QM/MM analysis of effect of divalent metal ions on OPRT action.

The role of Mg2+ cofactor in orotate phosphoribosyltransferase (OPRT) catalyzed synthesis of orotidine monophosphate (OMP) from phosphoribosyl pyrophosphate (PRPP) and orotate (OA) in substrate binding and the influence of the identity of the divalent metal ion on the reaction mechanism were addressed in this study using quantum mechanics/molecular mechanics framework. Energetics of migration and binding of different substrate complexes in the active site cavity was established. A quantitative analysis of various processes indicated the reaction pathway to consist of complexation of Mg2+ with PRPP, migration of Mg2+-PRPP and OA towards the active site, binding of OA to OPRT, and binding of Mg2+-PRPP complex to OA-OPRT complex. The mechanism of the reaction was unaltered by the change in the identity of divalent metal ion. Experimentally reported inhibiting character of Co2+ was explained on the basis of large Co2+-PRPP binding and migration energies. Mg2+, Ca2+, Mn2+, Co2+ and Zn2+ ions were screened computationally to assess their inhibiting/activating characteristics. Trends obtained by our computational investigations were in correspondence with experimentally reported trends.

Synthesis, spectroscopic and thermal studies of Mg(II), Ca(II), Sr(II) and Ba(II) diclofenac sodium complexes as anti-inflammatory drug and their protective effects on renal functions impairment and oxidative stress.

The main task of our present study is the preparation of newly complexes of Mg(II), Ca(II), Sr(II) and Ba(II) with diclofenac which succeeded to great extent in alleviating the side effects of diclofenac alone and ameliorating the kidney function parameters and antioxidant capacities with respect to diclofenac treated group alone. The Mg(II), Ca(II), Sr(II) and Ba(II) with diclofenac have been synthesized and characterized using infrared, electronic and (1)H NMR spectral, thermogravimetric and conductivity measurements. The diclofenac ligand has been found to act as bidentate chelating agent. Diclofenac complexes coordinate through the oxygen's of the carboxyl group. The molar ratio chelation is 1:2 (M(2+)-dic) with general formula [M(dic)2(H2O)2]⋅nH2O. Antibacterial screening of the alkaline earth metal complexes against Escherichia coli (Gram-ve), Bacillus subtilis (Gram+ve) and anti-fungal (Asperagillus oryzae, Asperagillus niger, Asperagillus flavus) were investigated. The kidney functions in male albino rats were ameliorated upon treatment with metal complexes of dic, which are represented by decreasing the levels of urea and uric acid to be located within normal values. The other looks bright spot in this article is the assessment of antioxidant defense system including SOD, CAT and MDA with the help of Sr(2+), Mg(2+) and Ca(2+)-dic complexes. The hormones related to kidney functions and stresses have been greatly ameliorated in groups treated with dic complexes in comparable with dic treated group.

Effect of metal ions on the hydrolytic and transesterification activities of Candida rugosa lipase.

In order to study the effect of metal ions on lipase activity, hydrolytic and transesterification activities of Candida rugosa lipase were investigated in presence of alkali (Na⁺ and K⁺), alkaline earth (Ca⁺² and Ba⁺²) and transition (Cr⁺³, Fe⁺³, Co⁺², Cu⁺² and Ni⁺²) metal ions. Maximum enhancement in hydrolytic activity of lipase was observed by Ca⁺², and in transesterification activity by Cr⁺³ and Co⁺². The kinetics of the lipase catalyzed transesterification (methanolysis and ethanolysis) reactions were also studied, and the activation energies of methanolysis and ethanolysis were reduced from 10.16 and 10.24 kcal mol⁻¹, respectively, to 5.41 and 7.55 kcal mol⁻¹, respectively, when reactions were performed in presence of Co⁺². Thus, in lipase catalyzed transesterification Cr⁺³ or Co⁺² could be added to the assay in order to produce the biodiesel in relatively shorter reaction duration.

CO2 gasification reactivity of biomass char: catalytic influence of alkali, alkaline earth and transition metal salts.

This study investigates the influence of alkali (Na, K), alkaline earth (Ca, Mg) and transition (Fe) metal nitrates on CO2 gasification reactivity of pistachio nut shell (PNS) char. The preliminary gasification experiments were performed in thermogravimetric analyzer (TGA) and the results showed considerable improvement in carbon conversion; Na-char>Ca-char>Fe-char>K-char>Mg-char>raw char. Based on TGA studies, NaNO3 (with loadings of 3-7 wt%) was selected as the superior catalyst for further gasification studies in bench-scale reactor; the highest reactivity was devoted to 5 wt% Na loaded char. The data acquired for gasification rate of catalyzed char were fitted with several kinetic models, among which, random pore model was adopted as the best model. Based on obtained gasification rate constant and using the Arrhenius plot, activation energy of 5 wt% Na loaded char was calculated as 151.46 kJ/mol which was 53 kJ/mol lower than that of un-catalyzed char.

Preparation and characterization of an acellular bovine pericardium intended for manufacture of valve bioprostheses.

Major problems with biological heart valves post-implantation are associated with progressive structural deterioration and calcification attributed to glutaraldehyde processing, dead cells, and cell fragments present in the native tissue. In spite of these problems, glutaraldehyde still is the reagent of choice. The results with acellular matrix xenograft usually prepared by detergent treatment in association with enzymes are rather conflicting because while preserving mechanical properties, tissue morphology and collagen structure are process dependent. This work describes a chemical approach for the preparation of an acellular bovine pericardium matrix intended for the manufacture of heart valve bioprostheses. Cell removal was performed by an alkaline extraction in the presence of calcium salts for periods ranging from 6 to 48 h. The results showed that cell removal was achieved after 12 h, with swelling and negative charge increasing with processing time. Nevertheless, collagen fibril structure, ability to form fibrils, and stability to collagenase were progressive after 24-h processing. There was no denaturation of the collagen matrix. A process is described for the preparation of acellular bovine pericardium matrices with preserved fibril structure and morphology for the manufacture of cardiac valve bioprostheses and may be used in other applications for tissue reconstruction.

Probing cationic selectivity of cardiac calsequestrin and its CPVT mutants.

CASQ (calsequestrin) is a Ca2+-buffering protein localized in the muscle SR (sarcoplasmic reticulum); however, it is unknown whether Ca2+ binding to CASQ2 is due to its location inside the SR rich in Ca2+ or due to its preference for Ca2+ over other ions. Therefore a major aim of the present study was to determine how CASQ2 selects Ca2+ over other metal ions by studying monomer folding and subsequent aggregation upon exposure to alkali (monovalent), alkaline earth (divalent) and transition (polyvalent) metals. We additionally investigated how CPVT (catecholaminergic polymorphic ventricular tachycardia) mutations affect CASQ2 structure and its molecular behaviour when exposed to different metal ions. Our results show that alkali and alkaline earth metals can initiate similar molecular compaction (folding), but only Ca2+ can promote CASQ2 to aggregate, suggesting that CASQ2 has a preferential binding to Ca2+ over all other metals. We additionally found that transition metals (having higher co-ordinated bonding ability than Ca2+) can also initiate folding and promote aggregation of CASQ2. These studies led us to suggest that folding and formation of higher-order structures depends on cationic properties such as co-ordinate bonding ability and ionic radius. Among the CPVT mutants studied, the L167H mutation disrupts the Ca2+-dependent folding and, when folding is achieved by Mn2+, L167H can undergo aggregation in a Ca2+-dependent manner. Interestingly, domain III mutants (D307H and P308L) lost their selectivity to Ca2+ and could be aggregated in the presence of Mg2+. In conclusion, these studies suggest that CPVT mutations modify CASQ2 behaviour, including folding, aggregation/polymerization and selectivity towards Ca2+.

Imaging calcium signals in vivo: a powerful tool in physiology and pharmacology.

The design and engineering of organic fluorescent Ca(2+) indicators approximately 30 years ago opened the door for imaging cellular Ca(2+) signals with a high degree of temporal and spatial resolution. Over this time, Ca(2+) imaging has revolutionized our approaches for tissue-level spatiotemporal analysis of functional organization and has matured into a powerful tool for in situ imaging of cellular activity in the living animal. In vivo Ca(2+) imaging with temporal resolution at the millisecond range and spatial resolution at micrometer range has been achieved through novel designs of Ca(2+) sensors, development of modern microscopes and powerful imaging techniques such as two-photon microscopy. Imaging Ca(2+) signals in ensembles of cells within tissue in 3D allows for analysis of integrated cellular function, which, in the case of the brain, enables recording activity patterns in local circuits. The recent development of miniaturized compact, fibre-optic-based, mechanically flexible microendoscopes capable of two-photon microscopy opens the door for imaging activity in awake, behaving animals. This development is poised to open a new chapter in physiological experiments and for pharmacological approaches in the development of novel therapies.

An update on the honesty of melanin-based color signals in birds.

The control mechanisms and information content of melanin-based color signals in birds have generated much recent interest and controversy among evolutionary biologists. Initial experimental studies on this topic manipulated coarse metrics of an individual's condition (i.e. food intake, disease state) and failed to detect significant condition-dependence of melanin ornament expression. However, three new lines of research appear profitable and target specific factors associated with the production of melanin pigments. These include the role of (i) metals, (ii) amino acids, and (iii) testosterone and social interactions in shaping the extent and intensity of melanin-colored plumage patches. Here, I review recent studies of and evidence for these honesty-reinforcing mechanisms.

Cofactor effects on the protein folding reaction: acceleration of alpha-lactalbumin refolding by metal ions.

About 30% of proteins require cofactors for their proper folding. The effects of cofactors on the folding reaction have been investigated with alpha-lactalbumin as a model protein and metal ions as cofactors. Metal ions accelerate the refolding of alpha-lactalbumin by lessening the energy barrier between the molten globule state and the transition state, mainly by decreasing the difference of entropy between the two states. These effects are linked to metal ion binding to the protein in the native state. Hence, relationships between the metal affinities for the intermediate states and those for the native state are observed. Some residual specificity for the calcium ion is still observed in the molten globule state, this specificity getting closer in the transition state to that of the native state. The comparison between kinetic and steady-state data in association with the Phi value method indicates the binding of the metal ions on the unfolded state of alpha-lactalbumin. Altogether, these results provide insight into cofactor effects on protein folding. They also suggest new possibilities to investigate the presence of residual native structures in the unfolded state of protein and the effects of such structures on the protein folding reaction and on protein stability.

Metal-dependent hydrolysis of myelin basic protein by IgGs from the sera of patients with multiple sclerosis.

Homogeneous IgG fractions were obtained by chromatography of the sera of patients with multiple sclerosis (MS) on Protein G-Sepharose under conditions that remove non-specifically bound proteins. These IgGs contained several chelated metals, the relative amount of which decreases in the order: Fe>or=Ca>Cu>or=Zn>or=Mg>or=Mn>or=Pb>or=Co>or=Ni. In contrast to homogeneous IgGs of healthy individuals, Abs of MS patients effectively hydrolyzed human myelin basic protein (MBP). A minor metal-dependent fraction was obtained by chromatography of highly purified IgGs from MS patient on Chelex-100. This IgG fraction did not hydrolyze human MBP in the absence of Me(2+) ions but was activated after addition of Me(2+) ions: Mg(2+)>Mn(2+)>Cu(2+)>Ca(2+). Proteolytic activities of IgGs from other MS patients were also activated by other metal ions (Ni(2+), Fe(2+), Co(2+), Zn(2+), Pb(2+), and Co(2+)) and especially Ni(2+). Ni(2+)-activated IgGs were separated into distinct MBP-hydrolyzing fractions by chromatography on HiTraptrade mark Chelating Sepharose charged with Ni(2+). Detection of Mg(2+)-dependent proteolytic activity in the SDS-PAGE area corresponding only to IgG provided direct evidence that IgG from sera of MS patients possesses metal-dependent human MBP-hydrolyzing activity. Observed properties of MS abzymes distinguish them from other known mammalian metalloproteases and demonstrate their pronounced catalytic diversity. Metal-dependent IgGs from MS patients represent the first example of abzymes with metal-dependent proteolytic activity.

Effects of calcium, strontium, and barium on isolated phrenic nerve-diaphragm preparation of rat and their interactions with diltiazem and nifedipine.

Calcium (Ca2+), strontium (Sr2+), and barium (Ba2+) are expected to exert similar chemical and pharmacological effects. The effects of barium, strontium and calcium were studied on the contractions of rat phrenic nerve-diaphragm preparations, following electrical stimulation and their interactions with nifedipine (nif) and diltiazem (DZM) were also studied. Low doses of strontium chloride (SrCl2), barium chloride (BaCl2) and calcium chloride (CaCl2) were able to increase the force of contraction of the rat diaphragm when actively stimulated. Diltiazem inhibited the stimulant effects of Sr2+, Ba2+, and Ca2+. On the other hand, nifedipine blocked the effects of Sr2+ and Ca2+ but potentiated the effects of Ba2+. Strontium, barium, and calcium restored the contractility of the muscle following electrical stimulation when the tissue was in biological fluid absolutely depleted of calcium. These findings suggest that Sr2+ and Ba2+ may be able to substitute Ca2+ in the rat diaphragm for its contractions and nifedipine and diltiazem may follow different mechanisms of actions or channels in their blocking effects.

The ionophore nigericin transports Pb2+ with high activity and selectivity: a comparison to monensin and ionomycin.

The K(+) ionophore nigericin is shown to be highly effective as an ionophore for Pb(2+) but not other divalent cations, including Cu(2+), Zn(2+), Cd(2+), Mn(2+), Co(2+), Ca(2+), Ni(2+), and Sr(2+). Among this group a minor activity for Cu(2+) transport is seen, while for the others activity is near or below the limit of detection. The selectivity of nigericin for Pb(2+) exceeds that of ionomycin or monensin and arises, at least in part, from a high stability of nigericin-Pb(2+) complexes. Plots of log rate vs log Pb(2+) or log ionophore concentration, together with the pH dependency, indicate that nigericin transports Pb(2+) via the species NigPbOH and by a mechanism that is predominately electroneutral. As with monensin and ionomycin, a minor fraction of activity may be electrogenic, based upon a stimulation of rate that is produced by agents which prevent the formation of transmembrane electrical potentials. Nigericin-catalyzed Pb(2+) transport is not inhibited by physiological concentrations of Ca(2+) or Mg(2+) and is only modestly affected by K(+) and Na(+) concentrations in the range of 0-100 mM. These characteristics, together with higher selectivity and efficiency, suggest that nigericin may be more useful than monensin in the treatment of Pb intoxication.

[The action of 232Th separately and in combination with heavy and alkaline metal salts on Tradescantia (clone 02)]. / Razdel'noe i v sochetanii s soliami tiazhelykh i shchelochnykh metallov deistvie 232Th na tradeskantsiiu (klon 02).

The action of 232Th-nitrate in concentration 0.09, 0.18 and 0.36 mg/l (counting on 232Th ion) on water cultures of Tradescantia (clone 02) was investigated. It was found that all investigated concentrations of 232Th showed statistically significant genotoxic effect and increased level of morphological abnormal cells in the stamen hairs of Tradescantia in the absence of modifying action of other metal ions. Synergistic toxic interaction was found between the 232Th in concentration 0.18 mg/l and metal ions for all samples of thawed water. Synergistic genotoxic effect of the combined action of these factors was revealed only at the low total contents of ions of heavy and alkaline metals in thawed water samples. The observed synergistic effects of the combined action of 232Th and metal ions should be taken into account when controlling the radionuclide level in the environment.

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase.

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).

Tuning the voltage dependence of tetraethylammonium block with permeant ions in an inward-rectifier K+ channel.

To understand the role of permeating ions in determining blocking ion-induced rectification, we examined block of the ROMK1 inward-rectifier K+ channel by intracellular tetraethylammonium in the presence of various alkali metal ions in both the extra- and intracellular solutions. We found that the channel exhibits different degrees of rectification when different alkali metal ions (all at 100 mM) are present in the extra- and intracellular solution. A quantitative analysis shows that an external ion site in the ROMK1 pore binds various alkali metal ions (Na+, K+, Rb+, and Cs+) with different affinities, which can in turn be altered by the binding of different permeating ions at an internal site through a nonelectrostatic mechanism. Consequently, the external site is saturated to a different level under the various ionic conditions. Since rectification is determined by the movement of all energetically coupled ions in the transmembrane electrical field along the pore, different degrees of rectification are observed in various combinations of extra- and intracellular permeant ions. Furthermore, the external and internal ion-binding sites in the ROMK1 pore appear to have different ion selectivity: the external site selects strongly against the smaller Na+, but only modestly among the three larger ions, whereas the internal site interacts quite differently with the larger K+ and Rb+ ions.

Identification of SNAREs involved in regulated exocytosis in the pancreatic acinar cell.

The molecular basis of exocytotic membrane fusion in the pancreatic acinar cell was investigated using an in vitro assay that measures both zymogen granule-plasma membrane fusion and granule-granule fusion. These two fusion events were differentially sensitive to Ca(2+), suggesting that they are controlled by different Ca(2+)-sensing mechanisms. Botulinum neurotoxin C (BoNT/C) treatment of the plasma membranes caused cleavage of syntaxin 2, the apical isoform of this Q-SNARE, but did not affect syntaxin 4, the basolateral isoform. BoNT/C also cleaved syntaxin 3, the zymogen granule isoform. BoNT/C treatment of plasma membranes abolished granule-plasma membrane fusion, whereas toxin treatment of the granules reduced granule-plasma membrane fusion and abolished granule-granule fusion. Tetanus toxin cleaved granule-associated synaptobrevin 2 but caused only a small reduction in both granule-plasma membrane fusion and granule-granule fusion. Our results indicate that syntaxin 2 is the isoform that mediates fusion between zymogen granules and the apical plasma membrane of the acinar cell. Syntaxin 3 mediates granule-granule fusion, which might be involved in compound exocytosis. In contrast, the major R-SNARE on the zymogen granule remains to be identified.

Bimodal action of alkaline earth cations on Dictyostelium discoideum ribonuclease P activity.

The ribonucleoprotein ribonuclease P (RNase P) cleaves all tRNA precursors endonucleolitically to produce the mature 5'-end. Dictyostelium discoideum RNase P displays an absolute requirement for Mg2+. Only the alkaline earth cations Ca2+, Sr2+, and Ba2+, under appropriate conditions can substitute to some extent for Mg2+. The transition metals Mn2+, Co2+, Ni2+, and Cd2+ are efficient inhibitors of the enzyme activity. Ca2+, Sr2+ and Ba2+, in the presence of Mg2+, exhibit a bimodal action at the kinetic phase of the reaction. Kinetic analysis of the activation phase revealed that Ca2+, Sr2+, or Ba2+ attached on a specific site of RNase P act as nonessential-noncompetitive activators. Further additions of Ca2+, Sr2+, or Ba2+ cause noncompetitive inhibition on the RNase P reaction, indicating that RNase P possesses a second binding site responsible for the inhibitory effect of Ca2+, Sr2+, and Ba2+. Both activator and inhibitory sites can be occupied by Ca2+, Sr2+, or Ba2+ at the same time.

Ca(2+)-permeability of muscle nicotinic acetylcholine receptor is increased by expression of the epsilon subunit.

We have expressed muscle embryonic (alpha beta gamma delta) and adult (alpha beta delta epsilon) nicotinic acetylcholine receptors in Xenopus Laevis oocytes and measured their current reversal potentials in the presence of extracellular Na+, Ca2+, Sr2+ or Ba2+ ions. The ionic permeability ratio PCa2+/PNa+ was increased about 3 fold by the change in the subunit composition of the nicotinic acetylcholine receptor (replacement of the gamma by the epsilon subunit). A similar increase was also found when permeability to Ba2+ and Sr2+ ions was studies. Comparison of the nicotinic Ca2+ currents recorded from oocytes injected with embryonic or adult receptor subunit combinations also showed that the Ca2+ influx was significantly increased by expressing the epsilon subunit. This increase was estimated to change the contribution of the Ca2+ current to the total net inward current from 0.8% (in the case of the alpha beta gamma delta receptor) to 2.5% (in the case of the alpha beta delta epsilon receptor). Taken together, these results suggest that important modifications in the acetylcholine-mediated Ca2+ influx occurred during muscle innervation and underline the role of the nicotinic receptor in the developmental regulation of Ca2+ influx.

Kinesin-driven microtubule motility in the presence of alkaline-earth metal ions: indication for a calcium ion-dependent motility.

We studied the effect of alkaline-earth metal ions on the kinesin-driven gliding of microtubules, using a narrow glass chamber enabling the exchange of buffer components without interrupting microscopic observation. Under standard conditions (0.5 mM Mg2+), microtubules were found to glide at a mean velocity of about 0.6 micron/s. Motility was widely ceased after removing Mg2+. Subsequent addition of Ca2+ restored motility (maximal mean gliding velocity measured: 0.26 micron/s at 2.5 mM Ca2+). Also in the presence of Sr2+ or Ba2+ a slow gliding could be observed (0.025 micron/s and 0.014 micron/s, respectively, at 0.5 mM). After removal of Ca2+, Sr2+, or Ba2+ and re-addition of Mg2+, the gliding velocities reached approximately the values determined under standard conditions. Motility was not changed when 0.5 mM Ca2+, Sr2+, or Ba2+ were applied together with Mg2+. Microtubule gliding stopped after substitution of 0.5 mM BeCl2 for Mg2+. When both BeCl2 and Mg2+ were present, the mean gliding velocity was reduced to 0.29 micron/s. In addition, many microtubules were released from the kinesin coated glass surface, indicating that the beryllium salt disorders the binding between kinesin and microtubules. Our results confirm that Mg2+ is the most suitable cofactor for kinesin driven microtubule motility. However, they also demonstrate that brain kinesin can generate motility when Ca2+ was substituted for Mg2+.

Intracellular inositol 1,3,4,5-tetrakisphosphate enhances the calcium current in hippocampal CA1 neurones of the gerbil after ischaemia.

1. To examine the role of the phosphoinositide cascade triggered by disturbed Ca2+ homeostasis in ischaemic neurones, inositol 1,3,4,5-tetrakisphosphate (InsP4) was applied to the cytoplasmic face of membrane patches isolated from CA1 pyramidal neurones in the gerbil hippocampus. 2. In outside-out recordings, InsP4 induced an inward current which was increased by raising the extracellular [Ca2+]. In contrast, no clear channel openings could be observed in patches from neurones of sham-operated gerbils. 3. Open probabilities of InsP4-activated channels were significantly decreased upon application of omega-conotoxin but were not affected by omega-agatoxin or nifedipine. 4. In inside-out patches using high concentrations of Ca2+, Ba2+ or Sr2+ in the pipette solution, InsP4 enhanced inward currents. 5. Application of the isomers of InsP4 slightly enhanced the currents, but inositol 1,4,5-trisphosphate (InsP3) had no effect. 6. In the absence of InsP4 there was a single main Ba2+ current peak of 4.0 pA in amplitude, whereas upon its application two main peaks of 3.0 and 7.2 pA were present. 7. The open probabilities of these channels were apparently increased by InsP4. 8. These findings support the view that a disturbed phosphoinositide cascade occurs in the hippocampal pyramidal neurones after ischaemia and the InsP4 thus formed plays an important role in promoting the Ca2+ accumulation which results in neuronal death.