Mixed ligand octahedral Co3+ β-diketonates,with acetylacetone,benzoylacetone and dibenzoylmethane. Synthesis,separation of isomers and characterization

Three series of complexes were studied: Co(acetylacetonate)_n(dibenzoylmethanato)_3-n, Co(acetylacetonate)_n(benzoylacetonate)_3-n and Co(dibenzoylmethanato)_n(benzoylacetonate)_3-n. The product mixtures were separated by thin-layer chromatography. Complete separation of all compounds and stereoisomers was obtained. No attempt was made to separate optical isomers which exist for each single stereoisomer. Characterization was by elemental analyses, NMR spectroscopy and the separation characteristics. Electronic absorption spectra were measured in acetonitrile in the 200–800 nm region. Interpretation of the spectral data is provided in terms of ∏ → ∏^*, d → ∏^*, d → d^* transitions. The influence of geometrical isomerism is discussed.     

Cloning,Expression, and Characterization of the Squid Na+–Ca2+ Exchanger (NCX-SQ1)

We have cloned the squid neuronal Na⁺–Ca²⁺ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na⁺–Ca²⁺ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca²⁺ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na⁺-dependent ⁴⁵Ca²⁺ uptake; the uptake was inhibited by injection of Ca²⁺ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na⁺-dependent inactivation, secondary activation by cytoplasmic Ca²⁺, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPγS, in the presence of F⁻ (0.2 mM) and vanadate (50 μM), and both effects reversed on application of a phosphatidylinositol-4′,5′-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPγS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4′,5′-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na⁺ and Ca²⁺ transport reactions. Outward current transients associated with Na⁺ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca²⁺ extrusion were much larger. For NCX-SQ1, charge movements of Ca²⁺ transport could be defined in voltage jump experiments with a low cytoplasmic Ca²⁺ (2 μM) in the presence of high extracellular Ca²⁺ (4 mM). The rates of charge movements showed “U”-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s⁻¹ at 0 mV) indicated an apparent valency of −0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.     

Preconcentration with Bacillus subtilis–Immobilized Amberlite XAD-16: Determination of Cu2+ and Ni2+ in River,Soil, and Vegetable Samples

Solid-phase extraction (SPE) method was developed for the preconcentration of Cu²⁺ and Ni²⁺ before their determination by inductively coupled plasma optical emission spectrometry (ICP-OES). Bacillus subtilis–immobilized Amberlite XAD-16 was used as biosorbent. Effects of critical parameters such as pH, flow rate of samples, amount of Amberlite XAD-16 and biosorbent, sample volume, eluent type, and volume and concentration of eluent on column preconcentration of Cu²⁺ and Ni²⁺ were optimized. Applicability of the method was validated through the analysis of the certified reference tea sample (NCS ZC73014). Sensitivity of ICP-OES was improved by 36.4-fold for Cu²⁺ and 38.0-fold for Ni²⁺ by SPE-ICP-OES method. Limit of quantitation (LOQ) was found to be 0.7 and 1.1 ng/ml for Cu²⁺ and Ni²⁺, respectively. Concentrations of Cu²⁺ and Ni²⁺ were determined by ICP-OES after application of developed method. Relative standard deviations (RSDs) were lower than 4.9% for Cu²⁺ and 7.9% for Ni²⁺. The Tigris River that irrigates a large agricultural part of Southeast Turkey is polluted by domestic and industrial wastes. Concentrations of Cu²⁺ and Ni²⁺ were determined in water, soil, and some edible vegetables as a biomonitor for heavy metal pollution.     

Regulation of Ca2+ Sparks by Ca2+ and Mg2+ in Mammalian and Amphibian Muscle. An RyR Isoform-specific Role in Excitation–Contraction Coupling?

Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks >100 microM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4-40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10(-7) in the frog or 10(-8) in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency.     

Co2+ binding to α-lactalbumin

α-Lactalbumin possesses multiple Zn²⁺ binding sites, with the strongest site having an affinity constant of 5×10⁵ M^?1 [Permyakovet al. (1991),J. Protein Chem. 100, 577]. The binding of zinc at secondary sites is accompanied by destabilization of the protein structure and progressive protein aggregation. This pronounced destabilization is reflected in a shift of the thermal denaturation transition temperature by more than 40°. The present work examines Co²⁺ binding to bovineα-lactalbumin, where for this analog of Zn²⁺, multiple binding sites were also found from spectrofluorimetric titrations. The strong site Co²⁺ binding constant was 1.3×10⁶ M^?1. However, in contrast to Zn²⁺ binding, Co²⁺ does not cause protein aggregation nor any significant thermal destabilization of the protein. Fluroescence energy transfer measurements between Tb³⁺ in the strong calcium site to Co²⁺ in the strong Zn²⁺ site gave a distance in the range of 14–18 Å, which was in excellent agreement with recent crystallographic data for humanα-lactalbumin [Renet al. (1993), J. Biol. Chem.268, 19292–19298] However, the X-ray structure did not identify the additional zinc sites found from earlier solution studies, presumably due to restrictive crystal packing interactions. The results from the current work confirm that the strong cobalt (zinc) site in solution is the same zinc site elucidated by X-ray crystallography.     

Thermoluminescence dosimetric attributes of Yb3+-doped BaO–ZnO–LiF–B2O3 glass material after Er3+ co-doping

Motivated by our previous study on Sm³⁺ ions as thermoluminescence (TL) sensitizers to the BaO–ZnO–LiF–B₂O₃–Yb₂O₃ glass system, in the current study we examined the effect of Er³⁺ ion co-doping on the TL characteristics of this glass system. The 4f–4f electronic transitions of the Er³⁺ and Yb³⁺ ions were confirmed via the optical absorption spectrum. Notably, the use of Yb³⁺–Er³⁺ ions failed to improve the TL intensity, sensitivity, and trap density. However, they enabled the glass system to function as an activator–quencher system. The linearity range and effective atomic number remained unaffected after co-doping. In addition, the problem of anomalous fading caused a remnant signal of just 58% after a week of storage of the Yb³⁺ monodoped glass. This was resolved by the optimum co-doping of Er³⁺ ions to achieve an 89% signal. The co-doping of Er³⁺ ions to the BaO–ZnO–LiF–B₂O₃–Yb₂O₃ glass system regulated its thermal stability and therefore supplemented its potential for radiation monitoring in food processing and retrospective dosimetry.     

Octanuclear {Co6W2} aggregate versus mixed valence {Co}3 cluster in the assembling of Co(phen)2Cl2 (phen = 1,10-phenanthroline) with octacyano metallates: A case of non-isostructurality between {W(CN)8}4− and {Nb(CN)8}4−

An unprecedented octanuclear aggregate, [{Co(phen)₂}₆{W(CN)₈}₂Cl₂] · 2Cl, 2, resulted from the assembling of {Co(phen)₂Cl₂}, 1, and {W(CN)₈}^4?. Surprisingly, the reaction with the paramagnetic {Nb(CN)₈}^4? unit did not afford the homologous {Co–Nb} cluster. Instead the latter building unit undergoes dissociation which led to the formation of a mixed-valence [{Co^II(phen)₂}{Co^III(phen)(CN)₄}₂], 3. This observation is in contrast to the usual trend that {Nb^IV(CN)₈}^4? forms compounds isostructural to that observed for {Mo^IV(CN)₈}^4? and {W^IV(CN)₈}^4?. The structures of the compounds 2 and 3 have been established by single crystal X-ray diffraction. Magnetic behaviors for compounds 1–3 are reported.     

高活性Co2+替代海因酶中Co2+的定量及酶学活性鉴定

利用产D-海因酶(HDT)的重组pE-hdt/E.coli菌株,在LB培养基中添加40μmol/L的Co2+,37℃培养10 h, 表达产物经Q-Sepharose 阴离子交换剂和Phenyl-Sepharose疏水层析,获得电泳纯Co2+D-海因酶(Co2+-HDT).该酶对底物DL-海因的比活性较HDT高约6倍,达21.8 U/mg.可见光光谱分析表明,在498 nm和568 nm处呈现Co2+海因酶络合物的特征性吸收峰.用ICP-AES测定纯酶金属离子含量,HDT每摩尔亚基含0.93摩尔Zn2+和0.04摩尔Co2+,而Co2+-HDT中每摩尔亚基中含0.17摩尔Zn2+ 和089摩尔Co2+.这一结果表明,HDT中的Zn2+ 已被Co2+所替代.此外,在动力学常数,pH和温度稳定性,金属螯合剂EDTA的影响等方面,HDT和Co2+-HDT也略有差异.     

Extra-matrix Mg2+ limits Ca2+ uptake and modulates Ca2+ uptake–independent respiration and redox state in cardiac isolated mitochondria

Cardiac mitochondrial matrix (m) free Ca²⁺ ([Ca²⁺]_m) increases primarily by Ca²⁺ uptake through the Ca²⁺ uniporter (CU). Ca²⁺ uptake via the CU is attenuated by extra-matrix (e) Mg²⁺ ([Mg²⁺]_e). How [Ca²⁺]_m is dynamically modulated by interacting physiological levels of [Ca²⁺]_e and [Mg²⁺]_e and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg²⁺]_e modulates Ca²⁺ uptake via the CU, it also alters bioenergetics in a matrix Ca²⁺–induced and matrix Ca²⁺–independent manner. To test this, we measured changes in [Ca²⁺]_e, [Ca²⁺]_m, [Mg²⁺]_e and [Mg²⁺]_m spectrofluorometrically in guinea pig cardiac mitochondria in response to added CaCl₂ (0–0.6 mM; 1 mM EGTA buffer) with/without added MgCl₂ (0–2 mM). In parallel, we assessed effects of added CaCl₂ and MgCl₂ on NADH, membrane potential (ΔΨ_m), and respiration. We found that >0.125 mM MgCl₂ significantly attenuated CU-mediated Ca²⁺ uptake and [Ca²⁺]_m. Incremental [Mg²⁺]_e did not reduce initial Ca²⁺uptake but attenuated the subsequent slower Ca²⁺ uptake, so that [Ca²⁺]_m remained unaltered over time. Adding CaCl₂ without MgCl₂ to attain a [Ca²⁺]_m from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca²⁺]_m did not additionally enhance NADH oxidation or respiration. Adding MgCl₂ did not increase [Mg²⁺]_m but it altered bioenergetics by its direct effect to decrease Ca²⁺ uptake. However, at a given [Ca²⁺]_m, state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg²⁺]_e. Thus, [Mg²⁺]_e without a change in [Mg²⁺]_m can modulate bioenergetics independently of CU-mediated Ca²⁺ transport.     

?-Blocker Timolol Prevents Arrhythmogenic Ca2+ Release and Normalizes Ca2+ and Zn2+ Dyshomeostasis in Hyperglycemic Rat Heart

Defective cardiac mechanical activity in diabetes results from alterations in intracellular Ca²⁺ handling, in part, due to increased oxidative stress. Beta-blockers demonstrate marked beneficial effects in heart dysfunction with scavenging free radicals and/or acting as an antioxidant. The aim of this study was to address how β-blocker timolol-treatment of diabetic rats exerts cardioprotection. Timolol-treatment (12-week), one-week following diabetes induction, prevented diabetes-induced depressed left ventricular basal contractile activity, prolonged cellular electrical activity, and attenuated the increase in isolated-cardiomyocyte size without hyperglycemic effect. Both in vivo and in vitro timolol-treatment of diabetic cardiomyocytes prevented the altered kinetic parameters of Ca²⁺ transients and reduced Ca²⁺ loading of sarcoplasmic reticulum (SR), basal intracellular free Ca²⁺ and Zn²⁺ ([Ca²⁺]_i and [Zn²⁺]_i), and spatio-temporal properties of the Ca²⁺ sparks, significantly. Timolol also antagonized hyperphosphorylation of cardiac ryanodine receptor (RyR2), and significantly restored depleted protein levels of both RyR2 and calstabin2. Western blot analysis demonstrated that timolol-treatment also significantly normalized depressed levels of some [Ca²⁺]_i-handling regulators, such as Na⁺/Ca²⁺ exchanger (NCX) and phospho-phospholamban (pPLN) to PLN ratio. Incubation of diabetic cardiomyocytes with 4-mM glutathione exerted similar beneficial effects on RyR2-macromolecular complex and basal levels of both [Ca²⁺]_i and [Zn²⁺]_i, increased intracellular Zn²⁺ hyperphosphorylated RyR2 in a concentration-dependent manner. Timolol also led to a balanced oxidant/antioxidant level in both heart and circulation and prevented altered cellular redox state of the heart. We thus report, for the first time, that the preventing effect of timolol, directly targeting heart, seems to be associated with a normalization of macromolecular complex of RyR2 and some Ca²⁺ handling regulators, and prevention of Ca²⁺ leak, and thereby normalization of both [Ca²⁺]_i and [Zn²⁺]_i homeostasis in diabetic rat heart, at least in part by controlling the cellular redox status of hyperglycemic cardiomyocytes.     

?-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca²⁺ and Mg²⁺ and the role of these changes in SR Ca²⁺ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N₂ to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca²⁺] <1 µM, ß-adrenergic stimulation increased luminal Ca²⁺ activation of single RyR channels, decreased luminal Mg²⁺ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg²⁺. At cytoplasmic [Ca²⁺] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg²⁺ and Ca²⁺ inhibition of RyRs. The K_a and maximum levels of cytoplasmic Ca²⁺ activation site were not affected by ß-adrenergic stimulation.Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca²⁺ binding to the luminal Ca²⁺ site and decreasing its affinity for luminal Mg²⁺ and 2) decreasing affinity of the low-affinity Ca²⁺/Mg²⁺ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.     

p34cdc2: the S and M kinase?

In the yeast cell cycle, the induction of two very different processes, DNA synthesis (S-phase) and mitosis (M-phase), requires the same serine/threonine-specific protein kinase p34cdc2, which has been highly conserved through evolution. On the basis of work conducted largely in multicellular eukaryotes, it has recently been suggested that p34cdc2 is able to perform these two mutually exclusive roles by phosphorylating different sets of substrates through a cell cycle-dependent association with other proteins that dictate the substrate specificity of the protein kinase. To recognize its mitotic substrates, p34cdc2 associates with one of the cyclins--a family of proteins of two distinct but related types (A and B) characterized by their periodic destruction at each mitosis. In interphase, the formation of a complex between p34cdc2 and another protein (or proteins) would allow the phosphorylation of a different set of proteins involved in the G1 to S transition. This review focuses on the evidence for this appealing simple model and the nature of the putative substrates proposed.     

Cisd2 mediates lifespan: is there an interconnection among Ca2+ homeostasis,autophagy, and lifespan?

Abstract

CISD2, an evolutionarily conserved novel gene, plays a crucial role in lifespan control and human disease. Mutations in human CISD2 cause type 2 Wolfram syndrome, a rare neurodegenerative and metabolic disorder associated with a shortened lifespan. Significantly, the CISD2 gene is located within a region on human chromosome 4q where a genetic component for human longevity has been mapped through a comparative genome analysis of centenarian siblings. We created Cisd2 knockout (loss-of-function) and transgenic (gain-of-function) mice to study the role of Cisd2 in development and pathophysiology, and demonstrated that Cisd2 expression affects lifespan in mammals. In the Cisd2 knockout mice, Cisd2 deficiency shortens lifespan and drives a panel of premature aging phenotypes. Additionally, an age-dependent decrease of Cisd2 expression has been detected during normal aging in mice. Interestingly, in the Cisd2 transgenic mice, we demonstrated that a persistent level of Cisd2 expression over the different stages of life gives the mice a long-lived phenotype that is linked to an extension in healthy lifespan and a delay in age-associated diseases. At the cellular level, Cisd2 deficiency leads to mitochondrial breakdown and dysfunction accompanied by cell death with autophagic features. Recent studies revealed that Cisd2 may function as an autophagy regulator involved in the Bcl-2 mediated regulation of autophagy. Furthermore, Cisd2 regulates Ca²⁺ homeostasis and Ca²⁺ has been proposed to have an important regulatory role in autophagy. Finally, it remains to be elucidated if and how the regulation in Ca²⁺ homeostasis, autophagy and lifespan are interconnected at the molecular, cellular and organism levels.     

Structural,photoluminescence, and optical characteristics of a Sm3+-doped lead borate–strontium–tungsten glass system: Evaluation of Judd–Ofelt intensity parameters and radiative properties

In this study, the melt quenching approach is used to synthesize a lead borate–strontium-based glass system doped with samarium ions. Modifications in the glass network structure arising from the addition of various concentrations of Sm³⁺ ions were investigated via Fourier transform infrared (FTIR) spectroscopy. FTIR analysis revealed B–O–B bridges, BO₃, and BO₄ units are present. UV–vis–NIR spectroscopic measurement was performed to study the optical absorption spectra. Optical constants such as optical bandgap energies, refractive indices, and other related parameters were evaluated. The lifetime fluorescence decay was measured and ranged between 1.04 and 1.88 ns. The photoluminescence spectra in the range 500–750 nm revealed four transitions from the ground state ⁶G_5/2 to the excited states ⁶H_5/2, ⁶H_7/2, ⁶H_9/2 and ⁶H_11/2 and J–O theory was utilized to study these optical transitions for Sm³⁺ ions. Calculations of the oscillator strengths and J–O intensity parameters were performed and the obtained J–O parameters followed the sequence Ω₄ > Ω₆ > Ω₂. The ratio O/R indicated a high lattice asymmetry around the samarium ions. The values of lifetimes and branching ratios for the fabricated samples emphasized their suitability to be used in laser applications. The current glass samples are good candidates for orange and red emission devices.     

Unravelling the interface of Co–Pi/Ag/Fe2O3 and Ni–Pi/Ag/Fe2O3 heterostructures for enhanced solar water splitting

Recently, a new type of haematite-based (a-Fe₂O₃) photoanode has achieved a considerable catalytic performance through simply depositing Ag and cobalt phosphate (Co–Pi) nanoparticles (NPs) onto haematite nanosheets. However, there is no detailed mechanism study on the reason for high-performance catalysis. In view of this, we conduct first-principles calculations and our results indicate that there is a further accumulation of positive charge on Ag NPs on the heterogeneous interfaces with the addition of Co–Pi NPs than that of haematite modified only by Ag NPs. Also, there is a slight increase of the adsorption energy of water molecules.