upon the addition of Ba2+ and further supports the conclusion that the cation requirement of this strain arises mainly from polymorphic regulation of lipid composition.

Currents carried by monovalent cations through calcium channels in mouse neoplastic B lymphocytes.

Membrane currents through the Ca2+ channel were studied in a hybridoma cell line (MAb-7B) constructed by fusion of S194 myeloma cells and splenic B lymphocytes from the mouse. The whole-cell variation of the patch-electrode voltage-clamp technique was used. When [Ca2+]o = 2.5 mM, [Na+]o = 150 mM and [Na+]i = 155 mM, the current reversed from inward to outward at 20.9 +/- 2.4 mV (mean +/- S.D., n = 62). Both inward and outward currents showed voltage-dependent inactivation with the same membrane potential dependence of steady-state inactivation. The decay time constant of the current decreased from about 27 ms at -44 mV to a saturation value of 16 ms at about -20 mV, and remained at this value even when the current became outward. From the above results both the inward and outward currents were considered to flow through Ca2+ channels. The inward current showed no change when the external Na+ was replaced with Cs+ or tetraethylammonium and increased when [Ca2+]o was increased. Also, the reversal potential became more positive with increasing [Ca2+]o with a slope of 29 mV/decade change of [Ca2+]o. Effects of different divalent cations examined at 10 mM concentration showed the reversal potential to become more positive in the order of Mn2+, Sr2+ approximately equal to Ba2+ and Ca2+ whereas the relative maximum amplitudes of peak inward current were 1.0 for Ca2+, 1.24 for Sr2+, 0.99 for Ba2+ and 0.07 for Mn2+. When [Ca2+]o or [Mg2+]o was reduced by chelators, monovalent cations became capable of carrying inward current through the Ca2+ channel. These monovalent currents share common kinetic properties with the Ca2+ current, as judged from the steady-state inactivation and the decay time constant of the current. The monovalent cation current was blocked by divalent cations in a voltage-dependent manner. The half-blocking concentrations of Ca2+ and Mg2+ at -45 mV were 2.0 X 10(-6) M and 3.0 X 10(-5) M respectively. The same voltage-dependent binding mechanism can explain the outward current carried by monovalent cations at large positive potentials at normal Ca2+ concentrations. The suppression of the monovalent currents by Ca2+ and Mg2+ showed different voltage dependences. The suppression by Ca2+ increased and then decreased as the membrane potential was made negative, whereas the suppression by Mg2+ increased monotonically. This difference can be explained by considering the fact the Ca2+ is permeant and Mg2+ is impermeant through the Ca2+ channel.

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

Secretory vesicles isolated from adrenal medulla were found to fuse in vitro in response to incubation with Ca 2+. Intervesicular fusion was detected by electron microscopy and was indicated by the appearance of twinned vesicles in freeze-fractured suspensions of vesicles and in thin-sectioned pellet. Two types of fusion could be distinguished: Type I, occurring between 10 −7 M and 10 −4 M Ca 2+, was specific for Ca 2+, was inhibited by other divalent cations and was abolished by pretreatment of vesicles with glutaraldehyde, neuraminidase or trypsin. Fusion type I was linear with temperature. A second type of intervesicular fusion was elicited by Ca 2+ in concentrations higher than 2.5 mM and was morphologically characterized by multiple fusions of secretory vesicles. This type of fusion was found to be similar to fusion of liposomes prepared from the membrane lipids of adrenal medullary secretory vesicles: Ca 2+ could be replaced by other divalent cations, the effect of different divalent cations was additive and pretreatments attacking membrane proteins were ineffective. Fusion type II of intact secretory vesicles as well as liposome fusion was discontinuous with temperature. Liposome fusion could be detected within 35 ms and persisted for 180 min. Using liposomes containing defined Ca 2+ concentrations we have not found a major influence of Ca 2+ asymmetry on fusion. Incorporation of the ganglioside GM 3, which is present in the membranes of intact adrenal medullary secretory vesicles did not change the properties of liposomes fusion. Using a Ca 2+-selective electrode we have identified in secretory vesicle membranes both high affinity binding sites for Ca 2+ ( K d = 1.6 · 10 −6 M ) and low affinity sites ( K d = 1.2 · 10 −4 M ).

A comparison of the effects of different divalent cations on a number of mitochondrial reactions linked to ion translocation

Ca 2+, Sr 2+, Mg 2+, Mn 2+, Ba 2+, and Be 2+ behave differently with respect to their effects on activation of respiration, proton ejection, and K + movements across the membrane of isolated rat liver mitochondria. The metal ions tested can be divided into three groups: (1) Mg 2+ and Be 2+ have no effect on either respiration, H + ejection, or K + movements. (2) Mn 2+ and Ba 2+ induce a slow activation of respiration and proton ejection. These ions exchange with mitochondrial K + also in the absence of valinomycin; this exchange is out of phase with respect to proton ejection. (3) Ca 2+ and Sr 2+ promote an extremely rapid extra-oxygen consumption accompanied by H + output. Ca 2+ can exchange with mitochondrial K + only when the membrane is rendered permeable to K + by valinomycin.