Alexey V. Agafonov

Ca2+-Induced Phase Separation in the Membrane of Palmitate-Containing Liposomes and Its Possible Relation to Membrane Permeabilization

A Ca2+-induced phase separation of palmitic acid (PA) in the membrane of azolectin unilamellar liposomes has been demonstrated with the fluorescent membrane probe nonyl acridine orange (NAO). It has been shown that NAO, whose fluorescence in liposomal membranes is quenched in a concentration-dependent way, can be used to monitor changes in the volume of lipid phase. The incorporation of PA into NAO-labeled liposomes increased fluorescence corresponding to the expansion of membrane. After subsequent addition of Ca2+, fluorescence decreased, which indicated separation of PA/Ca2+ complexes into distinct membrane domains. The Ca2+-induced phase separation of PA was further studied in relation to membrane permeabilization caused by Ca2+ in the PA-containing liposomes. A supposition was made that the mechanism of PA/Ca2+-induced membrane permeabilization relates to the initial stage of Ca2+-induced phase separation of PA and can be considered as formation of fast-tightening lipid pores due to chemotropic phase transition in the lipid bilayer.

Ca(2+)-dependent permeabilization of mitochondria and liposomes by palmitic and oleic acids: a comparative study.

In the present work, we examine and compare the effects of saturated (palmitic) and unsaturated (oleic) fatty acids in relation to their ability to cause the Ca(2+)-dependent membrane permeabilization. The results obtained can be summarized as follows. (1) Oleic acid (OA) permeabilizes liposomal membranes at much higher concentrations of Ca(2+) than palmitic acid (PA): 1mM versus 100μM respectively. (2) The OA/Ca(2+)-induced permeabilization of liposomes is not accompanied by changes in the phase state of lipid bilayer, in contrast to what is observed with PA and Ca(2+). (3) The addition of Ca(2+) to the PA-containing vesicles does not change their size; in the case of OA, it leads to the appearance of larger and smaller vesicles, with larger vesicles dominating. This can be interpreted as a result of fusion and fission of liposomes. (4) Like PA, OA is able to induce a Ca(2+)-dependent high-amplitude swelling of mitochondria, yet it requires higher concentrations of Ca(2+) (30 and 100μM for PA and OA respectively). (5) In contrast to PA, OA is unable to cause the Ca(2+)-dependent high-amplitude swelling of mitoplasts, suggesting that the cause of OA/Ca(2+)-induced permeability transition in mitochondria may be the fusion of the inner and outer mitochondrial membranes. (6) The presence of OA enhances PA/Ca(2+)-induced permeabilization of liposomes and mitochondria. The paper discusses possible mechanisms of PA/Ca(2+)- and OA/Ca(2+)-induced membrane permeabilization, the probability of these mechanisms to be realized in the cell, and their possible physiological role.

Light-induced reversible local fusions of thylakoid membranes in the presence of dibucaine or tetracaine.

The dynamics of structural changes in pea chloroplasts in the presence of 25-50 microM dibucaine or tetracaine has been examined using electron microscopy. The light-induced uptake of anesthetic cations by thylakoids is attended by the appearance of local fusions of stroma-exposed thylakoid membranes. The first membrane protrusions and interthylakoid contacts are observed after 4 s illumination and they become numerous by 10 s. As a result, a network of anastomoses is formed which is maintained during at least 10 min. These effects are reversible in the dark and can be reproduced several times. The formation of membrane fusions is inhibited by the addition of protonophore. It is supposed that the energy-dependent uptake of protonated anesthetics by thylakoids leads to an increase in positive surface charge and thus a lateral pressure on the inner side of the thylakoid membrane. The appearance of membrane protrusions (crinkles) having the positive curvature of their inner surface may be considered as a way of compensating for lateral pressure. Presumably, anastomoses result from the fusion of crinkles to adjacent thylakoids.

Dynamics of hybrid X-pinches

The dynamics of a new type of pinches—hybrid X-pinches (HXPs)—has been studied experimentally and numerically. The initial configuration of an HXP consists of a high-current diode with conical tungsten electrodes separated by a 1- to 3-mm-long gap and shunted with a 20- to 100-μm diameter wire. It was shown earlier that a hot spot (HS) with high plasma parameters also formed in the HXP, although its initial configuration is simpler than that of a standard X-pinch. Although details of the HXP dynamics still remain insufficiently studied, the main factors governing the HXP formation were investigated both experimentally and using magnetohydrodynamic simulations. The formation of a specific pressure profile in the electrode plasma after the wire explosion was investigated both experimentally and theoretically. It is shown that the effect of the pressure profile on the expanding wire plasma is similar for both standard X-pinches and HXPs, which allows one to assign them to the same class of loads of pulsed facilities. It is also established that the final stages of HS formation and the parameters of the HS plasma in standard X-pinches and HXPs are practically identical.

Effects of heterocyclic and tertiary permeant amines on the electron transfer in thylakoid membranes

The effect of low concentrations (up to 50 μM) of lipophilic permeant amines on the electron transfer in thylakoid membranes of pea chloroplasts has been investigated. In the presence of heterocyclic amines (9-aminoacridine and neutral red), the electron transfer, initiated from H2O to PS I acceptors, has been shown to be inhibited to a level amounting to less than 50% of control, this taking place for both the basal (at alkaline pH) and the gramicidin-uncoupled transport (at pH 6.5–8.5). Under the same conditions, tertiary amines (dibucaine, tetracaine) cause only a 10–15% inhibition of transport. With all the amines, the rate of electron transport from H2O to DCBQ, PS II acceptor is decreased to 80–90% of control at pH above 8.0, but this effect is completely removed when pH is lowered to 7.7–6.5. In the region of PS I, all the amines accelerate the basal transport, but do not influence the uncoupled electron transfer. A conclusion has been drawn that, parallel with uncoupling, heterocyclic and tertiary amines also cause an inhibition of PS II, appearing at alkaline pH values. Additionally, heterocyclic amines seem to brake electron flow at the level of plastoquinone reduction.

Involvement of palmitate/Ca2+(Sr2+)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca2+-induced (Pal/Ca2+) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca2+ with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca2+ from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca2+ influx, seems to be partly due to the opening of Pal/Ca2+ pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca2+-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca2+ (Sr2+) also induced an outflow of K+ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K+/H+ exchange blocker, and the reuptake was prevented by an ATP-dependent K+ channel inhibitor. It was also shown that the addition of Sr2+ to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr2+ and K+ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca2+-dependent phospholipase A2, and a new Sr2+ cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr2+. All of the Ca2+- and Sr2+-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca2+ pores in the maintenance of cell ion homeostasis.

Effect of surface-potential modulators on the opening of lipid pores in liposomal and mitochondrial inner membranes induced by palmitate and calcium ions.

The effect of surface-potential modulators on palmitate/Ca2+-induced formation of lipid pores was studied in liposomal and inner mitochondrial membranes. Pore formation was monitored by sulforhodamine B release from liposomes and swelling of mitochondria. ζ-potential in liposomes was determined from electrophoretic mobility. Replacement of sucrose as the osmotic agent with KCl decreased negative ζ-potential in liposomes and increased resistance of both mitochondria and liposomes to the pore inducers, palmitic acid, and Ca2+. Micromolar Mg2+ also inhibited palmitate/Ca2+-induced permeabilization of liposomes. The rate of palmitate/Ca2+-induced, cyclosporin A-insensitive swelling of mitochondria increased 22% upon increasing pH from 7.0 to 7.8. At below the critical micelle concentration, the cationic detergent cetyltrimethylammonium bromide (10 μM) and the anionic surfactant sodium dodecylsulfate (10-50 μM) made the ζ-potential less and more negative, respectively, and inhibited and stimulated opening of mitochondrial palmitate/Ca2+-induced lipid pores. Taken together, the findings indicate that surface potential regulates palmitate/Ca2+-induced lipid pore opening.

Transport and Measurements of High‐Current Electron Beams from X pinches

Generation of electron beams is an unavoidable property of X‐pinches and other pulsed‐power‐driven pinches of different geometry. Some issues concerning high‐current electron beam transport from the X pinch to the diagnostic system and measurements of the beam current by Faraday cups with different geometry’s are discussed. Of particular interest is the partially neutralized nature of the beam propagating from the X‐pinch to a diagnostic system. Two scenarios of electron beam propagation from X‐pinch to Faraday cup are analyzed by means of computer simulation using the PIC‐code KARAT. The first is longitudinal neutralization by ions extracted from plasma at an output window of the X‐pinch diode; the second is the beam transport through a plasma background between the diode and a diagnostic system.

Study of the prebreakdown stage of a gas discharge in a diode with point cathode by laser probing

The prebreakdown stage of a gas discharge in a diode with strongly overloaded cathode is studied by laser methods (by simultaneous use of multiframe interferometry and shadow and schlieren photographing) at atmospheric pressure. The spatial resolution of the methods is about 20 μm. A probing pulse of a laser (LS-2151 Nd: YAG laser with a half amplitude duration of 70 ps and a pulse energy of up to 40 mJ) is synchronized with a voltage pulse with accuracy of about 1 ns. High field strength at the cathode is achieved due to the use of thin individual metal tips on the electrodes. It is shown that the initial stage of breakdown of a discharge gap is accompanied by the emergence of a dense plasma cloud at the end of a tip with electron density of about 5 × 1019 cm–3 with a size of tens of microns, as well as by a sharp increase in the total current through the diode. After the emergence of a dense plasma cloud at the end of a cathode tip, a similar cloud is formed on the surface of the anode; sometime later, these clouds join together and form a tubular current channel. The dynamics of the breakdown, as well as the parameters of the plasma are studied by the abovementioned techniques in three independent optical channels.

Interaction of Phospholipase A of the E. coli Outer Membrane with the Inhibitors of Eucaryotic Phospholipases A 2 and Their Effect on the Ca 2+ -Induced Permeabilization of the Bacterial Membrane

Phospholipase A of the bacterial outer membrane (OMPLA) is a β-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A2—palmitoyl trifluoromethyl ketone (PACOCF3) and aristolochic acid (AA)—with OMPLA and considers a possible involvement of the enzyme in the Ca2+-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF3 and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine. It has also been shown that PACOCF3, AA, and bromophenacyl bromide inhibit the Ca2+-induced temperature-dependent changes in the permeability of the bacterial membrane for the fluorescent probe propidium iodide and suppressed the transformation of E. coli cells with plasmid DNA induced by Ca2+ and heat shock. The cell viability was not affected by the eucaryotic phospholipases A2 inhibitors. The study discusses a possible involvement of OMPLA in the mechanisms of bacterial transmembrane transport based on the permeabilization of the bacterial outer membrane.