Abstract

The influence of cholesterol fraction in the membranes of giant unilamellar vesicles (GUVs) on their size distributions and bending moduli has been investigated. The membranes of GUVs were synthesized by a mixture of two elements: electrically neutral lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol and also a mixture of three elements: electrically charged lipid 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), DOPC and cholesterol. The size distributions of GUVs have been presented by a set of histograms. The classical lognormal distribution is well fitted to the histograms, from where the average size of vesicle is obtained. The increase of cholesterol content in the membranes of GUVs increases the average size of vesicles in the population. Using the framework of Helmholtz free energy of the system, the theory developed by us is extended to explain the experimental results. The theory determines the influence of cholesterol on the bending modulus of membranes from the fitting of the proper histograms. The increase of cholesterol in GUVs increases both the average size of vesicles in population and the bending modulus of membranes.

Abstract

Electropermeabilization is a promising phenomenon that occurs when pulsed electric field with high frequency is applied to cells/vesicles. We quantify the required values of pulsed electric fields for the rupture of cell-sized giant unilamellar vesicles (GUVs) which are prepared under various surface charges, cholesterol contents and osmotic pressures. The probability of rupture and the average time of rupture are evaluated under these conditions. The electric field changes from 500 to 410 Vcm^-1 by varying the anionic lipid mole fraction from 0 to 0.60 for getting the maximum probability of rupture (i.e., 1.0). In contrast, the same probability of rupture is obtained for changing the electric field from 410 to 630 Vcm^-1 by varying the cholesterol mole fraction in the membranes from 0 to 0.40. These results suggest that the required electric field for the rupture decreases with the increase of surface charge density but increases with the increase of cholesterol. We also quantify the electric field for the rupture of GUVs containing anionic mole fraction of 0.40 under various osmotic pressures. In the absence of osmotic pressure, the electric field for the rupture is obtained 430 Vcm^-1, whereas the field is 300 Vcm^-1 in the presence of 17 mOsmL^-1, indicating the instability of GUVs at higher osmotic pressures. These investigations open an avenue of possibilities for finding the electric field dependent rupture of cell-like vesicles along with the insight of biophysical and biochemical processes.

Abstract

External tension in membranes plays a vital role in numerous physiological and physicochemical phenomena. In this review, recent developments in the constant electric- and mechanical-tension-induced rupture of giant unilamellar vesicles (GUVs) are considered. We summarize the results relating to the kinetics of GUV rupture as a function of membrane surface charge, ions in the bathing solution, lipid composition, cholesterol content in the membrane, and osmotic pressure. The mechanical stability and line tension of the membrane under these conditions are discussed. The membrane tension due to osmotic pressure and the critical tension of rupture for various membrane compositions are also discussed. The results and their analysis provide a biophysical description of the kinetics of rupture, along with insight into biological processes. Future directions and possible developments in this research area are included.

Abstract

A new purification technique is developed for obtaining distribution of giant unilamellar vesicles (GUVs) within a specific range of sizes using dual filtration. The GUVs were prepared using well known natural swelling method. For filtration, different combinations of polycarbonate membranes were implemented in filter holders. In our experiment, the combinations of membranes were selected with corresponding pore sizes–(i) 12 and 10 μm, (ii) 12 and 8 μm, and (iii) 10 and 8 μm. By these filtration arrangements, obtained GUVs size distribution were in the ranges of 6−26 μm, 5–38 μm and 5–30 μm, respectively. In comparison, the size distribution range was much higher for single filtration technique, for example, 6−59 μm GUVs found for a membrane with 12 μm pores. Using this technique, the water-soluble fluorescent probe, calcein, can be removed from the suspension of GUVs successfully. The size distributions were analyzed with lognormal distribution. The skewness became smaller (narrow size distribution) when a dual filtration was used instead of single filtration. The mode of the size distribution obtained in dual filtration was also smaller to that of single filtration. By continuing this process of purification for a second time, the GUVs size distribution became even narrower. After using an extra filtration with dual filtration, two different size distributions of GUVs were obtained at a time. This experimental observation suggests that different size specific distributions of GUVs can be obtained easily, even if GUVs are prepared by different other methods.

Abstract

Irreversible electroporation (IRE) is a nonthermal tumor/cell ablation technique in which a series of high-voltage short pulses are used. As a new approach, we aimed to investigate the rupture of giant unilamellar vesicles (GUVs) using the IRE technique under different osmotic pressures (Π), and estimated the membrane tension due to Π. Two categories of GUVs were used in this study. One was prepared with a mixture of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC) and cholesterol (chol) for obtaining more biological relevance while other with a mixture of DOPG and DOPC, with specific molar ratios. We determined the rate constant (k_p) of rupture of DOPG/DOPC/chol (46/39/15)-GUVs and DOPG/DOPC (40/60)-GUVs induced by constant electric tension (σ_c) under different Π. The σ_c dependent k_p values were fitted with a theoretical equation, and the corresponding membrane tension (σ_oseq) at swelling equilibrium under Π was estimated. The estimated membrane tension agreed well with the theoretical calculation within the experimental error. Interestingly, the values of σ_oseq were almost same for both types of synthesized GUVs under same osmotic pressure. We also examined the sucrose leakage, due to large osmotic pressure-induced pore formation, from the inside of DOPG/DOPC/chol(46/39/15)-GUVs. The estimated membrane tension due to large Π at which sucrose leaked out was very similar to the electric tension at which GUVs were ruptured without Π. We explained the σ_c and Π induced pore formation in the lipid membranes of GUVs.

Abstract

We have analyzed the purification of charged giant unilamellar vesicles (GUVs) prepared in a buffer containing various concentrations of salt using their size distribution. The membranes of GUVs were synthesized by a mixture of dioleoylphosphocholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) lipids. The DOPG mole fractions (X) in the membranes of GUVs were 0.10, 0.25, 0.40, 0.55, 0.70, 0.90 in a physiological buffer containing 162 mM salt. In addition, for a fixed value of X the concentrations of salt (C) in the buffer were 12, 62, 112, 162, 212, 312, 362 mM. The size distribution histograms of experimentally investigated unpurified and purified GUVs were fitted with the lognormal distribution and obtained the multiplication factor γ for mean (μ) and η for standard deviation (σ) of the lognormal distribution. The key parameters γ and η were responsible for changing the average size and size distribution of unpurified GUVs to purified ones. The theoretically fitting equation of experimentally obtained X- and C-dependent values of γ and η provided the calibration equation for estimating the average size of purified GUVs theoretically for any values of X and C. The estimated size of purified GUVs increased with the increase in electrostatic effect (i.e., increase in vesicle surface charge density or decrease in salt concentration in buffer). The estimated size of purified GUVs varied with X and C, which supported the previous report qualitatively. These investigations might be helpful in the field of cell/chemical biology for understanding the process of purification of vesicles/cells investigated by any other techniques.

Abstract

Irreversible electroporation (IRE) is a technique for the disruption of localized cells or vesicles by a series of short and high−frequency electric pulses which has been used for tissue ablation and treatment in certain diseases. It is well reported that IRE induces lateral tension in the membranes of giant unilamellar vesicles (GUVs). The GUVs are prepared by a mixture of anionic lipid dioleoylphosphatidylglycerol (DOPG) and neutral lipid dioleoylphosphatidylcholine (DOPC) using the natural swelling method. Here the influence of DOPG mole fraction, X_DOPG, on the critical tension of electroporation in GUVs has been investigated in sodium chloride-containing PIPES buffer. The critical tension decreases from 9.0 ± 0.3 to 6.0 ± 0.2 mN/m with the increase of X_DOPG from 0.0 to 0.60 in the membranes of GUVs. Hence an increase in X_DOPG greatly decreases the mechanical stability of membranes. We develop a theoretical equation that fits the X_DOPG dependent normalized critical tension, and obtain a binding constant for the lipid-ion interaction of 0.75 M⁻¹. The decrease in the energy barrier for formation of the nano−size nascent or prepore state, due to the increase in X_DOPG, is the main factor explaining the decrease in critical tension of electroporation in vesicles.

Abstract

Irreversible electroporation (IRE) is primarily a nonthermal ablative technology that uses a series of high-voltage and ultra-short pulses with high-frequency electrical energy to induce cell death. This paper presents the influence of cholesterol on the IRE-induced probability of pore formation and the rate constant of pore formation in giant unilamellar vesicles (GUVs). The GUVs are prepared by a mixture of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC) and cholesterol using the natural swelling method. An IRE signal of frequency 1.1 kHz is applied to the membranes of GUVs. The probability of pore formation and the rate constant of pore formation events are obtained using statistical analysis from several single GUVs. The time-dependent fraction of intact GUVs among all those examined is fitted to a single exponential decay function from where the rate constant of pore formation is calculated. The probability of pore formation and the rate constant of pore formation decreases with an increase in cholesterol content in the membranes of GUVs. Theoretical equations are fitted to the tension-dependent rate constant of pore formation and to the probability of pore formation, which allows us to obtain the line tension of membranes. The obtained line tension increases with an increase in cholesterol in the membranes. The increase in the energy barrier of the prepore state, due to the increase of cholesterol in membranes, is the main factor explaining the decrease in the rate constant of pore formation.

Abstract

The influence of electrostatic conditions (salt concentration of the solution and vesicle surface charge density) on the size distribution of self-assembled giant unilamellar vesicles (GUVs) is considered. The membranes of GUVs are synthesized by a mixture of dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine in a physiological buffer using the natural swelling method. The experimental results are presented in the form of a set of histograms. The log-normal distribution is used for statistical treatment of results. It is obtained that the decrease of salt concentration and the increase of vesicle surface charge density of the membranes increase the average size of the GUV population. To explain the experimental results, a theory using the Helmholtz free energy of the system describing the GUV vesiculation is developed. The size distribution histograms and average size of GUVs under various conditions are fitted with the proposed theory. It is shown that the variation of the bending modulus due to changing of electrostatic parameters of the system is the main factor causing a change in the average size of GUVs

Abstract

The interaction of anionic magnetite nanoparticles (MNPs) of size 18 nm with negatively charged giant unilamellar vesicles (GUVs) formed from a mixture of neutral dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylglycerol (DOPG) lipids has been investigated. It has been obtained that NPs induces the deformation of spherical GUVs. The reaction of other GUVs on NPs consists in the appearance of pores in their membranes. We focused the effect of electrostatics on the interaction of charged membranes with MNPs. To study the influence of the surface charge of GUVs on the processes under consideration, we varied the fraction of DOPG in the vesicles from 0 to 100%. We examined the influence of salt concentration in the range of 50−300 mM NaCl concentration. To describe the degree of deformation, a special parameter compactness was introduced. The pore formation in the membranes of GUVs was investigated by the leakage of sucrose. The compactness increases with time and also NPs concentration. The fraction of deformed GUVs increases with the increase of surface charge density of membranes as well as the decrease of salt concentration in buffer. The value of compactness for neutral membrane is 1.25 times higher than that of charged ones. The fraction of deformed GUVs become constant after 20 min, however it increases with NPs concentration. The time taken for stochastic pore formation is less for charged membrane than neutral one. The physical mechanism explaining the experimental results obtained in these investigations.

Abstract

Cell penetrating peptide transportan 10 and antimicrobial peptide melittin formed submicron pores in the lipid membranes of vesicles which are explained by the leakage of water-soluble fluorescent probes from the inside of vesicles to the outside. It is hypothesized that these submicron pores induce submicron discontinuities in the membranes. Considering this hypothesis, a technique has developed to locate the submicron discontinuities in the membranes of giant unilamellar vesicles (GUVs) using ImageJ. In this technique, at first the edges of membrane of a ‘single GUV’ are detected and then these edges are used to locate the submicron discontinuities. Two continuous rings are observed after applying the ImageJ in GUVs which indicated the edges of membrane. In contrast, the submicron discontinuations are detected at the edges of transportan 10 and melittin induced pore formed membranes. This investigation might be helpful for the elucidation of mechanism of the peptide-induced pore formation in the membranes of vesicles.

Abstract

Stretching in the plasma membranes of cells and lipid membranes of vesicles plays important roles in various physiological and physicochemical phenomena. Irreversible electroporation (IRE) is a minimally invasive non-thermal tumor ablation technique where a series of short electrical energy pulses with high frequency is applied to destabilize the cell membranes. IRE also induces lateral tension due to stretching in the membranes of giant unilamellar vesicles (GUVs). Here, the kinetics of irreversible pore formation under constant electrical tension in GUVs has been investigated. The GUVs are prepared by a mixture of dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine using the natural swelling method. An IRE signal of frequency 1.1 kHz is applied to the GUVs through a gold-coated electrode system. Stochastic pore formation is observed for several ‘single GUVs’ at a particular constant tension. The time course of the fraction of intact GUVs among all the examined GUVs is fitted with a single-exponential decay function from which the rate constant of pore formation in the vesicle, k_p, is calculated. The value of k_p increases with an increase of membrane tension. An increase in the proportion of negatively charged lipids in a membrane gives a higher k_p. Theoretical equations are fitted to the tension-dependent k_p and to the probability of pore formation, which allows us to obtain the line tension of the membranes. The decrease in the energy barrier for formation of the nano-size nascent or prepore state, due to the increase in electrical tension, is the main factor explaining the increase of k_p.

Abstract

Irreversible electroporation (IRE) is a new technique in which a series of short pulses with high frequency electrical energy is applied on the targeted regions of cells or vesicles for their destruction or rupture formation. IRE induces lateral tension in the membranes of vesicles. We have investigated the electrostatic interaction effects on the constant electrical tension-induced rate constant of irreversible pore formation in the membranes of giant unilamellar vesicles (GUVs). The electrostatic interaction has been varied by changing the salt concentration in buffer and the surface charge density of membranes. The membranes of GUVs are synthesized by a mixture of negatively charged lipid dioleoylphosphatidylglycerol (DOPG) and neutral lipid dioleoylphosphatidylcholine (DOPC) using the natural swelling method. The rate constant of pore formation increases with the decrease of salt concentration in buffer along with the increase of surface charge density of membranes. The tension dependent probability of pore formation and the rate constant of pore formation are fitted to the theoretical equation, and obtained the line tension of membranes. The decrease in energy barrier of a prepore due to electrostatic interaction is the key factor causing an increase of rate constant of pore formation.

Abstract

Lipid membranes of giant unilamellar vesicles (GUVs) with diameters greater than 10 μm are promising model membranes for investigating the physical and biological properties of the biomembranes of cells. These are extensively used for the study of the interaction of various membrane-active agents, where purified and similar-size oil-free GUVs are necessary. Although the existing membrane filtering method provides the required quality and quantity of GUVs, it includes a relatively expensive double-headed peristaltic pump. In our proposed non-electromechanical technique, gravity is used to maintain the flow of buffer, wherein the flow rate of buffer with the suspension of GUVs is controlled by a locally available low cost roller clamp regulator. We have characterized the results of this non-electromechanical approach in terms of size distribution, average size, flow rate and efficiency for dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC)-GUVs prepared by the natural swelling method. The technique purifies the GUVs by removing the non-entrapped solutes at an optimum flow rate 1.0–2.0 mL/min. In addition, it gives similar results to the pump-driven membrane filtering method. Therefore, it might be a cost effective technique for the purification of GUVs without employing any electromechanical devices.

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Antimicrobial peptide magainin 2 forms pores in lipid bilayers, a property that is considered the main cause of its bactericidal activity. Recent data suggest that tension or stretching of the inner monolayer plays an important role in magainin 2-induced pore formation in lipid bilayers. Here, to elucidate the mechanism of magainin 2-induced pore formation, we investigated the effect on pore formation of asymmetric lipid distribution in two monolayers. First, we developed a method to prepare giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC), and lyso-PC (LPC) in the inner monolayer and of DOPG/DOPC in the outer monolayer. We consider that in these GUVs, the lipid packing in the inner monolayer was larger than that in the outer monolayer. Next, we investigated the interaction of magainin 2 with these GUVs with an asymmetric distribution of LPC using the single GUV method, and found that the rate constant of magainin 2-induced pore formation, k_p, decreased with increasing LPC concentration in the inner monolayer. We constructed a quantitative model of magainin 2-induced pore formation, whereby the binding of magainin 2 to the outer monolayer of a GUV induces stretching of the inner monolayer, causing pore formation. A theoretical equation defining k_p as a function of magainin 2 surface concentration, X, reasonably explains the experimental relationship between k_p and X. This model quantitatively explains the effect on k_p of the LPC concentration in the inner monolayer. On the basis of these results, we discuss the mechanism of the initial stage of magainin 2-induced pore formation.

Abstract

Osmotic pressure (Π) induces the stretching of plasma membranes of cells or lipid membranes of vesicles, which plays various roles in physiological functions. However, there have been no experimental estimations of the membrane tension of vesicles upon exposure to Π. In this report, we estimated experimentally the lateral tension of the membranes of giant unilamellar vesicles (GUVs) when they were transferred into a hypotonic solution. First, we investigated the effect of Π on the rate constant, k_p, of constant-tension (σ_ex)-induced rupture of dioleoylphosphatidylcholine (DOPC)-GUVs using the method developed by us recently. We obtained the σ_ex dependence of k_p in GUVs under Π and by comparing this result with that in the absence of Π, we estimated the tension of the membrane due to Π at the swelling equilibrium, . Next, we measured the volume change of DOPC-GUVs under small Π. The experimentally obtained values of and the volume change agreed with their theoretical values within the limits of the experimental errors. Finally, we investigated the characteristics of the Π-induced pore formation in GUVs. The corresponding to the threshold Π at which pore formation is induced is similar to the threshold tension of the σ_ex-induced rupture. The time course of the radius change of GUVs in the Π-induced pore formation depends on the total membrane tension, σ_t; for small σ_t, the radius increased with time to an equilibrium one, which remained constant for a long time until pore formation, but for large σ_t, the radius increased with time and pore formation occurred before the swelling equilibrium was reached. Based on these results, we discussed the and the Π-induced pore formation in lipid membranes.

Abstract

The cell-penetrating peptide R₉, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R₉ (CF-R₉) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R₉-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R₉ with dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) GUVs containing AF647 and small DOPG/DOPC vesicles. The fluorescence intensity of the GUV membrane due to CF-R₉ (i.e., the rim intensity) increased with time to a steady-state value, and then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R₉ entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which AF647 could leak. The fraction of entry of CF-R₉ at 6 min in the absence of pore formation, P_entry (6 min), increased with an increase in CF-R₉ concentration, but the CF-R₉ concentration in the lumen was low. We obtained similar results for dilauroyl-PG (DLPG)/ditridecanoyl-PC (DTPC) (2/8) GUVs. The values of P_entry (6 min) of CF-R₉ for DLPG/DTPC (2/8) GUVs were larger than those obtained with DOPG/DOPC (2/8) GUVs at the same CF-R₉ concentrations. In contrast, a high concentration of CF-R₉ induced pores in DLPG/DTPC (4/6) GUVs through which CF-R₉ entered the GUV lumen, so the CF-R₉ concentration in the lumen was higher. However, CF-R₉ could not enter DOPG/DOPC/cholesterol (2/6/4) GUVs. Analysis of the rim intensity showed that CF-R₉ was located only in the outer monolayer of the DOPG/DOPC/cholesterol (2/6/4) GUVs. On the basis of analyses of these results, we discuss the elementary processes by which CF-R₉ enters GUVs of various lipid compositions.

Abstract

The stretching of biomembranes and lipid membranes plays important roles in various physiological and physicochemical phenomena. Here we analyzed the rate constant k_p of constant tension-induced rupture of giant unilamellar vesicles (GUVs) as a function of tension σ using their activation energy U_a. To determine the values of k_p, we applied constant tension to a GUV membrane using the micropipette aspiration method and observed the rupture of GUVs, and then analyzed these data statistically. First, we investigated the temperature dependence of k_p for GUVs of charged lipid membranes composed of negatively charged dioleoylphosphatidylglycerol (DOPG) and electrically neutral dioleoylphosphatidylcholine (DOPC). By analyzing this result, the values of U_a of tension-induced rupture of DOPG/DOPC-GUVs were obtained. U_a decreased with an increase in σ, supporting the classical theory of tension-induced pore formation. The analysis of the relationship between U_a and σ using the theory on the electrostatic interaction effects on the tension-induced rupture of GUVs provided the equation of U_a including electrostatic interaction effects, which well fits the experimental data of the tension dependence of U_a. A constant which does not depend on tension, U₀, was also found to contribute significantly to U_a. The Arrhenius equations for k_p using the equation of U_a and the parameters determined by the above analysis fit well to the experimental data of the tension dependence of k_p for DOPG/DOPC-GUVs as well as for DOPC-GUVs. On the basis of these results, we discussed the possible elementary processes underlying the tension-induced rupture of GUVs of lipid membranes. These results indicate that the Arrhenius equation using the experimentally determined U_a is useful in the analysis of tension-induced rupture of GUVs.

Abstract

Tension plays a vital role in pore formation in biomembranes, but the mechanism of pore formation remains unclear. We investigated the temperature dependence of the rate constant of constant tension (σ)–induced pore formation in giant unilamellar vesicles of lipid membranes using an experimental method we developed. By analyzing this result, we determined the activation energy (U_a) of tension-induced pore formation as a function of tension. A constant (U₀) that does not depend on tension was found to contribute significantly to U_a. Analysis of the activation energy clearly indicated that the dependence of U_a on σ in the classical theory is correct, but that the classical theory of pore formation is not entirely correct due to the presence of U₀. We can reasonably consider that U₀ is a nucleation free energy to form a hydrophilic pre-pore from a hydrophobic pre-pore or a region with lower lateral lipid density. After obtaining U₀, the evolution of a pre-pore follows a classical theory. Our data provide valuable information that help explain the mechanism of tension-induced pore formation in biomembranes and lipid membranes.

Abstract

We investigated the effects of electrostatic interactions on the rate constant $\left(k_p\right)$ for tension-induced pore formation in lipid membranes of giant unilamellar vesicles under constant applied tension. A decrease in salt concentration in solution as well as an increase in surface charge density of the membranes increased $k_p$. These data indicate that $k_p$ increases as the extent of electrostatic interaction increases. We developed a theory on the effect of the electrostatic interactions on the free energy profile of the membrane containing a prepore and also on the values of $k_p$; this theory explains the experimental results and fits the experimental data reasonably well in the presence of weak electrostatic interactions. Based on these results, we conclude that a decrease in the free energy barrier of the prepore state due to electrostatic interactions is the main factor causing an increase in $k_p$.

Abstract

Antimicrobial peptide magainin 2 forms pores in lipid membranes and induces membrane permeation of the cellular contents. Although this permeation is likely the main cause of its bactericidal activity, the mechanism of pore formation remains poorly understood. We therefore investigated in detail the interaction of magainin 2 with lipid membranes using single giant unilamellar vesicles (GUVs). The binding of magainin 2 to the lipid membrane of GUVs increased the fractional change in the area of the membrane, δ, which was proportional to the surface concentration of magainin 2, X. This indicates that the rate constant of the magainin 2-induced two-state transition from the intact state to the pore state greatly increased with an increase in δ. The tension of a lipid membrane following aspiration of a GUV also activated magainin 2-induced pore formation. To reveal the location of magainin 2, the interaction of carboxyfluorescein (CF)-labeled magainin 2 (CF-magainin 2) with single GUVs containing a water-soluble fluorescent probe, AF647, was investigated using confocal microscopy. In the absence of tension due to aspiration, after the interaction of magainin 2 the fluorescence intensity of the GUV rim due to CF-magainin 2 increased rapidly to a steady value, which remained constant for a long time, and at 4–32 s before the start of leakage of AF647 the rim intensity began to increase rapidly to another steady value. In contrast, in the presence of the tension, no increase in rim intensity just before the start of leakage was observed. These results indicate that magainin 2 cannot translocate from the outer to the inner monolayer until just before pore formation. Based on these results, we conclude that a magainin 2-induced pore is a stretch-activated pore and the stretch of the inner monolayer is a main driving force of the pore formation.

Abstract

Fe₆₉V₆P₁₅C₁₀ amorphous metallic alloy was prepared by the standard melt spinning technique and characterize their structural, transport, thermal and magnetic properties. The composition of the as prepared alloy was confirmed by energy dispersive X-ray (EDX) and the surface morphology was carried out by scanning electron microscopy (SEM). The structure of the as prepared and annealed sample was studied by X-ray diffraction (XRD). The XRD patterns of annealed sample shows that contain a BCC structure for temperatures between 400ºC and 450ºC and a hexagonal structure for temperatures between 500ºC and 650ºC. The grain size of the sample is found to vary from 20 to 53 nm. Hall resistivity and magnetoresistance (MR) was measured using four-probe technique. Resistivity remains constant upto 400ºC and then decreases with the increase of temperature. The crystallization behavior of the sample was performed by differential thermal analysis (DTA). Magnetization was measured by using vibrating sample magnetometer (VSM) at room temperature and the measured value of the saturation magnetization was 82.6 emu/g. Both the magnitude of impedance and phase angle remained constant upto 10 MHz and then remarkably increased with frequency.

Abstract

We recently developed the single giant unilamellar vesicle (GUV) method for investigating the functions and dynamics of biomembranes. The single GUV method can provide detailed information on the elementary processes of physiological phenomena in biomembranes, such as their rate constants. Here we describe the process of pore formation induced by the antimicrobial peptide (AMP), magainin 2, and the pore-forming toxin (PFT), lysenin, as revealed by the single GUV method. We obtained the rate constants of several elementary steps, such as peptide/protein-induced pore formation in lipid membranes and the membrane permeation of fluorescent probes through the pores. Information on the entry of the cell-penetrating peptide (CPP), transportan 10 (TP10), into a single GUV and its induced pore formation in lipid membranes was also obtained. We compare the single GUV method with other methods for investigating the interaction of peptides/proteins with lipid membranes (i.e., the large unilamellar vesicle (LUV) suspension method, the GUV suspension method, and single channel recording), and discuss the pros and cons of the single GUV method. On the basis of these data, we discuss the advantages of the single GUV method.

Abstract

The amorphous (Fe_1-xMn_x)₇₅P₁₅C₁₀ (0 ≤ x ≥ 0.30) alloys were prepared by the standard melt spinning technique and investigated their crystallization, thermal, transport and magnetic properties. Crystallization was observed from 400℃ to 650℃ with an interval 50℃within 30 minutes annealing time by XRD. The as-cast samples were amorphous in nature. Annealing 400℃ to 450℃ samples showed the mixed bcc Fe and amorphous structures. The lattice parameter ‘a’ was varied from 2.855 to 2.859 ? but above 450℃, samples contained hexagonal, FeP and FeC structures. The lattice parameters ‘a’ and ‘c’ were varied from (5.016-5.036) ? and (13.575-13.820) ? , respectively. Average crystallite size was found to vary from 8 to 48 nm. Crystallization temperature and weight change were observed by differential thermal analysis and thermogravimetric analysis, respectively. Crystallization temperature was increased with increasing Mn content. Resistivity was increased above and bellows the Curie temperature. Real permeability remained almost constant upto around 10⁶ Hz for of all samples after that it was decreased with increasing frequency and it was also decreased with Mn, whereas imaginary permeability was increased sharply above frequency 10⁷ Hz. The value of saturation magnetization was found to decrease with increment Mn.

Abstract

(Fe100-xVx)75P15C10 (x=0, 5, 10 and 15) amorphous alloys in the form of ribbon were prepared by the standard melt spinning technique and studied their transport and magnetic properties. The resistivity follows ‘Mooij correlation’ at low temperature (300-93)K. The Hall resistivity and the magnetoresistance (MR) were measured in an applied magnetic field upto 0.6T at room temperature (RT=300 K). Anomalous Hall effect was observed in the Hall resistivity measurement and MR was found to vary 0-8%. The saturation magnetization gradually decreasedwith the increase of V in the alloys at RT.

Abstract

(Fe100-xVx)75P15C10[x=1.5, 3, 9 and 15] semi-amorphous alloys (partially crystalline) in the form of ribbon were prepared by the standard melt spinning technique and studied their transport, magnetic and thermal properties. The nature of the as prepared samples was studied by x-ray diffraction (XRD). The resistivity of the samples was investigated fromtemperature 93K to 800 K. The resistivity followed ‘Mooij Correlation’ at low temperature (93 K-300 K). The resistivity athigher temperature (300 K-800 K) remained constant upto a certain temperature and then decreased with temperature rise. The Hall resistivity and the magnetoresistance (MR) were measured in an applied magnetic field upto 0.6 T at room temperature (RT=300 K). Anomalous Hall effect was observed in the Hall resistivity measurement and MR was found to vary 0-8%. The saturation magnetization gradually decreases with the increase of the substitution of Fe by Vat RT. Both the impedance magnitude and phase angle remained constant upto 106 Hz and then remarkably increased with frequency. The thermal properties associated with crystallization temperature and weight changes were measured by using the differential thermal analyzer (DTA) and the thermo gravimetric (TG) techniques respectively.

Abstract

This work presents the results of an empirical study of the sensitivity of a four-electrode FIM
technique developed by us earlier, for objects of different conductivity. FIM has potential for the
characterization of biological tissue in physiological study and diagnosis. Experimental
measurements were performed on a 2D phantom made up of saline with a focused square zone at the
centre. Three cylindrical objects of different conductivities (an insulator, a conductor, and a piece of
potato offering an intermediate conductivity) were used for the sensitivity measurements. Adjacent
square zones had sensitivities of about 22% of that at the center for the insulator, about 13% for the
conductor and about 10% for potato, showing a better focusing in the last case. The outer locations
had negligible sensitivities. Inverse (negative) sensitivity, which is unavoidable in tetrapolar
impedance measurements, was small and negligible in all the cases. Degree of perturbation of
equipotential lines has been suggested to be the cause of the above differences in focusing, less
perturbation giving rise to better focusing, which would apply for most biological objects to be
studied using FIM.

Abstract

Zn1-x-yCdxLiyOδ (x=0.30; y=0.05, 0.10, 0.15, 0.20 and δ= 0.975, 0.95, 0.925, 0.90)samples have been prepared byconventional solid state reaction method and studied their electrical, magnetic and structural properties. The dc electrical resistivity measurement shows that all samples are highly resistive (~106 ohm-cm) upto a transition temperature (Tt), above which resistivity falls drastically that reveals the samples are semiconducting in nature and Ttdecreases with increase of Li. The activation energies vary from (0.74-0.51) eV depending on doping concentrations, i.e. the activation energy is lower for higher concentration of Li in the solution. Magnetic mass susceptibility measurement shows negative sign that indicate the samples are diamagnetic. From XRD analysis, there exist two phases one is hexagonal ZnO and another is cubic CdO which suggests the formation of superlattice structure of the system. Crystallite size at planes (100), (002), (101), (102), (110), (103), (200), (112) and (111), (200), (220) for ZnO and CdOranges from 20 to 50 nm and for Li ranges from 27 to 40 nm.

Abstract

Zn_1-x-yCd_xLi_yO (x=0.30 and y=0.05, 0.10, 0.15, 0.20) have been prepared by solid state reaction method. The prepared samples have been characterized by structural and dielectric measurements. X-ray diffraction (XRD) patterns show a good crystalline nature having double crystal structure and indicates the phase mixing of the constituent components. The hexagonal phase corresponding to ZnO and cubic phase to CdO is well defined and the lattice parameters are consistent with the published values (Grant in Aid report 1987). The estimated lattice parameters, bond length and crystallite size are quite consistent corresponding to the hexagonal ZnO and cubic CdO which suggests the formation of super lattice structure of the system. Crystallite size at different crystallographic planes analyzed from XRD for both ZnO and CdO lie between (15-50) nm. The variation of the dielectric constant of the samples with frequency is systematic and the dielectric constant increases with the increase of Li in the solution.

Abstract

Abstract

A recently developed Focused Impedance Measurement (FIM) system (by the authors’ group) uses six electrodes to localize a zone of interest. Because of 3D sensitivity it could give physiological information on large organs like stomach, lungs, etc. using surface electrodes in the frontal plane. This paper presents a modified FIM technique using four electrodes placed at the corners of a square matrix. Firstly current is driven through an adjacent electrode pair while the potential is measured across the opposite pair from which an impedance value is obtained. Then a similar measurement is made at 90° to the above by changing connections to the electrode pairs appropriately. The sum of these two impedance values has a dominant contribution from the central region within the square matrix, giving the desired focusing. Experimental sensitivity maps obtained from a 2D phantom have verified the focusing effect. Compared to the previous six-electrode FIM system the focusing effect is slightly less, but this new technique has less negative artifacts in the periphery. This new FIM method can be applied both in the frontal plane and in the transverse plane of the human thorax, giving a further advantage besides requiring fewer electrodes.