Engineering a subcellular targetable, red-emitting, and ratiometric fluorescent probe for Ca2+ and its bioimaging applications

A new, visible-light-excited and red-emitting fluorescent Ca²⁺ probe, STDBT, was synthesized, which consists of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid as a Ca²⁺-chelating moiety and two benzothiazolium hemicyanine dyes as fluorophores. The spectral profiles of its free and Ca²⁺-bound forms were studied. Upon addition of Ca²⁺, the fluorescence spectra of STDBT displayed a significant enhancement (about 48-fold) in fluorescence intensity and a 20-nm blueshift (from 600 to 580 nm) in the emission spectrum. Both the absorption and the excitation spectra of STDBT showed a very large (more than 100 nm) hypsochromic shift in the long-wavelength maxima upon binding with Ca²⁺. Interestingly, in contrast with the commonly used Ca²⁺ indicator Fluo-3, when the acetoxymethyl ester of STDBT enters into cells, it distributes both in the cytosol and the nucleus, but displays a very clear boundary between the two compartments. This allows STDBT to be used as a double targetable Ca²⁺ probe that can be used to report cytoplasmic Ca²⁺ and nuclear Ca²⁺ simultaneously.     

H2O2: a Ca2+ or Mg2+‐sensing function in statin passive diffusion

In a previous paper Guillaume's group demonstrated that magnesium (Mg²⁺ concentration range 0.00–2.60 mm ) increased the passive diffusion of statins and thus played a role in their potential toxicity. In order to confirm an increase in this passive diffusion by divalent salt cations, the role of calcium chloride (CaCl₂) on the statin–immobilized artificial membrane (IAM) association was studied. It was demonstrated that calcium supplementation (Ca²⁺ concentration range 0.00–3.25 mm ) increases the statin passive diffusion. In addition, it was shown that the Ca²⁺ effect on the statin–IAM association is higher than that of Mg²⁺. These results show that Ca²⁺ enhances the passive diffusion of drugs into biological membranes and thus their potential toxicity. Also, addition of H₂O₂ to the medium showed a hyperbolic response for the statin passive diffusion and this effect was enhanced for the highest Ca²⁺ or Mg²⁺ concentrations in the medium. H₂O₂ is likely to interact with the polar head groups of the IAM through dipole–dipole interactions. The conformational changes in H₂O₂–IAM result in a higher degree of exposure of hydrophobic areas, thus explaining why the binding of pravastatin, which showed the lowest logP value, was less affected by H₂O₂. This result shows the significant contribution of H₂O₂ and thus the oxidative stress on the statin passive diffusion. Much of the sensitivity derives from the action of Ca²⁺ or Mg²⁺, in turn supported the idea that H₂O₂ may serve a Ca²⁺ or Mg²⁺ sensing function in statin passive diffusion Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis of Novel Dibenzo‐18‐crown‐6‐ ether‐Functionalized Benzimidazoles and its Applications in Colorimetric Recognition to Hg2+ and as Antifungal Agents

New crown ether‐functionalized benzimidazoles was designed and synthesized via formylation of dibenzo‐18‐crown‐6 followed by condensation with different o‐phenylene diamines. The complexation properties of crown ether‐functionalized benzimidazoles with various metals (K⁺, Ca²⁺, Ba²⁺, Co²⁺, Hg²⁺) were examined using UV–vis spectroscopy. Hg²⁺ showed a well‐defined peculiar absorption maximum at 366 nm exclusively. All these newly synthesized compounds were screened for antifungal activity against Aspergillus niger and Aspergillus oryzae, respectively.     

Complexation Properties of a Carbon-Bridged Cryptand with Metal Ions in Aqueous Solution at 25°C

Abstract

Thermodynamic quantities (log K, ΔH, and ΔS) for the interactions of a carbon-bridged cryptand with Li⁺, Na⁺, K⁺, Ca²⁺, Sr²⁺, Ba²⁺, and Pb²⁺ were determined at 25° C by calorimetric titration in aqueous solution. The cryptand forms complexes with Na⁺, Sr²⁺, Ba²⁺, and Pb²⁺ with log K ≤ 2. Complexation was not detected for Li⁺, K⁺, and Ca²⁺. Weak interactions with Li⁺ and K⁺ and a log K value of 2.4 for Na⁺ suggest that the cavity size of the cryptand is close to that of Na⁺ but too small for K⁺ and too large for Li⁺. The carbon-bridged cryptand selectively binds Sr²⁺ (log K = 3.2) over Ca²⁺ and Ba²⁺ by more than one order of magnitude.     

Purification and Characterization of Extracellular Inulinase from a Marine Yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and Inulin Hydrolysis by the Purified Inulinase

The extracellular inulinase in the supernatant of the cell culture of the marine yeast Cryptococcus aureus G7a was purified to homogeneity with a 7.2-fold increase in specific inulinase activity compared to that in the supernatant by ultrafiltration, concentration, gel filtration chromatography (Sephadex™ G-75), and anion exchange chromatography (DEAE sepharose fast flow anion exchange). The molecular mass of the purified enzyme was estimated to be 60.0 kDa. The optimal pH and temperature of the purified enzyme were 5.0 and 50 °C, respectively. The enzyme was activated by Ca²⁺, K⁺, Na⁺, Fe²⁺, and Zn²⁺. However, Mg²⁺, Hg²⁺, and Ag⁺ acted as inhibitors in decreasing the activity of the purified inulinase. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, EDTA, and 1,10-phenanthroline. The K _m and V _max values of the purified enzyme for inulin were 20.06 mg/ml and 0.0085 mg/min, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin with the purified inulinase, indicating the purified inulinase had a high exoinulinase activity.     

HPLC micro-fractionation coupled to a cell-based assay for automated on-line primary screening of calcium antagonistic components in plant extracts

High performance liquid chromatography (HPLC) micro-fractionation was successfully coupled to an automated ⁴⁵Ca²⁺ uptake assay using GH₄C₁ cells for the separation of natural product extracts and for the primary detection of their calcium antagonistic components. The reliability of the procedure was first established with a reference solution consisting of pure compounds with a known effect on the Ca²⁺ uptake. No loss of activity was observed to occur after HPLC micro-fractionation. Extracts of Peucedanum palustre and Pinus sylvestris, showing high and no inhibition of Ca²⁺ uptake as total extracts, respectively, were analysed and the inhibitory activity of the P. palustre extract could be traced to two components, identified as columbianadin and isoimperatorin. As expected, no significant inhibition was observed with the micro-fractionated P. sylvestris samples. In summary, the procedure was found to be applicable for primary detection of calcium antagonistic components in complex matrices and to significantly reduce the time previously needed for bioactivity-guided isolation.     

Free Ca<Superscript>2+</Superscript> as an early intracellular biomarker of exposure of cyanobacteria to environmental pollution

Calcium functions as a versatile messenger in a wide variety of eukaryotic and prokaryotic cells. Cyanobacteria are photoautotrophs which have a great ecological impact as primary producers. Our research group has presented solid evidence of a role of calcium in the perception of environmental changes by cyanobacteria and their acclimation to these changes. We constructed a recombinant strain of the freshwater cyanobacterium Anabaena sp. PCC 7120 that constitutively expresses the calcium-binding photoprotein apoaequorin, enabling in-vivo monitoring of any fluctuation in the intracellular free calcium concentration of the cyanobacterium in response to any environmental stimulus. The “Ca²⁺ signature” is the combination of changes in all Ca²⁺ signal properties (magnitude, duration, frequency, source of the signal) produced by a specific stimulus. We recorded and analyzed the Ca²⁺ signatures generated by exposure of the cyanobacterium to different groups of environmental pollutants, for example cations, anions, organic solvents, naphthalene, and pharmaceuticals. We found that, in general, each group of tested chemicals triggered a specific calcium signature in a reproducible and dose-dependent manner. We hypothesize that these Ca²⁺ signals may be related to the cellular mechanisms of pollutant perception and ultimately to their toxic mode of action. We recorded Ca²⁺ signals triggered by binary mixtures of pollutants and a signal induced by a real wastewater sample which could be mimicked by mixing its main constituents. Because Ca²⁺ signatures were induced before toxicity was evident, we propose that intracellular free Ca²⁺ may serve as an early biomarker of exposure to environmental pollution.     

Antifungal activity of mango peel and seed extracts against clinically pathogenic and food spoilage yeasts

The antioxidant and antifungal (antiyeast) properties of mango (Mangifera indica) peel and seed by-products were investigated. Nine extracts were obtained using three cultivars and two extraction methods. Significant differences between cultivars and extraction methods were detected in their bioactive compounds and antioxidant activity. The antifungal property was determined using agar diffusion and broth micro-dilution assays against 18 yeast species of the genera Candida, Dekkera, Hanseniaspora, Lodderomyces, Metschnikowia, Pichia, Schizosaccharomyces, Saccharomycodes and Zygosaccharomyces. All mango extracts showed antifungal activity. The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) values were lower for seed than for peel extracts. MICs and MFCs ranged from values <0.1 to 5 and 5 to >30 mgGAE/mL, respectively. The multivariate analysis showed a relationship between antifungal activity, the capacity to inhibit lipid peroxidation and total phenol content. These properties were associated with high levels of proanthocyanidins, gallates and gallotannins in the extracts.     

Role of Calcium in Photodynamically Induced Cell Damage of Human Fibroblasts

Photodynamically induced changes in the cytoplasmic free calcium concentration ([Ca²⁺]_i) and its role in cell damage were investigated in human skin fibroblasts using confocal laser microscopy. Fluorescence and absorbance spectrophotometry measurements indicate that the photosensitizer aluminum phthalocyanine tetrasulfonate (AlPcS₄) binds to the plasma membrane and only after irradiation is able to enter the cells, causing massive morphologic alterations. Upon irradiation of sensitizer-treated cells, the increase in [Ca²⁺]_i is related to the amount of light and extracellular [Ca²⁺]_e. The increase in [Ca²⁺]_i was substantially reduced in the absence of [Ca²⁺]_e. Cell damage or death after photodynamic treatment was prevented and shifted toward higher fluence by increasing [Ca²⁺]_i at high [Ca²⁺]_e and was greater at low [Ca²⁺]_e. Application of Ca²⁺ channel blockers, such as Co²⁺, Cd²⁺ or verapamil, could not prevent the increase of [Ca²⁺]_i. Our results indicate that activation of the photosensitizer, AlPcS₄, causes an influx of Ca²⁺, which protects cells from photodamage. At low [Ca²⁺]_e and high fluence values, release of Ca²⁺ from internal stores probably as a protective measure occurs in order to increase the [Ca²⁺]_i.     

Release of Ca2+ and Mg2+ from yeast mitochondria is stimulated by increased ionic strength

Background  

Divalent cations are required for many essential functions of mitochondrial metabolism. Yet the transporters that mediate the flux of these molecules into and out of the mitochondrion remain largely unknown. Previous studies in yeast have led to the molecular identification of a component of the major mitochondrial electrophoretic Mg²⁺ uptake system in this organism as well as a functional mammalian homolog. Other yeast mitochondrial studies have led to the characterization of an equilibrative fatty acid-stimulated Ca²⁺ transport activity. To gain a deeper understanding of the regulation of mitochondrial divalent cation levels we further characterized the efflux of Ca²⁺ and Mg²⁺ from yeast mitochondria.     

Multicomponent Titration of Calcium + Magnesium Mixtures Employing a Potentiometric Electronic‐Tongue

Abstract

A fast novel potentiometric titration procedure is proposed, in which the detection system is formed by a potentiometric electronic tongue. The titration consists in a reduced number of fixed titrant additions to the sample and the recording of the potentials of an array of Ion Selective Electrodes. The obtained data matrix is entered to an Artificial Neural Network response model, previously trained to furnish concentrations of a multicomponent mixture. The principle is demonstrated with automated EDTA titration of mixtures of Ca²⁺ and Mg²⁺ at fixed pH 8.5. In this case, five 2 ml fixed volume additions plus the readings of four sensors were adequate for the resolution of the mixture. In the conditions used, titration was feasible up to 3.3 mM total ion concentration, the precision was estimated as 3.20% RSD for Ca²⁺ and 2.76% RSD for Mg²⁺ (n=5), and the detection limits 0.16 mM Ca²⁺ and 0.26 mM Mg²⁺. The procedure was applied to mineral waters and compared with reference methods (correlation=0.92 for Ca²⁺ and correlation=0.89 for Mg²⁺, n=14).     

PHOTOPHYSICS OF THE FLUORESCENT Ca2+ INDICATOR QUIN-2

The photophysics of the complex forming reaction between Quin-2 and Ca²⁺ were investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with EGTA as Ca²⁺ buffer: k₀₁= 8.6 times 10⁸ s^?1, k₂₁= 1 times 10¹¹M^?1 s^?1, k₀₂= 8.8 times 10⁷ s^?1, k₁₂= 4 times 10⁴ s^?1. k₀₁ and k₀₂ denote the respective deactivation rate constants of the Ca²⁺ free and bound forms of Quin-2 in the excited state. The constant k₂₁ represents the second-order rate constant of binding of Ca²⁺ and Quin-2 in the excited state while k₁₂ is the first-order rate constant of dissociation of the excited Ca²⁺:Quin-2 complex. From the estimated values of k₁₂ and k₂₁ the dissociation constant K_d* in the excited state was calculated. It was found that pK_d* (6.4) is slightly smaller than pK_d (7.2). There was no interference of the excited-state complex forming reaction with the determination of K_d. Intracellular Ca²⁺ concentrations can thus accurately be determined from fluorometric measurements using Quin-2 as Ca²⁺ indicator.     

Parametrization of calcium binding site in proteins and molecular dynamics simulation on phospholipase A2

The empirical energy parameters for a calcium ion and its ligands in proteins were determined within a pairwise additive framework. The interaction energies of Ca²⁺-water, Ca²⁺-peptide group and Ca²⁺-carboxyl group systems were calculated using the ab initio molecular orbital method with basis sets of double zeta quality including polarization or diffuse functions. The resulting potential energy surfaces served as references for the determination of the nonbonded parameters in the empirical energy function. The nonadditive corrections for the Ca²⁺-ligand pair potentials are incorporated implicitly in the nonbonded paremeters by treating three-body (1:2 complex) or seven-body (1:6 complex) systems in reference calculations. Ligand polarizations induced by Ca²⁺ are estimated from the partial atomic charges of two-body (1:1 complex) systems. The charge sets were determined by scaling so as to reproduce the reference potential energy surfaces. The newly determined parameter set was used in a stochastic boundary molecular dynamics simulation of phospholipase A₂. The solvated structure of the Ca²⁺-binding site obtained from an X-ray crystallographic study is well reproduced by the parameter set.     

Ca2+‐Induced Oxygen Generation by FeO42− at pH 9–10

Although FeO₄^2? (ferrate(IV)) is a very strong oxidant that readily oxidizes water in acidic medium, at pH 9–10 it is relatively stable (<2 % decomposition after 1 h at 298 K). However, FeO₄^2? is readily activated by Ca²⁺ at pH 9–10 to generate O₂. The reaction has the following rate law: d[O₂]/dt=k_Ca[Ca²⁺][FeO₄^2?]². ¹⁸O‐labeling experiments show that both O atoms in O₂ come from FeO₄^2?. These results together with DFT calculations suggest that the function of Ca²⁺ is to facilitate O–O coupling between two FeO₄^2‐ions by bridging them together. Similar activating effects are also observed with Mg²⁺ and Sr²⁺.     

Calix[4]arene-based fluorescent probe and the adsorption capacity of its electrospun nanofibrous film for the cesium cation as an adsorbent

Calix[4]arene-based cation receptor 1 has been synthesised by following a multi-step synthetic procedure. The fluorescence properties of 1 upon the addition of various metal ions were investigated by fluorescence spectroscopy. As a result, it was revealed that 1 displayed dramatic quenching effect upon the exposure to Cs⁺. In contrast, no significant quenching effects were observed upon the addition of other metal ions such as Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ag⁺, Zn²⁺ and Ni²⁺. Compound 1 was also found by Job plot to form a 1:1 complex with Cs⁺. In addition, we also prepared 1-embedded electrospun nanofibrous film (NF-1) as an adsorbent for Cs⁺. NF-1 is proved to adsorb Cs⁺ effectively from an aqueous solution, indicating that it would be usefully utilised as an adsorbent to remove Cs⁺.     

A Novel Aspect of Photodynamic Action: Induction of Recurrent Spikes in Cytosolic Calcium Concentration

Effects of photodynamic action of gadolinium porphyrin-like macrocycle B (PLMGdB) on cytosolic Ca²⁺ concentration, [Ca²⁺]_c, was investigated in isolated rat pancreatic acini. The PLMGdB alone or light alone (2 min) had no effect on [Ca²⁺]_c. Cell-bound PLMGdB upon brief (0.5–2.0 min) light activation triggered recurrent spikes in [Ca²⁺]_c. At lower PLMGdB concentration (100 nM) the spikes continued during the whole period of monitoring [Ca²⁺]_c. At a higher concentration of 500 nM, the spikes continued for the first 40 min, followed by a gradual increase in basal [Ca²⁺]_c upon which smaller spikes were superimposed. At 1, the spikes continued for the first 20 min, after that spiking gradually degenerated into a plateau phase. In many aspects, photodynamically triggered spikes resembled spikes generated by physiological concentrations of cholecystokinin. The spikes triggered by photodynamic action were likely to be the result of the ignition of a physiological “chain reaction”, because functional inositol-1,4,5-trisphosphate (IP₃) receptors were required for spiking to occur. Two-aminoe-thoxydiphenylborate, an inhibitory modulator of IP₃-triggered Ca²⁺ release from intracellular stores, effectively inhibited photodynamically generated spikes. Therefore photodynamic action appears to be able to permanently transfix a physiological process, leading to long-lasting pharmacological or therapeutic effects.     

Alendronate zwitterions bind to calcium cations arranged in columns

Alendronate is used clinically in the treatment of skeletal disorders, the mode of action depending on the adsorption to calcium hydroxy­apatite crystals (bone). In the title compound, calcium 4‐ammonium‐1‐hydroxy­butyl­idene‐1,1‐bis­phospho­n­ate, Ca²⁺·2C₄H₁₂NO₇P₂⁻, alendronate is a zwitterion, possessing one negative charge on each PO₃ group and a protonated N atom. The zwitterion is disposed with its negative end facing the Ca²⁺ ion, while its positive end is stretched in the opposite direction. The geometry of the carbon chain is all‐trans, while the hydroxy group is approximately gauche. The Ca²⁺ ion lies on a twofold axis parallel to b. The coordination sphere around the metal cation is octahedral and is determined by monodentate‐ and bidentate‐coordinated alendronate zwitterions. The O⋯O bite distance is 3.080 (2) Å. Coordinated Ca²⁺ metal cations are arranged at the centre of a column running along c.     

A Thermodynamic Study Between 18-Crown-6 with Mg2+, Ca2+, Sr2+and Ba2+ Cations in Water–Methanol and Water–Ethanol Binary Mixtures Using The Conductometric Method

The complexation reactions between Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺ cations with the macrocyclic ligand, 18-Crown-6 (l8C6) in water–methanol (MeOH) binary systems as well as the complexation reactions between Ca²⁺ and Sr²⁺ cations with 18C6 in water–ethanol (EtOH) binary mixtures have been studied at different temperatures using conductometric method. The conductance data show that the stoichiometry of all the complexes is 1:1. It was found that the stability of 18C6 complexes with Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺ cations is sensitive to solvent composition and in all cases, a non-linear behaviour was observed for the variation of log K _f of the complexes versus the composition of the mixed solvents. In some cases, the stability order is changed with changing the composition of the mixed solvents. The selectivity order of 18C6 for the metal cations in pure methanol is: Ba²⁺ > Sr²⁺ > Ca²⁺ > Mg²⁺. The values of thermodynamic parameters (Δ H _c ° and Δ S _c °) for formation of 18C6–Mg²⁺, 18C6–Ca²⁺, 18C6–Sr²⁺ and 18C6–Ba²⁺complexes were obtained from temperature dependence of the stability constants. The obtained results show that the values of (Δ H _c ° and Δ S _c °) for formation of these complexes are quite sensitive to the nature and composition of the mixed solvent, but they do not vary monotonically with the solvent composition.This revised version was published online in July 2005 with a corrected issue number.     

The crystal and magnetic structures of Ca2NdRuO6, Ca2HoRuO6, and Sr2ErRuO6

The crystal structure of Sr₂ErRuO₆ has been refined from neutron powder diffraction data collected at room temperature; space group P2₁/n, A = 5.7626(2), B = 5.7681(2), C = 8.1489(2) Å, β = 90.19(1)°. The structure is that of a distorted perovskite with a 1:1 ordered arrangement of Ru⁵⁺ and Er³⁺ over the 6-coordinate sites. Data collected at 4.2 K show the presence of long range antiferromagnetic order involving both Ru⁵⁺ and Er³⁺. The temperature dependence of the sublattice magnetizations is described. The crystal structure of Ca₂NdRuO₆ is also that of a distored perovskite (P2₁/n, A = 5.5564(1), B = 5.8296(1), C = 8.0085(1) β = 90.19(1)°. The β = 90.07(1)°) with a random distribution of Ca²⁺ and Nd³⁺ on the A site and a 1:1 ordered arrangement of Ca²⁺ and Ru⁵⁺ on the 6-coordinate B sites. The Ru⁵⁺ sublattice is antiferromagnetic at 4.2 K but there is no evidence for magnetic ordering of the Nd³⁺ ions. Ca₂HoRuO₆ is also a distorted perovskite (P2₁/n, A = 5.4991(1), B = 5.7725(1), C = 7.9381(2), β = 90.18(1)° at 4.2 K) with a cation distribution best represented as Ca_1.46Ho_0.54[Ca_0.54Ho_0.46Ru]O₆. There is no ordering among the Ca³⁺ or Ho³⁺ ions on either the A or the B sites, but the Ca/Ho ions form a 1:1 ordered arrangement with Ru⁵⁺ on the B sites. At 4.2 K the Ru⁵⁺ ions adopt a Type I antiferromagnetic arrangement but there is no evidence of long range magnetic ordering among the Ho³⁺ ions.     

Direct Synthesis of Nanocrystalline Hydroxyapatite by Hydrothermal Hydrolysis of Alkylphosphates

Summary.  The influence of reaction conditions (temperature, type of catalyst, time) on the base-catalyzed reaction of mono-, di-, and trialkylphosphates (alkyl = methyl, ethyl, i-propyl, n-butyl) with Ca²⁺ ions and on the structure and composition of the reaction products was studied. The composition of the calcium phosphates depends mainly on the reaction temperature. At temperatures below 100°C, a nanocrystalline solid product transforming into dicalcium phosphate by heating (calcination) was found. Pure nanocrystalline hydroxyapatite was prepared by hydrothermal synthesis at 160°C from mono- and dialkylphosphates. The size of hydroxyapatite crystallites was about 1 nm, the particle size about 150 nm. Agglomerated particles of hydroxyapatite larger than 2 μm were prepared at 200°C. Hydrothermal reaction of trialkylphosphates with Ca²⁺ ions at 200°C produced CaHPO₄. The experimental results were used to propose a reaction mechanism for base-catalyzed hydrothermal reactions of alkylphosphates with Ca²⁺ ions. Received October 4, 2001. Accepted (revised) November 19, 2001