Influence of triphenyllead chloride on biological and model membranes

The physiological and hemolytic toxicities of triphenyllead chloride (TPhL) as well as its modifying influence on model lipid membranes were studied. The experiments allowed the determination of TPhL concentrations causing 50% inhibition of growth of Spirodela oligorrhiza, Lemna minor and Salvinia natans (EC50), 100% hemolysis of pig erythrocytes (C100) and destabilization of planar lipid membranes (CC). Also, fluidity of erythrocyte ghosts was measured by fluorescence technique and osmotic sensitivity of erythrocytes to the presence of TPhL. All parameters studied were found to be dependent on pH, of experimental solutions and the concentration of TPhL. Acidic conditions increased EC50 C100 and CC concentrations of TPhL. Fluorescence and osmotic measurements showed that osmotic stability and fluidity decreased with increasing trimethyllead concentration. A possible mechanism of TPhL toxicity is discussed. It is assumed that TPhL is interacting with the lipid phase of the models used. It is also assumed that there may exist various, ionic and nonionic, forms of TPhL as a result of its speciation under different experimental conditions. These species, due to their differentiated lipophilicity, may exert different effects on the model membranes studied.     

The effect of rimantadine on the structure of model and biological membranes

The action of the antiviral drug rimantadine on the structure of bilayer lipid membranes (BLM) and RBC membranes was investigated. Structural changes in BLM were recorded by ionophore conductivity changes and by changes in the third harmonic of capacity current signal due to lateral compression of BLM in an electric field. It was shown that the adsorption of rimantadine on BLM results in an increase in ionophore mobility in bilayer membranes of dioleolyllecithin (DOL) and common lipids of bovine brain (CL) and in a decrease in those of azolectin (A). Relative changes in the third harmonic signal also depend on the membrane composition and have different signs. The results may be explained by the rimantadine action on the lipid bilayer structure: "rigidification" of A-membranes and "fluidization" of BLM from DOL and CL. Structural reorganization of RBC membranes as investigated by the ability of the cells to enter a micropipette (inner diameter greater than or equal to 3 microns) thereby undergoing deformation. It was shown that rimantadine influences RBC deformability due to drug induced inhomogenous mechanical membrane properties. Also, rimantadine accelerated the process of artificially induced aggregation of erythrocytes. The relation of the effects on artificial and biological membranes, and the structural changes in the lipid phase of membrane are discussed.     

Antioxidative activity of some phenoxy and organophosphorous compounds

Experiments were performed in order to check whether biological activity of some organophosphorous compounds widely applied as herbicides: 2,4-dichlorophenoxyacetic acid (1) and its sodium salt (2), N-phosphonomethylglycine acid (3) and its sodium salt (4), diethyl 1-butylamino-1-cyclohexanephosphonate (5) and diethyl 9-butylamino-9-fluorenephosphonate (6) followed from their oxidative activity. The compounds studied differed in their polarity and hydrophobicity. On the contrary, it was found that all herbicides protected erythrocyte membranes against partial peroxidation induced by UV irradiation. The effect was somewhat differentiated and followed the sequence: 5 >1 >2 >6 >3 >4. The observed differences between the antioxidative activities of the compounds are probably related to differences in their ability to incorporate into the lipid phase of the erythrocyte membrane. Once incorporated, they change fluidity of the membranes. The extent of the changes was determined in fluorescence measurements. Polarization and anisotropy coefficients of erythrocyte membranes modified by micromolar concentrations of herbicides at different temperatures were measured for that purpose. Generally, they followed the sequence found for antioxidative activity of the herbicides studied, which confirms the assumption of close correlation between the depth of incorporation of a herbicide into the erythrocyte membrane and its protective efficiency.     

The hemolytic and physiological activities of mixtures of some phenoxy and organophosphorous herbicides

Experiments were performed investigating the potential to improve the biological activity of some phenoxy and organophosphorous compounds by using them in binary mixtures. The compounds were: 2,4-dichlorophenoxyacetic acid (1) and its sodium salt (2), dibutyl 1-butylamino-1-cyclohexanephosphonate (3) and diethyl 9-butylamino-9-fluorenephosphonate (4), all widely used as herbicides. There were two test methods: the inhibition of cucumber (Cucumis sativus) growth induced by one single herbicide or by equimolar binary mixtures of herbicides; and, in parallel, the hemolytic efficiency of separate compounds or their mixtures. The hemolytic properties of the compounds were studied as hemolysis is generally a good measure of their toxicity, especially in the case of lipophilic compounds. Pig erythrocytes were used as good models for the determination of toxicity and the kinetics of red blood cell hemolysis. In the plant-based experiments, binary mixtures were found to display additive type toxicity. The compounds' hemolytic activities were of additive or antagonistic types. In some combinations, the addition of a second component did not change the hemolytic efficiency of the first component, and vice versa.     

Insulin-induced changes in mechanical characteristics of lipid bilayers modified by liver plasma membrane fragments

Insulin interaction with BLM with incorporated fragments of rat liver plasma membranes, containing hormone receptors, was studied by determining Young modulus of elasticity of bilayer lipid membranes in direction perpendicular to the surface, E. The presence of membrane proteins in a concentration of 60 micrograms.ml-1 induced a significant decrease in parameter E (to approx. 50%) as compared with values obtained in non-modified membranes during insulin action (concentration interval 10(-11)-10(-9) mol.l-1). The extent of the effect was dependent on the initial phase state of the membrane, on cholesterol content in BLM as well as on membrane proteins concentration in lipid bilayer.     

Elasticity, strength and stability of bilayer lipid membranes and their changes due to phospholipid modification

Elasticity measurements of bilayer lipid membranes (BLM) based on registration of the third harmonic of the membrane current during the application of a periodic tension to the membrane was used to study the effects of lipid peroxidation (LPO) and phospholipase A on BLM. LPO resulted in decreased values of the Young modulus for BLM, while some products of LPO and phospholipid hydrolysis (linolenic acid) were able to increase drastically the modulus. The presence of individual products of LPO and phospholipid hydrolysis in BLM produced non-additive effects on the elasticity, strength and stability of BLM. Lysolecithine strongly affected both the strength and stability of BLM. without changing its elasticity modulus. It was found that the lower the rate of structural changes in lecithine BLM, the longer its lifetime. Membranes having a heterogeneous polar composition form more stable BLM as compared to chemically homogeneous membranes.     

Interaction of phospholipid bilayers with polyamines of different length

The tip-dip patch clamp method was used to study the effect of three polyamines, putrescine, spermidine and spermine, on bilayer lipid membrane (BLM) stability. Two kinds of mixed lipid-polyamine membranes were investigated. The poration voltage (V _p), and the closed (σ_cl) and open (σ_op) state conductances for pure and polyamine-treated lipid membranes were determined by the method of current-voltage surfaces. It was demonstrated that putrescine and spermidine destabilized lipid membranes under all circumstances. BLM stabilization by spermine was observed when it was added to preformed membranes. Received: 19 November 1999 / Revised version: 25 February 2000 / Accepted: 25 February 2000     

Importance of the cell membrane on the mechanism of action of cyclotides

Their distinctive structures, diverse range of bioactivities, and potential for pharmaceutical or agricultural applications make cyclotides an intriguing family of cyclic peptides. Together with the physiological role in plant host defense, cyclotides possess antimicrobial, anticancer, and anti-HIV activities. In all of the reported activities, cell membranes seem to be the primary target for cyclotide binding. This article examines recent literature on cyclotide-membrane studies and highlights the hypothesis that the activity of cyclotides is dependent on their affinity for lipid bilayers and enhanced by the presence of specific lipids, i.e., phospholipids containing phosphatidylethanolamine headgroups. There is growing evidence that the lipid composition of target cell membranes dictates the amount of cyclotides bound to the cell and the extent of their activity. After membrane targeting and insertion in the bilayer core, cyclotides induce disruption of membranes by a pore formation mechanism. This proposed mechanism of action is supported by biophysical studies with model membranes and by studies on natural biological membranes of known lipid compositions.     

Effects of some cyclic elements containing amphiphilic compounds on stability and transport properties of model lecithin membranes

The paper presents results of studies on changes in model membrane properties induced by some single-chain amphiphilic quaternary ammonium salts with fungicidal activity, N-dodecyl-N-methylmorpholinium chloride (IVC), N-dodecyloxymethylene-N-methylmorpholinium chloride (IVD), N-dodecyl-N-methylpiperidinium chloride (VIC), N-dodecyloxymethylene-N-methylpiperidinium chloride (VID) and N-dodecyl-N,N-dimethyl-N-benzy-lammonium chloride (IB). Two different lipid systems were used, namely, unilamellar liposomes and black planar membranes (BLM). All the compounds studied interfered with sulphate ion transport across liposomal membranes as well as with calcium ion desorption from the membranes. The compounds were found also to change the stability of black lecithin membranes. Generally the sequence of effectiveness of the salts studied on both models used was: IB greater than VID greater than IVD greater than VIC greater than IVC, although there were some differences for particular processes. The results obtained are discussed in view of a possible mechanism of the interaction between the salts studied and the lipid model including the role the salt head group structure and charge distribution can play in this interaction.     

Rimantadine effects on the elasticity of bilayer lipid membranes and on ion transport through gramicidin D channels.

The effect of the antiviral preparation rimantadine on lipid bilayer membranes (BLM) was studied by measuring the modulus of elasticity in the direction normal to the surface (E perpendicular) and by estimating the conductance lambda, the lifetime tau of single gramicidin D channels (GRD), and the coefficient of nonlinearity beta of current voltage characteristics (IVC) of GRD-modified BLM. Rimantadine induced a nonmonotonic change in E perpendicular of BLM prepared from a mixture of egg lecithin with cholesterol: at relatively low rimantadine concentrations (0-40 micrograms/ml) E perpendicular first increased, reached a maximum and started to decrease. The effectivity of rimantadine was dependent on the cholesterol concentration in the BLM. Changes in E perpendicular suggest an increased ordering of the lipid bilayer at low rimantadine concentrations and formation of clusters of the preparation at concentrations exceeding those necessary to obtain maximal values of E perpendicular for the given BLM lipid composition. Rimantadine concentrations lifetime by approximately 20 percent, affected the degree of IVC nonlinearity and superlinearity of GRD-modified membranes, which suggests some effect on the height of the barrier at the ionic channel mouth and in its centre.     

hERG ion channel pharmacology: cell membrane liposomes in porous-supported lipid bilayers compared with whole-cell patch-clamping

The purpose of this study was to obtain functional hERG ion channel protein for use in a non-cell-based ion channel assay. hERG was expressed in Sf9 insect cells. Attempts to solubilise the hERG protein from Sf9 insect cell membranes using non-ionic detergents (NP40 and DDM) were not successful. We therefore generated liposomes from the unpurified membrane fraction and incorporated these into porous Teflon-supported bilayer lipid membranes. Macroscopic potassium currents (1 nA) were recorded that approximated those in whole-cell patch-clamping, but the channels were bidirectional in the bilayer lipid membrane (BLM). Currents were partially inhibited by the hERG blockers E4031, verapamil, and clofilium, indicating that the protein of interest is present at high levels in the BLM relative to endogenous channels. Cell liposomes produced from Sf9 insect cell membranes expressing voltage-gated sodium channels also gave current responses that were activated by veratridine and inhibited by saxitoxin. These results demonstrate that purification of the ion channel of interest is not always necessary for liposomes used in macro-current BLM systems.     

Stalk dynamics and lipid flow upon membrane hemifusion

A hydrodynamic theory describing the stalk dynamics and lipid flows upon BLM hemifusion was developed. The value of intermonolayer viscosity, etar, for membranes formed from azolectin mixed with lysophosphatidylcholine (7.10(-4) mg/ml) in n-decane, etar approximately 10(-9) g/s, was determined from comparison of the theoretical calculations and literature data. For membranes formed in squalene, the values etar approximately 10(-7) g/s for phosphatidylethanolamine and etar approximately 2-10(-7) g/s for azolectin were obtained. The calculated values are close to the published results of independent experiments which shows that the developed theory describes well the stalk growth and lipid flow.     

Pore formation in lipid bilayer membranes made of phosphatidylinositol and oxidized cholesterol followed by means of alternating current.

The kinetics of porin incorporation into black lipid membranes (BLM) made of phosphatidylinositol (PI) or oxidized cholesterol (Ox Ch) were studied by means of alternating current; the set-up was able to acquire resistance and capacitance simultaneously by means of a mixed double-frequency approach at 1 Hz and 1 KHz, respectively. Conductance was dependent on the interaction between protein-forming pores and lipids. For PI membranes below a porin concentration of 12.54 ng/ml, there was no membrane conductivity, whereas at 200 ng/ml a steady-state value was reached. Different behavior was displayed by Ox Ch membranes, in which a concentration of 12.54 ng/ml was sufficient to reach a steady state. The incorporation kinetics when porin was added after membrane formation were sigmoidal. When porin was present in the medium before membrane formation, the kinetics were sigmoidal for PI membranes but became exponential for Ox Ch membranes. Furthermore, for BLM made of PI, the conductance-versus-porin concentration relationship is sigmoidal, with a Hill coefficient of 5.6 +/- 0.07, which is functional evidence corroborating the six-channel repeating units seen previously. For BLM made of Ox Ch, this relationship followed a binding isotherm curve with a Hill coefficient of 0.934 +/- 0.129.     

Interaction of human immunodeficiency virus (HIV-1) fusion peptides with artificial lipid membranes

The interaction of 11 overlapping synthetic peptides corresponding to N-terminal segment of HIV transmembrane glycoprotein gp41 (fusion domain) with artificial lipid membranes has been studied. For this purpose the increase of a bilayer lipid membrane (BLM) conductivity and the changes in ESR spectra of spin-labelled liposomes were registrated. Peptide fragment 523-532 gp160 (BRU strain) had the critical length with regard to channel-forming activity on BLM. The degree of such membranotropic action increased simultaneously with the growth of peptide length and the temperature in the cell. Peptides 518-532 and 517-532 lysed TEMPOcholine-containing liposomes at 37 degrees C. The significance of observed effects for explanation of the mechanism of HIV-induced membrane fusion is discussed.     

Lipoic acid decreases lipid peroxidation and protein glycosylation and increases (Na(+) + K(+))- and Ca(++)-ATPase activities in high glucose-treated human erythrocytes

Lipoic acid supplementation has been found to be beneficial in preventing neurovascular abnormalities in diabetic neuropathy. Insufficient (Na(+) + K(+))-ATPase activity has been suggested as a contributing factor in the development of diabetic neuropathy. This study was undertaken to test the hypothesis that lipoic acid reduces lipid peroxidation and glycosylation and can increase the (Na(+) + K(+))- and Ca(++)-ATPase activities in high glucose-exposed red blood cells (RBC). Washed normal human RBC were treated with normal (6 mM) and high glucose concentrations (45 mM) with 0-0.2 mM lipoic acid (mixture of S and R sterioisomers) in a shaking water bath at 37 degrees C for 24 h. There was a significant stimulation of glucose consumption by RBC in the presence of lipoic acid both in normal and high glucose-treated RBC. Lipoic acid significantly lowered the level of glycated hemoglobin (GHb) and lipid peroxidation in RBC exposed to high glucose concentrations. High glucose treatment significantly lowered the activities of (Na(+) + K(+))- and Ca(++)-ATPases of RBC membranes. Lipoic acid addition significantly blocked the reduction in activities of (Na(+) + K(+))- and Ca(++)-ATPases in high glucose- treated RBC. There were no differences in lipid peroxidation, GHb and (Na(+) + K(+))- and Ca(++)-ATPase activity levels in normal glucose-treated RBC with and without lipoic acid. Thus, lipoic acid can lower lipid peroxidation and protein glycosylation, and increase (Na(+) + K(+))- and Ca(++)-ATPase activities in high-glucose exposed RBC, which provides a potential mechanism by which lipoic acid may delay or inhibit the development of neuropathy in diabetes.     

Influence of counterions on the interaction of pyridinium salts with model membranes

The interaction of pyridinium salts (PS) with red blood cells and planar lipid membranes was studied. The aim of the work was to find whether certain cationic surfactant counterion influence its possible biological activity. The counterions studied were Cl-, Br-, I-, ClO4-, BF4- and NO3-. The model membranes used were erythrocyte and planar lipid membranes (BLM). At high concentration the salts caused 100% erythrocyte hemolysis (C100) or broke BLMs (CC). Both parameters describe mechanical properties of model membranes. It was found that the efficiency of the surfactant to destabilize model membranes depended to some degree on its counterion. In both, erythrocyte and BLM experiments, the highest efficiency was observed for Br-, the lowest for NO3-. The influence of all other anions on surfactant efficiency changed between these two extremities; that of chloride and perchlorate ions was similar. Some differences were found in the case of BF4- ion. Its influence on hemolytic possibilities of PS was significant while BLM destruction required relatively high concentration of this anion. Apparently, the influence of various anions on the destructive action of PS on the model membrane used may be attributed to different mobilities and radii of hydrated ions and hence, to different possibilities of particular anions to modify the surface potential of model membranes. This can lead to a differentiated interaction of PS with modified bilayers. Moreover, the effect of anions on the water structure must be taken into account. It is important whether the anions can be classified as water ordering kosmotropes that hold the first hydration shell tightly or water disordering chaotropes that hold water molecules in that shell loosely.     

Bilayer lipid membranes (BLM): Study of antigen-antibody interactions

Previous work of del Castillo and co-workers has shown that bilayer lipid membranes (BLM) can be used as transducers for detection of antigen-antibody reactions. The present experiments extend the previous work by incorporating complement into the BLM system. The results indicate that the antigen-antibody complex or the complement has no ability to affect the BLM system separately, but when carefully combined they will destabilize the BLM even at a much reduced concentration. Further development using the BLM as a tool for investigating immunological reactions is suggested.     

Cholesterol-dependent gramicidin A channel inactivation in red blood cell membranes and lipid bilayer membranes

The exchange diffusions of tracer cations (22Na+, 86Rb+) are studied on gramicidin-A-treated red blood cell (RBC) membranes. A time-dependent decrease in cation permeability has been observed and has been considered to be the result of a channel inactivation process. The channel inactivation appears at 20 and 30 degrees C but not at a temperature as low as 6 degrees C. The gramicidin A channel inactivation can be monitored by a conductivity decay of molecular lipid membranes (BLM) prepared either from cholesterol or from a mixture of cholesterol and phospholipids but not of pure phosphatidylethanolamine. The role of cholesterol in the channel inactivation is discussed.     

The interaction of bee melittin with lipid bilayer membranes

The influence of melittin and the related 8-26 peptide on the stability and electrical properties of bilayer lipid membranes is reported. Melittin, unlike the 8-26 peptide, has a dramatic influence on lipid membranes, causing rupture at dilute concentrations. The circular dichroism of melittin demonstrated that under physiological conditions, in water, melittin is in extended conformation, which is enhanced in aqueous ethanol. However in 'membrane-like' conditions it is essentially alpha-helical. Secondary structure predictions were used to locate possible alpha-helical nucleation centres and a model of melittin was built according to these predictions. It is postulated that melittin causes a wedge effect in membranes.     

Probing the stability of S-layer-supported planar lipid membranes

Isolated protein subunits of the crystalline bacterial cell surface layer (S-layer) of Bacillus coagulans E38-66 have been recrystallized on one side of planar black lipid membranes (BLMs) and their influence on the electrical properties, rupture kinetics and mechanical stability of the BLM was investigated. The effect on the boundary potential, the capacitance or the conductance of the membrane was negligible whereas the mechanical properties were considerably changed. The mechanical stability was characterized by applying voltage pulses or ramps to induce irreversible rupture. The amplitude of the voltage pulse leading to rupture allows conclusions on the ability of membranes to resist external forces. Surprisingly, these amplitudes were significantly lower for composite S-layer/lipid membranes compared to undecorated BLMs. In contrast, the delay time between the voltage pulse and the appearance of the initial defect was found to be drastically longer for the S-layer-supported lipid bilayer. Furthermore, the kinetics of the rupture process was recorded. Undecorated membranes show a fast linear increase of the pore conductance in time, indicating an inertia-limited defect growth. The attachment of an S-layer causes a slow exponential increase in the conductance during rupture, indicating a viscosity-determined widening of the pore. In addition, the mechanical properties on a longer time scale were investigated by applying a hydrostatic pressure across the BLMs. This causes the BLM to bulge, as monitored by an increase in capacitance. Compared to undecorated BLMs, a significantly higher pressure gradient has to be applied on the S-layer face of the composite BLMs to observe any change in capacitance. Received: 4 May 1999 / Revised version: 1 July 1999 / Accepted: 1 July 1999