β<Subscript>2</Subscript>-Integrin-Mediated Adhesion and Intracellular Ca<Superscript>2+</Superscript> Release in Human Eosinophils

Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO₃-buffered HybriCare medium maintained in a humidified 5% CO₂ incubator at 37°C. Impedance increased by more than 1 kΩ within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca²⁺] that precedes adhesion with BAPTA-AM (10 μM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca²⁺] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with β₂-integrins, or jasplakinolide (2 μM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 μM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca²⁺] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion.     

PA1b,a plant peptide,induces intracellular [Ca<Superscript>2+</Superscript>] increase via Ca<Superscript>2+</Superscript> influx through the L-type Ca<Superscript>2+</Superscript> channel and triggers secretion in pancreatic β cells

Using alginic acid to adsorb polypeptides at pH 2.7, we isolated a peptide pea albumin 1b (PA1b) from pea seeds. The PA1b is a single chain peptide consisting of 37 amino acid residues with 6 cysteines which constitutes the cystine-knot structure. Using microfluorometry and patch clamp techniques, we found that PA1b significantly elevated the intracellular calcium level ([Ca²⁺]_i) and elicited membrane capacitance increase in the primary rat pancreatic β cells. The PA1b effect on [Ca²⁺]_i elevation was abolished in the absence of extracellular Ca²⁺ or in the presence of L-type Ca²⁺ channel blocker, nimodipine. Interestingly, we found that PA1b significantly depolarized membrane potential, which could lead to the opening of voltage-dependent L-type Ca²⁺ channels and influx of extracellular Ca²⁺, and then evoke robust secretion. In this study we identified the plant peptide PA1b which is capable of affecting the excitability and function of mammalian pancreatic β cell.     

Ca<Superscript>2+</Superscript>-dependent aggregation and permeabilization of erythrocytes by ω-hydroxypalmitic and α, ω-hexadecandioic acids

This paper presents the data and describes the Ca²⁺-dependent effect of the products of ω-oxidation of palmitic acid, as well as ω-hydroxypalmitic and α, ω-hexadecandioic acids, on rat erythrocytes. It is shown that in the presence of Ca²⁺ these acids induce aggregation of erythrocytes, which is accompanied by a reduction in the number of single cells in suspension. As well, a release of K⁺ from the cells occurs, which indicates the permeabilization of the plasma membrane. However, ω-hydroxypalmitic and α, ω-hexadecandioic acids are inferior to palmitic acid in their ability to induce Ca²⁺-dependent erythrocyte permeabilization. Bovine serum albumin and blood serum inhibit the effects of palmitic acid. At the same time, the influence of these agents on the effects of ω-hydroxypalmitic and α, ω-hexadecandioic acids appears to be much weaker. It is shown that ω-hydroxypalmitic and α, ω-hexadecandioic acids in the presence of Ca²⁺ induce an increase in the hydrodynamic diameter of single-walled lecithin liposomes, which indicates their fusion and (or) aggregation. The mechanisms of ω-hydroxypalmitic acid/Ca²⁺- and α, ω-hexadecandioic acid/Ca²⁺-induced effects on rat erythrocytes are discussed.     

Effect of different methods of Ca<Superscript>2+</Superscript> extraction from PSII oxygen-evolving complex on the Q<Subscript>A</Subscript><Superscript>−</Superscript> oxidation kinetics

Lumenal extrinsic proteins PsbO, PsbP, and PsbQ of photosystem II (PSII) protect the catalytic cluster Mn₄CaO₅ of oxygen-evolving complex (OEC) from the bulk solution and from soluble compounds in the surrounding medium. Extraction of PsbP and PsbQ proteins by NaCl-washing together with chelator EGTA is followed also by the depletion of Ca²⁺ cation from OEC. In this study, the effects of PsbP and PsbQ proteins, as well as Ca²⁺ extraction from OEC on the kinetics of the reduced primary electron acceptor (Q_A ^?) oxidation, have been studied by fluorescence decay kinetics measurements in PSII membrane fragments. We found that in addition to the impairment of OEC, removal of PsbP and PsbQ significantly slows the rate of electron transfer from Q_A ^? to the secondary quinone acceptor Q_B. Electron transfer from Q_A ^? to Q_B in photosystem II membranes with an occupied Q_B site was slowed down by a factor of 8. However, addition of EGTA or CaCl₂ to NaCl-washed PSII did not change the kinetics of fluorescence decay. Moreover, the kinetics of Q_A ^? oxidation by Q_B in Ca-depleted PSII membranes obtained by treatment with citrate buffer at pH 3.0 (such treatment keeps all extrinsic proteins in PSII but extracts Ca²⁺ from OEC) was not changed. The results obtained indicate that the effect of NaCl-washing on the Q_A ^? to Q_B electron transport is due to PsbP and PsbQ extrinsic proteins extraction, but not due to Ca²⁺ depletion.     

The cluster [Re<Subscript>6</Subscript>Se<Subscript>8</Subscript>I<Subscript>6</Subscript>]<Superscript>3−</Superscript> penetrates biological membranes: drug-like properties for CNS tumor treatment and diagnosis

Tumorigenic cell lines are more susceptible to [Re₆Se₈I₆]^3? cluster-induced death than normal cells, becoming a novel candidate for cancer treatment. Still, the feasibility of using this type of molecules in human patients remains unclear and further pharmacokinetics analysis is needed. Using coupled plasma optical emission spectroscopy, we determined the Re-cluster tissue content in injected mice, as a biodistribution measurement. Our results show that the Re-cluster successfully reaches different tissues, accumulating mainly in heart and liver. In order to dissect the mechanism underlying cluster biodistribution, we used three different experimental approaches. First, we evaluate the degree of lipophilicity by determining the octanol/water partition coefficient. The cluster mostly remained in the octanol fraction, with a coefficient of 1.86?±?0.02, which indicates it could potentially cross cell membranes. Then, we measured the biological membrane penetration through a parallel artificial membrane permeability assays (PAMPA) assay. The Re-cluster crosses the artificial membrane, with a coefficient of 122 nm/s that is considered highly permeable. To evaluate a potential application of the Re-cluster in central nervous system (CNS) tumors, we analyzed the cluster’s brain penetration by exposing cultured blood–brain-barrier (BBB) cells to increasing concentrations of the cluster. The Re-cluster effectively penetrates the BBB, reaching nearly 30% of the brain side after 24 h. Thus, our results indicate that the Re-cluster penetrates biological membranes reaching different target organs—most probably due to its lipophilic properties—becoming a promising anti-cancer drug with high potential for CNS cancer’s diagnosis and treatment.     

Effects of Calmodulin and Ca<Superscript>2+</Superscript> Channel Blockers on ω-conotoxin GVI A Binding to Crude Membranes from α<Subscript>1B</Subscript> Subunit (Ca<Subscript>v</Subscript>2.2) Expressed BHK Cells and Mice Brain Lacking the α<Subscript>1B</Subscript> Subunits

Characteristics for the specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to crude membranes from BHKN101 cells expressing the α_1B subunits of Ca_v2.2 channels and from mice brain lacking the α_1B subunits of Ca_v2.2 channels, particularly, the effects of CaM and various Ca²⁺ channel blockers on these specific bindings were investigated. Specific binding of ¹²⁵I-ω-CTX GVIA to the crude membranes from BHKN101 cells was observed, but not from control BHK6 cells. ω-CTX GVIA, ω-CTX MVIIC and ω-CTX SVIB inhibited the specific binding of ¹²⁵I-ω-CTX GVIA to crude membranes from BHKN101 cells, and the IC₅₀ values for ω-CTXGVIA, ω-CTX MVIIC and ω-CTX SVIB were 0.07, 8.5 and 1.7 nM, respectively. However, ω-agatoxin IVA and calciseptine at concentrations of 10⁻⁹–10⁻⁶ M did not inhibit specific binding. Specific binding was also about 80% inhibited by 20 μg protein/ml CaM. The amount of ¹²⁵I-ω-CTX GVIA (30 pM) specifically bound to membranes from brain of knockout mice lacking α_1B subunits of Ca_v2.2 channels was about 30% of that to the crude membranes from brain of wild-type. On the other hand, specific binding of ¹²⁵I-ω-CTX MVIIC (200 pM) was observed on the crude membranes of both BHKN101 and control BHK6 cells. The specific binding of ¹²⁵I-ω-CTX MVIIC (200 pM) was not inhibited by ω-CTX GVIA and ω-CTX SVIB, and also ω-Aga IVA and calciseptine at concentrations of 10⁻⁹–10⁻⁷ M, although specific binding was almost completely dose dependently inhibited by non-radiolabeled ω-CTX MVIIC (IC₅₀ value was about 0.1 nM). 20 μg protein/ml CaM did not inhibit specific binding. Therefore, these results suggest that BHKN101 cells have a typical Ca_v2.2 channels which are also inhibited by CaM and have not specific binding sites for ω-CTX MVIIC, although ω-CTX MVIIC is a blocker for both Ca_v2.1 (α_1A; P/Q-type) and Ca_v2.2 channels.     

Different effects of SNP and GSNO on mitochondrial O<Stack><Subscript>2</Subscript><Superscript>.−</Superscript></Stack>/H<Subscript>2</Subscript>O<Subscript>2</Subscript> production

Sodium Nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) differently affect mitochondrial H₂O₂ release at Complex-I. mM SNP increases while GSNO decreases the release induced by succinate alone or added on top of NAD-linked substrates. Stimulation likely depends on Nitric Oxide ( ^. NO) (released by SNP but not by GSNO) inhibiting cytochrome oxidase and mitochondrial respiration. Preincubations with SNP or high GSNO (10 mM plus DTE to increases its ^. NO release) induces an inhibition of the succinate dependent H₂O₂ production consistent with a ^. NO dependent covalent modification. However maximal inhibition of the succinate dependent H₂O₂ release is obtained in the presence of low GSNO (20–100 μM), but not with SNP. This inhibition appears independent of ^. NO release since μM GSNO does not affect mitochondrial respiration, or the H₂O₂ detection systems and its effect is very rapid. Inhibition may be partly due to an increased removal of O₂^.− since GSNO chemically competes with NBT and cytochrome C in O₂^.− detection.     

Membranotropic effects of ω-hydroxypalmitic acid and Ca<Superscript>2+</Superscript> on rat liver mitochondria and lecithin liposomes. Aggregation and membrane permeabilization

The paper examines membranotropic Ca²⁺-dependent effects of ω-hydroxypalmitic acid (HPA), a product of ω-oxidation of fatty acids, on the isolated rat liver mitochondria and artificial membrane systems (liposomes). It was established that in the presence of Ca²⁺, HPA induced aggregation of liver mitochondria, which was accompanied by the release of cytochrome c from the organelles. It was further demonstrated that the addition of Ca²⁺ to HPA-containing liposomes induced their aggregation and/or fusion. Ca²⁺ also caused the release of the fluorescent dye sulforhodamine B from liposomes, indicating their permeabilization. HPA was shown to induce a high-amplitude swelling of Ca²⁺-loaded mitochondria, to decrease their membrane potential, to induce the release of Ca²⁺ from the organelles and to result in the oxidation of the mitochondrial NAD(P)H pool. Those effects of HPA were not blocked by the MPT pore inhibitor CsA, but were suppressed by the mitochondrial calcium uniporter inhibitor ruthenium red. The effects of HPA were also observed when Ca²⁺ was replaced with Sr²⁺ (but not with Ba²⁺ or Mg²⁺). A supposition is made that HPA can induce a Ca²⁺-dependent aggregation of mitochondria, as well as Ca²⁺dependent CsA-insensitive permeabilization of the inner mitochondrial membrane – with the subsequent lysis of the organelles.     

Tunable paramagnetic relaxation enhancements by [Gd(DPA)<Subscript>3</Subscript>]<Superscript>3−</Superscript> for protein structure analysis

Paramagnetic relaxation enhancements (PRE) present a powerful source of structural information in nuclear magnetic resonance (NMR) studies of proteins and protein–ligand complexes. In contrast to conventional PRE reagents that are covalently attached to the protein, the complex between gadolinium and three dipicolinic acid (DPA) molecules, [Gd(DPA)₃]^3?, can bind to proteins in a non-covalent yet site-specific manner. This offers straightforward access to PREs that can be scaled by using different ratios of [Gd(DPA)₃]^3? to protein, allowing quantitative distance measurements for nuclear spins within about 15 Å of the Gd³⁺ ion. Such data accurately define the metal position relative to the protein, greatly enhancing the interpretation of pseudocontact shifts induced by [Ln(DPA)₃]^3? complexes of paramagnetic lanthanide (Ln³⁺) ions other than gadolinium. As an example we studied the quaternary structure of the homodimeric GCN4 leucine zipper.     

Effect of NO<Subscript>3</Subscript><Superscript>−</Superscript>:NH<Subscript>4</Subscript><Superscript>+</Superscript> ratios on growth,root morphology and leaf metabolism of oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) seedlings

The effect of NO₃ ^?:NH₄ ⁺ ratio (14:1, 9:6, 7.5:7.5, 1:14, total 15 mmol/L N) in the nutrient solution on biomass, root morphology, and C and N metabolism parameter in hydroponically grown oilseed rape (Brassica napus L.) was evaluated. The dry weights of leaves and roots were significantly largest at the equal NO₃ ^?:NH₄ ⁺ ratio (7.5:7.5) compared with those of high NO₃ ^?:NH₄ ⁺ ratio (14:1) or low NO₃ ^?:NH₄ ⁺ ratio (1:14). Additionally, low NO₃ ^?:NH₄ ⁺ ratio (1:14) reduced total root length and root surface area compared with the equal NO₃ ^?:NH₄ ⁺ ratio (7.5:7.5), while high NO₃ ^?:NH₄ ⁺ ratio (14:1) did not show any significant effect on root morphology except average diameter. The maximum of chlorophyll a, chlorophyll b and carotenoid were obtained under 7.5:7.5 treatment, whereas the maximum of the leaf net photosynthetic (P _n), stomatal conductance (G _s) and transpiration rate (T _r) were increased with increase in NH₄ ⁺ concentration in the nutrient solution. The activity of nitrate reductase (NR) showed a significant difference at different NO₃ ^?:NH₄ ⁺ ratios and ranged 9:6 > 7.5:7.5 > 14:1 > 1:14, whereas the range of soluble sugar and soluble protein was 7.5:7.5 > 1:14 > 9:6 > 14:1. Our study reveals that oilseed rape growth is greater under 7.5:7.5 treatment than that under three other treatments. Oilseed rape growth at high or low NO₃ ^?:NH₄ ⁺ ratios was inhibited by decreased pigments, NR activity, soluble sugar, and soluble protein, whereas subdued root growth should be apprehended considerate under high NH₄ ⁺ condition.     

The involvement of Ca<Superscript>2+</Superscript> gradients,Ca<Superscript>2+</Superscript> fluxes,and CaM kinase II in polarization and germination of<Emphasis Type="Italic"> Silvetia compressa</Emphasis> zygotes

Previous work has shown that distinct Ca²⁺ gradients precede and predict the loci of germination of the zygotes of the brown alga, Silvetia compressa (J. Agardh) E. Serrão, T.O. Cho, S.M. Boo et Brawley, that are polarized by unilateral blue light. We show here that dark-grown S. compressa zygotes also form cytosolic Ca²⁺ gradients prior to germination and then germinate from the site of elevated Ca²⁺. In no case did germination occur without a prior formation of a Ca²⁺ gradient. Using the self-referencing Ca²⁺-selective probe, we measured highly localized influx of Ca²⁺ during photopolarization, indicating that extracellular stores supply at least some of the Ca²⁺ needed to construct a gradient. Finally, we find that germination was inhibited by a bath-applied inhibitor of calcium/calmodulin-dependent kinase II (CaM kinase II), KN-93 (but not by its inactive analog, KN-92), and by an injected inhibitory peptide for the kinase. KN-93 did not interfere with the photopolarization of the zygotes, consistent with the view that calmodulin is not involved in the initial response to light. The KN-93 results indicate that the requirement for active CaM kinase II for germination ends about 2 h before overt germination. We conclude that Ca²⁺ gradients, generated in part by localized calcium entry from the seawater, are an essential part of the process of polarity development and expression in these cells, regardless of the nature of the external cue that directs the orientation of the axis. Calmodulin and CaM kinase II are involved in interpreting (but not in establishing) the calcium gradient, allowing germination to occur at the site of elevated calcium, but CaM kinase II appears not to be involved in the initiation of germination.     

Expression and Developmental Regulation of the Cystine/Glutamate Exchanger (x<Stack><Subscript>c</Subscript><Superscript>−</Superscript></Stack>) in the Rat

The cystine/glutamate exchanger (antiporter x_c⁻) is a membrane transporter involved in the uptake of cystine, the rate-limiting amino acid in the synthesis of glutathione. Recent studies suggest that the antiporter plays a role in the slow oxidative excitotoxity and in the pathological effects of β-N-oxalylamino-l-alanine, the molecule responsible for neurolathyrism, a neurotoxic upper motor neuron disease. The mouse cystine/glutamate exchanger has been cloned and showed to be composed of two distinct proteins, one of which being a novel protein, named xCT, of 502 amino acids and 12 putative trans-membrane domains. We have generated and purified a polyclonal antibody to mouse xCT and studied its expression in rat brain and in different cultured cells (astrocytes, fibroblasts and neurons) using Western blot and immunocytochemical techniques. Expression of xCT was also studied in rat brain and muscle at different developmental stages. Parallel experiments were carried out with antibodies to the heavy chain of 4F2 surface antigen, the non-specific subunit of the antiporter x_c⁻. xCT antibody detected in all cell and tissue extracts a specific band of about 40 kDa. Subcellular fractionation demonstrated that xCT is concentrated mainly in the microsomal-mitochondrial fraction, in accord with its structure as transmembrane protein. Immunocytochemical analysis showed a strong staining in all cells examined, included neurons. Furthermore, both xCT and the heavy chain of 4F2 surface antigen increased in the brain during development, reaching the highest expression in adulthood. The study of the expression and developmental profile of xCT represents a first step towards a better characterization of its biochemical properties and function, which in turn may help to understand the relative contribution of the x_c⁻ antiporter in the pathogenesis of certain neurodegenerative diseases.     

Volume-dependent Glutamate Permeation Depends on Transmembrane Ionic Strength and Extracellular Cl<Superscript>−</Superscript>

Many mammalian cells regulate their volume by the osmotic movement of water directed by anion and cation flux. Ubiquitous volume-dependent anion currents permit cells to recover volume after swelling in response to a hypotonic environment. This study addressed competition between glutamate (Glu) and Cl^– permeation in volume-activated anion currents in order to provide insight into the ionic requirements for volume regulation, volume-dependent anion channel activity and to the architecture of the channel pore. The effect of changing the intracellular molar fraction (MF) of Glu and Cl^– on conductance and relative anion permeability was evaluated as a function of the extracellular permeant anion and/or the ionic strength. Relative permeability of Glu to Cl^– was determined by measuring reversal potentials under defined ionic conditions. Under conditions with high (150 mM) or low (50 mM) ionic strength solutions on both sides of the membrane, Cl^– was always more permeable than Glu. When a transmembrane ionic strength gradient (150 mM extracellular: 50 mM intracellular) was set to drive water into the cell, and in the presence of extracellular Cl^–, Glu became up to 16-fold more permeable than Cl^–. Replacement of extracellular Cl^– with Glu abolished this effect. These results indicate that it is possible for Glu to move into the extracellular environment during volume-regulatory events and they support the emerging role of glutamate as a modulator of anion channel activity.     

Molecular Weight and Subunit Composition of the Sensitive to GABA-Ergic Ligands Cl<Superscript>−</Superscript>/HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>-Stimulated Mg<Superscript>2+</Superscript>-ATPase from Plasma Membrane of Rat Brain

The molecular weight and subunit composition of Cl-,HCO3(-)- and picrotoxin-stimulated Mg2+-ATPase from rat brain plasma membrane solubilized in sodium deoxycholate were studied by gel filtration chromatography. The enzyme activity eluted from a Sephacryl S-300 column in a single peak associated with a protein of molecular weight approximately 300 kD and a Stokes radius of 5.4 nm. The enzyme-enriched fraction, concentrated and denatured by SDS, migrated through a Sephacryl S-200 column as three peaks with molecular weights of approximately 57, 53, and 45 kD. SDS-PAGE also showed three major protein bands with molecular weights of about 57, 53, and 48 kD. The molecular weight and subunit composition of the Cl- and HCO3(-)-stimulated Mg2+-ATPase from neuronal membrane of rat brain are similar with the molecular properties of GABA(A)-benzodiazepine receptor complex from mammalian brain but are different from those of P-type transport ATPases.     

Effect of Vitamin В<Subscript>1</Subscript> Alimentary Deficiency on Spontaneous and Evoked Transmitter Release in Murine Neuromuscular Synapses

Using a microelectrode technique, we studied the effects of alimentary vitamin В₁ deficiency on synaptic transmission in isolated phrenico-hemidiaphragmatic murine preparations. Animals of group І (control) were on a standard thiamine-controlled diet (16 mg/kg thiamine) with no limitations. Animals of group II (control with alimentary limitation) were on the same diet, but daily consumption in these animals was limited and made similar to the amount of food consumed by the animals of group ІІІ within idential periods of cage housing (for differentiation of the effects of anorexia related to the thiamine-deficient state in group III and proper effects of В₁ hypovitaminosis). Animals of group ІІІ (thiamine-deficient) were on a standard diet (with no limitations) mostly analogous to that in group І but containing no thiamine. In phrenicohemidiaphragmatic preparations obtained from animals of group ІІІ, the amplitude of end-plate potentials (EPPs) and miniature EPPs (mEPPs) on the 10th day of consumption of the thiamine-defficient diet and the quantum composition of EPPs on the 20th day became significantly (Р < 0.01) smaller than in preparations obtained from animals of both groups І and ІІ. The frequency of mEPPs and membrane potential of muscle fibers in group ІІІ remained unchanged. Two processes, a decrease in the dimension of the transmitter quantum (which is observed within rather early stages of the development of thiamine-defficient state) and a decrease in the quantum composition of evoked EPPs (at later stages) underlie a gradual decrease in the amplitude of EPPs related to the development of alimentary vitamin В₁ deficiency. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 322–331, July–August, 2008.     

The Protective Effect of Mitochondrial ATP-Sensitive K<Superscript>+</Superscript> Channel Opener,Nicorandil, Combined With Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> Exchange Blocker KB-R7943 on Myocardial Ischemia–Reperfusion Injury in Rat

To study the protective effect of mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP channel) opener, nicorandil, combined with Na⁺/Ca²⁺ exchange blocker KB-R7943 on myocardial ischemia–reperfusion injury in isolated rat hearts; the isolated rat heart was perfused by modified Langendorff device, after 15-min balanced perfusion, 45-min ischemia (about left and right coronary perfusion flow reduced to 5% of the original irrigation flow), and 2-h reperfusion were performed. Forty Wistar rats were randomly divided into four groups: control group, nicorandil group, KB-R7943 group, and the combination of nicorandil and KB-R7943 group. After 45-min ischemia and then 2-h reperfusion, the myocardial infarct size was 34.31% in control group, 26.35% in nicorandil group, 28.74% in KB-R7943 group, and 19.23% in combination of nicorandil and KB-R7943 group. SOD activity in coronary perfusion fluid was the highest in the combination of nicorandil and KB-R7943 group, and MDA content was the lowest. In the combination drug group compared with the control group, myocardial ultrastructural injury was significantly reduced. The combination of nicorandil and KB-R7943 significantly reduced myocardial infarct size, significantly reduced myocardial ultrastructural damage, could increase coronary perfusion fluid SOD activity, and reduced MDA levels.     

The effect of MII α-conotoxin and its N-terminal derivatives on Ca<Superscript>2+</Superscript>- and Na<Superscript>+</Superscript>-signals induced by nicotine in SH-SY5Y neuroblastoma cells

Nicotinic acetylcholine receptors (nAChRs) are involved in the regulation of intracellular Ca²⁺-dependent processes both in normal and pathological states. α-Conotoxins from the venom of Conus marine mollusks are a valuable tool for the investigation of the pharmacological action and functional role of nAChRs. Analogues of α-conotoxin MII labeled by Bolton-Hunter reagent (BH-MII) or fluorescein isothiocyanate (FITC-MII) on the N-terminal glycine residue have been synthesized in the present work. Fluorescence microscopy studies of SH-SY5Y neuroblastoma cells loaded with Ca²⁺ indicator Fura-2, or by both Ca²⁺ indicator Fluo-4 and Na⁺ indicator SBFI, were used to test the effect of MII modification on its ability to block Ca²⁺ and Na⁺ signals induced by nicotine. Measurements in SH-SY5Y cells showed that kinetics of the increase and recovery of the concentration of free Ca²⁺ ([Ca²⁺] _i ) upon nicotine application and washout was different from that for free Na⁺ ([Na⁺] _i ), this being evidence of differences in the mechanism of Ca²⁺ and Na⁺ homeostasis regulation. MII suppressed the nicotine-induced increase of [Ca²⁺] _i and [Na⁺] _i in a concentration-dependent manner. An additional tyrosine residue added to the N-terminus of one of the MII derivatives caused a significant decrease in the inhibitory action of MII; this decrease was even more pronounced when a large FITC label was introduced into MII. The BH-MII derivative had an inhibitory effect similar to that of unmodified α-conotoxin. MII and its iodinated derivatives are promising tools for radioligand assays.     

Singlet molecular oxygen [O<Subscript>2</Subscript>(<Superscript>1</Superscript>Δ<Subscript>g</Subscript>)]-mediated photodegradation of tyrosine derivatives in the presence of cationic and neutral micellar systems

The kinetics of rose bengal-sensitized photooxidation of tyrosine and several tyrosine-derivatives (tyr-D) named tyrosine methyl ester, tyrosine ethyl ester and tyrosine benzyl ester was studied in buffered pH 11 water, and buffered pH 11 micellar aqueous solutions of 0.01 M cetyltrimethylammonium chloride (CTAC) and 0.01 M-octylphenoxypolyethoxyethanol [triton X100 (TX100)]. Through time-resolved phosphorescence detection of singlet molecular oxygen (O(2)((1)Delta(g))) and polarographic determination of oxygen consumption, the respective bimolecular rate constants for reactive (k(r)) and overall (k(t)) quenching of the oxidative species by tyr-D were evaluated. Both rate constants behave in different fashion depending on the particular reaction medium. k(r)/k(t) values, increase in the sense CTAC相似文献   

Methyl-β-cyclodextrin inhibits Ca<Superscript>2+</Superscript>-responses induced by glutoxim and molixan in macrophages

Using Fura-2AM microfluorimetry, we have shown for the first time that methyl-β-cyclodextrin, inducing cholesterol extraction from membranes and raft disruption, significantly inhibits glutoxim- and molixan-induced Ca²⁺-responses in rat peritoneal macrophages. The results suggest that intact rafts are necessary for signaling cascade induced by glutoxim or molixan and leading to intracellular Ca²⁺ concentration increase in macrophages.