The Mechanisms of Lipid Vesicle Fusion Inhibition by Extracts of Chaga and Buckthorn Leaves.

The ability of extracts of grapefruit seeds (ESG), sea buckthorn leaves (ESBL), and chaga (EC) to inhibit membrane fusion was evaluated. It was found that ESBL and EC inhibited Ca2+-mediated fusion of phosphatidylglycerol-enriched lipid vesicles; the inhibition indexes were about 90 and 100%, respectively. ESG did not inhibit the fusion of negatively charged liposomes induced by calcium. In addition to calcium-mediated liposome fusion, EC inhibited the fusion of vesicles from a mixture of phosphatidylcholine and cholesterol under the action of polyethylene glycol with a molecular weight of 8000 Da (the inhibition index was 80%). The other two extracts had no effect on polymer-induced fusion of uncharged membranes. The effect of some major components of the tested extracts on the fusion of vesicles was evaluated. It has been shown that flavonols, quercetin and myricetin, which are major components of ESBL, inhibited the fusion of negatively charged membranes under the action of calcium (the inhibition indexes were about 85 and 60%, respectively). Another flavonol of ESBL, the glycoside of quercetin rutin, did not have such an effect. The data obtained made it possible to relate the ESBL suppression of calcium-induced fusion of lipid vesicles with the presence of quercetin and myricetin in its composition. These flavonols had virtually no effect on polyethylene glycol-induced vesicle fusion, which is consistent with the absence of ESBL action on liposome fusion under the action of polymer. The ability of quercetin and myricetin to reduce the melting temperature of phosphatidylglycerol with saturated hydrocarbon chains and to increase the half-width of the peak corresponding to melting has been demonstrated. The observed correlation between the parameters characterizing the thermotropic behavior of the lipid in the presence of quercetin and myricetin and the index of inhibition of calcium-mediated liposome fusion by these compounds may indicate a relationship between the ability of flavonols to influence the packaging of membrane lipids and inhibit vesicle fusion. Pentacyclic triterpenoids, betulin and lupeol, which are part of EC, did not inhibit the fusion of vesicles under the action of both calcium and polyethylene glycol, and their presence in EC cannot be responsible for the antifusogenic activity of EC.

The mechanisms of action of water-soluble aminohexanoic and malonic adducts of fullerene C60 with hexamethonium on model lipid membranes.

In an attempt to understand the possibility of applications of the fullerene-based systems for transporting various polar compounds like hexamethonium through the blood-brain barrier, we studied the influence of a series of derivatives of fullerene C60 in the form of salts with hexamethonium bis-anion, namely the adducts of fullerenols with 6-aminohexanoic acid (IEM-2197), and two bis-adduct malonic acid derivatives of fullerene with addents bound in two hemispheres (IEM-2143) and in equatorial positions (IEM-2144), on model membranes. We showed that IEM-2197 induced the disintegration of the bilayers composed of DOPC at the concentrations more than 2 mg/ml. IEM-2144 and IEM-2143-induced ion-permeable pores at concentrations of 0.3 and 0.02 mg/ml, respectively; herewith, IEM-2143 was characterized by the greater efficiency than IEM-2144. IEM-2197 did not significantly affect the phase behavior of DPPC, while the melting temperature significantly decreased with addition of IEM-2144 and IEM-2143. The increase in the half-width of the main transition peaks by more than 2.0 °C in the presence of IEM-2144 and IEM-2143 was observed, along with the pronounced peak deconvolution. We proposed that the immersion of IEM-2144 and IEM-2143 into the polar region of the DOPC or DPPC bilayers led to an increase in the relative mobility of tails and formation of ion-permeable defects. IEM-2197 demonstrated the more pronounced effects on the melting and ion permeability of PG- and PS-containing bilayers compared to PC-enriched membranes. These results indicated that IEM-2197 preferentially interacts with the negatively charged lipids compared to neutral species.

The Mechanisms of Action of Triindolylmethane Derivatives on Lipid Membranes.

The effects of new synthetic antibacterial agents - tris(1-pentyl-1H-indol-3-yl)methylium chloride (LCTA-1975) and (1-(4-(dimethylamino)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indol-3-yl)bis(1-propyl- 1H-indol-3-yl)methylium chloride (LCTA-2701 - on model lipid membranes were studied. The ability of the tested agents to form ion-conductive transmembrane pores, influence the electrical stability of lipid bilayers and the phase transition of membrane lipids, and cause the deformation and fusion of lipid vesicles was investigated. It was established that both compounds exert a strong detergent effect on model membranes. The results of differential scanning microcalorimetry and measuring of the threshold transmembrane voltage that caused membrane breakdown before and after adsorption of LCTA-1975 and LCTA-2701 indicated that both agents cause disordering of membrane lipids. Synergism of the uncoupling action of antibiotics and the alkaloid capsaicin on model lipid membranes was shown. The threshold concentration of the antibiotic that caused an increase in the ion permeability of the lipid bilayer depended on the membrane lipid composition. It was lower by an order of magnitude in the case of negatively charged lipid bilayers than for the uncharged membranes. This can be explained by the positive charge of the tested agents. At the same time, LCTA-2701 was characterized by greater efficiency than LCTA-1975. In addition to its detergent action, LCTA-2701 can induce ion-permeable transmembrane pores: step-like current fluctuations corresponding to the opening and closing of individual ion channels were observed. The difference in the mechanisms of action might be related to the structural features of the antibiotic molecules: in the LCTA-1975 molecule, all three substituents at the nitrogen atoms of the indole rings are identical and represent n-alkyl (pentyl) groups, while LCTA-2701 contains a maleimide group, along with two alkyl substituents (n-propyl). The obtained results might be relevant to our understanding of the mechanism of action of new antibacterial agents, explaining the difference in the selectivity of action of the tested agents on the target microorganisms and their toxicity to human cells. Model lipid membranes should be used in further studies of the trends in the modification and improvement of the structures of new antibacterial agents.

Dipole Modifiers Regulate Lipid Lateral Heterogeneity in Model Membranes.

In this study we report on experimental observations of giant unilamellar liposomes composed of ternary mixtures of cholesterol (Chol), phospholipids with relatively low Tmelt (DOPC, POPC, or DPoPC) and high Tmelt (sphingomyelin (SM), or tetramyristoyl cardiolipin (TMCL)) and their phase behaviors in the presence and absence of dipole modifiers. It was shown that the ratios of liposomes exhibiting noticeable phase separation decrease in the series POPC, DOPC, DPoPC regardless of any high-Tmelt lipid. Substitution of SM for TMCL led to increased lipid phase segregation. Taking into account the fact that the first and second cases corresponded to a reduction in the thickness of the lipid domains enriched in low- and high-Tmelt lipids, respectively, our findings indicate that the phase behavior depends on thickness mismatch between the ordered and disordered domains. The dipole modifiers, flavonoids and styrylpyridinium dyes, reduced the phase segregation of membranes composed of SM, Chol, and POPC (or DOPC). The other ternary lipid mixtures tested were not affected by the addition of dipole modifiers. It is suggested that dipole modifiers address the hydrophobic mismatch through fluidization of the ordered and disordered domains. The ability of a modifier to partition into the membrane and fluidize the domains was dictated by the hydrophobicity of modifier molecules, their geometric shape, and the packing density of domain-forming lipids. Phloretin, RH 421, and RH 237 proved the most potent among all the modifiers examined.

DIPOLE-MODIFYING EFFECT OF STYRYLPYRIDINIUM DYES AND FLAVONOIDS ON THE MODEL MEMBRANES OF DIFFERENT LIPID COMPOSITIONS.

The changes in the dipole potential of lipid bilayers (jd) mimicking cell membranes induced by the adsorption of low molecular weight amphiphiles, flavonoids (phloretin and quercetin) and styrylpyridinium dyes (RH 421 and RH 237) were measured. The method based on the determination of ionophore-induced transmembrane current was used to evaluate the changes in jd after the addition of the modifiers. The characteristic parameters of the Langmuir adsorption isotherm, the maximum changes in jd at an infinitely large concentration of flavonoid and its desorption constant, which reflects the affinity of the flavonoid to the lipid phase, were determined. The slopes of linear dependencies of increasing in jd on the concentration of the styrylpiridinium dyes in membrane bathing solution were defined. It was found that the dipole-modifying effect of phloretin depends on the charge of the lipids forming the membranes, while the ability of quercetin to reduce jd turned on the initial hydration of the bilayers. The results indicated on the different mechanisms of the decrease in jd at the adsorption of tested flavonoids. It was shown that the changes in jd at the incorporation of styrylpyridinium dyes into bilayers is determined by the interaction of the modifiers with the membrane components.

NCREASING OF THE FLUIDITY OF MODEL LIPID MEMBRANES UNDER THE INFLUENCE OF LOCAL ANESTHETICS.

We have studied the effect of local anesthetics on the permeability of phospholipid liposomes of various composition to calcein. We have tested amides (lidocaine, prilocaine, mepivacaine and bupivacaine) and ester anesthetics (benzocaine, procaine and tetracaine). The permeability of large unilamellar liposomes to calcein has been determined by measuring the relative fluorescence intensity of calcein released from the phospholipid vesicles. We have shown that all tested amide anesthetics have little of feet on the leakage of calcein from dioleoylphosphocholine (DOPC) liposomes. The most effective amide was did bupivacaine. Substitution in the membranes of 20 mol % DOPC on tetraoleoylcardiolipin (TOCL) was did accompanied by increased activity of amides. Benzocaine and procaine at concentration up to 100 mM practically did not change DOPC liposome permeability. Addition of tetracaine up to concentration of 2 mM did not affect the permeability of vesicl. Further increas in the anesthetic concentration up to 50 mM led to increase in the intensity of calcein fluorescence and, at a concentration of 100 mM, a complete engagement of fluorescent marker was observed. In the case of vesicles including 20 mol % TOCL, the threshold concentration of tetracaine and the concentration that corresponded to 100% leakage of calcein were been 7 and 20 mM, respectively. Using confocal fluorescence microscopy of giant unilamellar vesicles formed from an equimolar mixture of DOPC and tetramyristoylcardiolipin (TMCL), we have shown that anesthetics destroy solid ordered domains, and this ability increases in the order: procaine d mepivacaine < bupivacaine n tetracaine. The revealed differences in the effect of local anesthetics might be caused by different depth of immersion of the anesthetics into the membrane.

[The influence of dipole modifiers on the channel-forming activity of amyloid and amyloid-like peptides in lipid bilayers].

We have studied the steady-state transmembrane current induced by amyloid and amyloid-like peptides in lipid bilayers in the presence of dipole modifiers. It has been shown that the addition of dipole modifier, phloretin, to the membrane bathing solutions leads to an increase in the multichannel activity of amyloid beta-peptide fragment 25-35, [Gly35]-amyloid beta-peptide fragment 25--35, prion protein fragment 106-126 and amyloid-like peptides myr-BASP1 (1--13), myr-BASP1(1--19) and GAP-43(1--40). We have found that the effect of phloretin is not the result of dipole potential changes due to adsorption of this modifier on the membrane. Using the various fragments of amyloid beta-peptide, presenilin, prion protein and neuronal proteins BASP1 and GAP-43 allowes to conclude that the steady-state peptide-induced transmembrane current in the case of addition of phloretin is due to the electrostatic interaction between the positively charged channel-forming agents and negatively charged dipole modifier. The results obtained by electron microscopy have demonstrated that this interaction increases degree of peptide oligomerization.

Modifiers of the Dipole Potential of Lipid Bilayers.

This paper assesses the magnitude of change in the dipole potential (φd) of membranes caused by the adsorption of modifiers on lipid bilayers of various compositions. We tested flavonoids, muscle relaxants, thyroid hormones, and xanthene and styrylpyridinium dyes in order to assess their dipole-modifying properties. A quantitative description of the modifying action of flavonoids, muscle relaxants, thyroid hormones, and xanthene dyes is shown as the ratio of the maximum change in the bilayer dipole potential upon saturation and the absolute φd value of the unmodified membrane. The slopes of the linear relationship between the increase in the dipole potential of phospholipid bilayers and the concentration of styrylpyridinium dyes in membrane-bathing solutions were found. We described the relationships between the change in φd and the chemical structure of modifiers, as well as the charge and spontaneous curvature of lipid monolayers.

Investigation of channel-forming activity of polyene macrolide antibiotics in planar lipid bilayers in the presence of dipole modifiers.

The role of membrane components, sterols, phospholipids and sphingolipids in the formation and functioning of ion-permeable nanopores formed by antifungal macrolide antibiotics, amphotericin B, nystatin and filipin in planar lipid bilayers was studied. Dipole modifiers, flavonoids and styryl dyes, were used as a tool to study the molecular mechanisms of polyene channel-forming activity. The introduction of dipole modifiers into the membrane bathing solutions was shown to change the conductance of single channels and the steadystate transmembrane current induced by polyene antibiotics in the sterol-containing phospholipid-bilayers. The conductance of single amphotericin B channels was found to depend on the dipole potential of the membrane. The experiments with various phospholipids, sterols, and polyenes led to the assumption that the shape of a phospholipid molecule, the presence of double bonds at the positions 7 and 22 of a sterol molecule, the number of conjugated double bonds, and the presence of an amino sugar in the polyene antibiotic molecule are important factors impacting the stability of polyene-lipid complexes forming ion-permeable pores. Experimental and literature data presented in the paper suggest that the channel-forming activity of polyene antibiotics is also affected by the physicochemical properties of polyene-enriched ordered membrane domains.

[Changes of dipole potential of phospholipid membranes resulted from flavonoid adsorption].

The effects of flavonoids, phloridzin, quercetin, myricetin and biochanin A on the dipole potential of planar lipid bilayers formed from dioleylphosphoethanolamine, dioleylphosphoserine, dioleoylphosphocholine, and diphytanoylphosphocholine are investigated. The characteristic parameters of the Langmuir adsorption isotherm, the maximum changes in the membrane dipole potential at an infinitely large concentration of flavonoid and its dissociation constant, which reflects the affinity of flavonoid to the membrane lipids, are determined. Modifying effects of chalcones, flavonols and isoflavones are compared. The influence of the surface charge of the lipid bilayer and the spontaneous curvature of the membrane-forming phospholipids on the adsorption of flavonoids on the model membranes is discussed.

[The effects of RH 421 on the activity of amphotericin B in cell and model membranes].

The combined action of polyene macrolide antibiotic amphotericin B and styryl dye RH 421 in model lipid membranes and in yeast Saccharomyces cerevisiae has been studied. RH 421 insertion inti the ergosterol-containing lipid bilayers led to the increase of the steady-state number of open amphotericin channels by 8.5 +/- 3.2 times. Paper disk agar diffusion method has demonstrated that RH 421 enhanced the antimicrobial effect of amphotericin B leading to an increase in the zone of growth inhibition of S. cerevisiae strain by 1.5 times. Our data suggest that RH 421 is a potential synergist of amphotericin B and can be used in new formulations with improved pharmacological properties.

[Transport of large organic ions through syringomycin channels in the membranes containing dipole modifiers].

The effect of the membrane dipole potential (Phid) on a conductance and a steady-state number of functioning channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes made from phosphocholine and bathed in 0.4 M solution of sodium salts of aspartate, gluconate and chloride was shown. The magnitude of Phid was varied with the introduction to membrane bathing solutions of phloretin, which reduces the Phid, and RH 421, increasing the Phid. It was established that in all studied systems the increase in the membrane dipole potential cause a decrease in the steady-state number of open channels. In the systems containing sodium salts of aspartate (Asp) or gluconate (Glc), changes in the number of functioning channels are in an order of magnitude smaller than in systems containing sodium chloride. At the same time, the conductance (g) of single SRE-channels on the membranes bathed in NaCI solution increases with the increase in Phid, and in the systems containing NaAsp or NaGlc the conductance of single channels does not depend on the Phid. The latter is due to the lack of cation/anion selectivity of the SRE-channels in these systems. The different channel-forming activity of SRE in the experimental systems is defined by the gating charge of the channel and the partition coefficient of the dipole modifiers between the lipid and aqueous phases.

[Conductance of phytotoxin channels in the presence of large organic ions].

In this study we evaluated the effect of penetrating anion size on the properties of the channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes. Conductance and the mean lifetime of the SRE channels have been measured in 0.4 M solutions of sodium chloride, aspartate, and gluconate. Comparison of the results of conductometrical and electrophysiological measurements has shown: 1) ratio of mobilities of aspartate anions in the channel and in the water solution is 5 times lower than that for chlorine anions; 2) conductivity of the channels in the presence of sodium gluconate is due mainly to cations. The results obtained indicate binding of the penetrating anions to the selective filter of the SRE pore. The radius of the SRE channel selective filter has been established (r - 0.3 nm) and its localization in the cis-mouth of the pore has been found.