The mechanoelectrical response of droplet interface bilayer membranes.

Mechanotransduction and interfacial properties in unsupported liquid biomimetic membranes are explored using the droplet-interface bilayer technique. The fluidic monolayer-membrane system afforded by this technique allows for dynamic control over the membrane dimensions and curvature, which under periodic deformations generates capacitive currents (akin to a Kelvin probe), and permits a detailed electrostatic characterization of the boundary layers as well as observation of flexoelectric effects. Both high and low displacement frequency regimes are examined, and the results show that the mechanoelectric signals generated by the membranes may be linked to the membrane electrostatic structure. In addition, we show that periodic membrane bending in a high-frequency regime generates tension sufficient to activate reconstituted mechanosensitive channels.

200 TW 45 fs laser based on optical parametric chirped pulse amplification.

200 TW peak power has been achieved experimentally using a Cr:forsterite master oscillator at 1250 nm, a stretcher, three optical parametrical amplifiers based on KD*P (DKDP) crystals providing 14.5 J energy in the chirped pulse at 910 nm central wavelength, and a vacuum compressor. The final parametrical amplifier and the compressor are described in detail. Scaling of such architecture to multipetawatt power is discussed.

In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA.

The skin cells of newborn mice were stably transformed in vivo with the aid of electroporation. The plasmid DNA was introduced subcutaneously followed by high-voltage pulses applied to the skin pleat. NEO-resistant colonies were found in primary cell cultures obtained from the treated skin. The experiments show that in vivo electroporation can be used for the introduction of plasmid DNA into skin cells of mouse.

Helicity, membrane incorporation, orientation and thermal stability of the large conductance mechanosensitive ion channel from E. coli.

In this report, we present structural studies on the large conductance mechanosensitive ion channel (MscL) from E. coli in detergent micelles and lipid vesicles. Both transmission Fourier transform infrared spectroscopy and circular dichroism (CD) spectra indicate that the protein is highly helical in detergents as well as liposomes. The secondary structure of the proteins was shown to be highly resistant towards denaturation (25-95 degrees C) based on an ellipticity thermal profile. Amide H+/D+ exchange was shown to be extensive (ca. 66%), implying that two thirds of the protein are water accessible. MscL, reconstituted in oriented lipid bilayers, was shown to possess a net bilayer orientation using dichroic ratios measured by attenuated total-reflection Fourier transform infrared spectroscopy. Here, we present and discuss this initial set of structural data on this new family of ion-channel proteins.

The electrical breakdown of cell and lipid membranes: the similarity of phenomenologies.

The current responses of human erythrocyte and L-cell membranes being subject to rectangular voltage pulses of 150-700 mV amplitude and 5 X 10(-3)-10 s duration were recorded by means of the patch-clamp method. The behaviour of planar lipid bilayer membranes of oxidized cholesterol and UO2(2+)-modified bilayers of azolectin in a high electric field was investigated for comparison. The gradual growth in the conductance (reversible electrical breakdown) was found for both the cell membranes and lipid bilayers of the compositions studied, with the application of voltage pulses of sufficient duration, to be completed by its drastic enhancement (irreversible breakdown). The time interval preceding the irreversible breakdown and the rate of increase in conductance during the reversible breakdown are determined by the amplitude of the voltage applied. The recovery of the initial properties of the membrane following the reversible breakdown consists of the two stages, the latter substantially differing by their characteristic times. The first very rapid stage (tau much less than 1 ms) reflects the lowering of the conductance of small pores with decreasing voltage across the membrane. The diminishing of the number and mean radii of the pores resulting in their complete disappearance occurs only at the second stage of membrane healing, which lasts several seconds or even minutes. The phenomenological similarity of the cell and lipid membrane breakdown indicates that pores developed during the electrical breakdown of biological membranes arise in their lipid matrices. The structure and the properties of the pores are discussed.

Electrical properties of cell pellets and cell electrofusion in a centrifuge.

A new approach is proposed for studying cell deformability by centrifugal force, electrical properties of cell membranes in a high electric field, and for performing efficient cell electrofusion. Suspensions of cells (L929 and four other cell types examined) are centrifuged in special chambers, thus forming compact cell pellets in the gap between the electrodes. The setup allows measurement of the pellet resistance and also the high-voltage pulse application during centrifugation. The pellet resistance increases sharply with the centripetal acceleration, which correlates with reduction of the cell pellet porosity due to cell compression and deformation. Experiments with cells pretreated with cytochalasin B or colcemid showed that cell deformability depends significantly on the state of cytoskeleton. When the voltage applied to the cell pellet exceeds a 'critical' value, electrical breakdown (poration) of cell membranes occurs. This is seen as a deflection in the I(V) curve for the cell pellet. The electropores formed during the breakdown reseal in several stages: the fastest takes 0.5-1 ms while the whole process completes in minutes. A novel effect of colloid-osmotic compression of cell pellets after electric cell permeabilization is described. Supercritical pulse application to the cell pellet during intensive centrifugation leads to massive cell fusion. The fusion index grows with the increase of centripetal acceleration, and drops drastically when the pulse is applied after the centrifuge is stopped. The colloid-osmotic pellet compression enhances the fusion efficiency. No fusion occurs when cells are brought in contact after the pulse treatment. The data suggest that tight intermembrane contact formed prior to pulse application is a prerequisite condition for efficient cell electrofusion. The capacities of the technique proposed and the mechanism of membrane electrofusion are discussed.

Electrofusion of fibroblasts on the porous membrane.

Electric fusion of cells is usually performed in two steps: the first is the creation of tight intercellular contact, the second is an application of electric pulses which induce membrane fusion proper. In the present work a new technique of cell electrofusion on the porous film is described. It consists of preliminary cultivation of cell monolayer on the porous film (protein-coated cellophane). Then cells of the same or any other type are added from above to form a second cell layer upon the first one. The pulses of the electric field are applied normally to the plane of the double cell layer to induce cell fusion. After pulse application a picture of mass polynucleation was observed. At the same time we did not obtain fusion of L cells by means of dielectrophoretic electrofusion technique. This difference in efficiency could be explained by the formation of broad zones of membrane contact between the cells adherent to the film, while during intensive dielectrophoresis only the point contacts were revealed. The high-conducting medium for electric treatment providing an efficient fusion on the film and high cell viability was composed. Neither cytochalasin B nor colcemid affected cell fusion noticeably; however the sodium azide (added with 2-deoxyglucose) inhibited fusion completely. The short hypotonic shock after electric treatment enhanced the rate of polycaryon formation.

Role of proteases in activation of apoptosis.

Programmed cell death, or apoptosis, is a physiological cell suicide mechanism, which is triggered in the cells by different stimuli. It has been shown that proteases play a significant role both in the target cell killing by cytotoxic lymphocytes and in the TNF- or anti-Fas-induced cell death. The proteases involved in the early (induction) and late (cell self-destruction) stages of apoptosis are reviewed. It is suggested that the late stages are connected with the activation of a cascade of intracellular proteases, which leads to massive protein destruction. It is likely that the protein destruction is mainly designed for preventing autoimmune response to proteins released from dying cells.

Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL.

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was DeltaE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (DeltaA), the free energy, TDeltaA, would correspond to DeltaA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest DeltaA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to DeltaA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.

The control of DNA replication in hybrids between neutrophils and fibroblasts.

DNA synthesis regulation in heterokaryons between mouse neutrophils and cultured cells of various proliferative potentials has been studied. The following features have been found. Both immortalized and non-immortalized cells can reactivate DNA synthesis in neutrophil nuclei. The reactivation ability of cultured cells increases after immortalization and is not changed by further transformation. Neutrophils inhibit the entry of cultured cell nuclei into S phase and have no effect on ongoing DNA synthesis. Malignant cells are much less sensitive to the inhibitory action of neutrophils than non-malignant ones. Non-malignant immortalized cells are as sensitive to this effect as non-immortalized cells. Neutrophil karyoplasts do not influence DNA synthesis in partner cultured cell nuclei. Cycloheximide pretreatment of neutrophils drastically diminishes their inhibitory effect.

Study of protein carbonyls in subcellular fractions isolated from liver and spleen of old and gamma-irradiated rats.

Age- and gamma-irradiation-dependent accumulation of oxidatively modified proteins (measured as carbonyl level) was studied in cytoplasm, mitochondria and nuclei isolated from spleen and liver of 4- and 26-month-old rats. The protein carbonyl levels significantly increased with age in all fractions studied. The carbonyl content was found to be two times higher in the nuclei than in the mitochondria and cytoplasm, which may be related to an extensive modification of lysine and arginine residues in histone molecules. Gamma-Irradiation of rats with 10 Gy caused a rise of protein carbonyls only in their cytoplasm and mitochondria, which was prevented in the animals fed with antioxidants and vitamins for a month before the irradiation. We observed an activation of histone-specific proteases in the nuclei of gamma-irradiated rats. The lack of carbonyl accumulation in the nuclear proteins isolated from tissues of gamma-irradiated animals may be explained by the degradation of oxidized histones by these proteases.

Age- and radiation-dependent changes in carbonyl content, susceptibility to proteolysis, and antigenicity of soluble rat liver proteins.

Soluble liver proteins (SLP) from old and gamma-irradiated young rats were studied with respect to their carbonyl content, the rates of autolysis and degradation by proteinase K, and their antigenicity for mice and compared with SLP from non-irradiated young animals. A significant increase in the carbonyl level was found in SLP from old and gamma-irradiated young rats as compared to SLP from intact young rats. The rates of SLP autolysis and proteolysis by proteinase K were increased in the same animal groups but did not correlate the carbonyl level. At the same time, whereas the antigenicity for mice of SLP from old rats was significantly higher than that of SLP from young rats, the antigenicity of SLP from gamma-irradiated rats did not differ from non-irradiated animals. Enrichment of the diet with antioxidant and vitamin supplements (AVS) during one month before the irradiation caused a decrease in the radiation-induced carbonyl level in rat SLP. However, this raised antioxidant level in animal diet did not influence the rates of SLP autolysis and degradation by proteinase K and also did not alter the antigenicity of these proteins. The data allow us to suggest that the increase in autolysis, degradation by the exogenous proteinase, and antigenicity of SLP from old rats are determined not only by carbonyl formation in these proteins due to action of oxygen radicals but also by other age-specific protein modifications.

A mechanosensitive channel protein and its gene in E. coli.

Receptor molecules that respond directly to gravity, touch, vibration, or osmotic pressure are inferred from their functions but not yet characterized as isolated proteins or products of cloned genes. These receptors are often in low abundance and in animal and plant tissues that are inaccessible, thus making biochemical analysis difficult. Yet, the application of the sensitive patch-clamp technique to measure transmembrane currents has demonstrated the ubiquity of ion channels whose opening is favored by membrane stretch forces. We have discovered in E. coli the activity of a mechanosensitive ion channel of large conductance (MscL), and have successfully isolated the corresponding protein and gene (Sukharev et al. 1994a). MscL channel appears to respond directly to stretch force in the lipid bilayer since it is active in artificial patches having only highly enriched MscL protein and lipids. Structurally, MscL is an integral membrane protein of only 136 amino-acid residues. Each channel pore is likely to be enclosed by six assembled MscL subunits. Hydropathy analysis suggests that the protein is largely hydrophobic with a more hydrophilic carboxyl tail. Targeted deletions and substitutions show that not all regions of the molecule contribute to channel function; however, strategic single amino-acid changes can alter channel kinetics and mechanosensitivity. MscL and its gene now form the first tangible system to study mechanosensing using a combination of genetic, biochemical, and biophysical techniques.

[Fusion of Erwinia chrysanthemi spheroplasts in an electric fields]. / Sliianie sferoplastov Erwinia chrysanthemi v élektricheskom pole.

A new approach has been elaborated for electrofusion of Erwinia chrysanthemi spheroplasts. The new approach consists of superimposition of high voltage impulses on the pellet of tightly contacting cells in the course of centrifugation. The mixture of spheroplasts of two genetically marked strains was placed into the special centrifuge chambers and spinned for 15 min at 2500 g to get a compressed pellet between chamber electrodes. Three successive pulses of 6.6 kv/cm amplitude and 30 microseconds duration were applied to spheroplast pellet during centrifugation. Fusion products were viable and after plating on the surface of hypertonic medium regenerated to the rod forms. As a result, the hybrid clones carrying the markers of both parents were isolated.

[DNA synthesis in heterokaryons obtained by the fusion of polymorphonuclear leukocytes and cultured cells possessing different proliferative potentials]. / Sintez DNK v geterokarionakh, poluchennykh pri sliianii segmentoiadernykh leikotsitov i kul'tiviruemykh kletok, obladaiushchikh razlichnym proliferativnym potentsialom.

Heterokaryons between terminally differentiated polymorphonuclear leukocytes (PL) and culture cells of different proliferative potentials: mouse and rat embryo fibroblasts (EFM, EFR); immortal cells NIH 3T3 and E2; malignant cells NCC2, L929, He239 and SV 3T3,--were obtained by means of electrofusion. Radioautographic study of 3H-thymidine incorporation in the nuclei of heterokaryons showed that all the cells taken for fusion were able to induce reactivation of DNA synthesis in PL nuclei, however, with different rates: 7-37% for EFM and NIH 3T3 and 20-40% for malignant cells. The presence of oncogenes Elan in E2 cells and ras in NCC2 cells increased the rate of PL reactivation approximately twice as compared with the cells of original lines (EFR and NIH 3T3, correspondingly). In parallel to reactivation of DNA synthesis in PL nuclei inhibition of the synthesis in culture cell nuclei in the same heterokaryons was found. The rate of inhibition was about 70% for non-malignant and 23, 40 and 18% for NCC2, L and SV 3T3 cells, respectively. He239 cells, transformed by a temperature-dependent mutant of virus SV40 showed at permissive temperature the increased capacity of inducing reactivation of PL nuclei, though He239 cells susceptibility to inhibitory action of PL nuclei did not change with temperature. According to the behaviour in heterokaryons PL were found to be similar to chick erythrocytes, but differing from them by a pronounced inhibiting effect upon DNA synthesis in the nuclei of malignant cells.

[Reversible electrical breakdown of cholesterol-containing lipid bilayer membranes modified with holothurin A]. / Obratimyi élektricheskii proboi kholesterinsoderzhashchikh bisloinykh lipidnykh membran, modifitsirovannykh goloturinom A.

Reversible electrical breakdown of cholesterol-containing BLM modified with holothurin A, i. e. a significant reversible increase of membrane conductivity under the effect of the electrical field was described. So when the voltage approximately 0.3 V was applied to the BLM for 10 ms its conductivity was reversibly increased by 4 orders as compared to the initial one. Reproducibility of current oscillograms, non-linear current breakdown--potential relationship and non-linear spasmodic decrease of conductivity at step-like reduction of the potential on the membrane show similarity of this phenomenon with the reversible breakdown of the membranes of oxidized cholesterol and of asolectin BLM in the presence of UO22+ ions. The mechanism of reversible electrical breakdown of BLM is discussed in terms of the development of a great number of local conductive defects in the membrane under the effect of the electrical field.

Hydration properties of mechanosensitive channel pores define the energetics of gating.

Opening of ion channels directly by tension in the surrounding membrane appears to be the most ancient and simple mechanism of gating. Bacterial mechanosensitive channels MscL and MscS are the best-studied tension-gated nanopores, yet the key physical factors that define their gating are still hotly debated. Here we present estimations, simulations and experimental results showing that hydration of the pore might be one of the major parameters defining the thermodynamics and kinetics of mechanosensitive channel gating. We associate closing of channel pores with complete dehydration of the hydrophobic gate (occlusion by 'vapor lock') and formation of two water-vapor interfaces above and below the constriction. The opening path is the expansion of these interfaces, ultimately leading to wetting of the hydrophobic pore, which does not appear to be the exact reverse of the closing path, thus producing hysteresis. We discuss specifically the role of polar groups (glycines) buried in narrow closed conformations but exposed in the open states that change the wetting characteristics of the pore lining and stabilize conductive states of the channels.