melitten and fluorexon

Antimicrobial peptides are one of the most promising alternatives to antibiotics for targeting pathogens without developing resistance. In this study, pore formation in 1,2-Dimyristoyl-snglycero-3-phosphocholine (DMPC) / cholesterol liposome induced by native melittin, its two mutant variants (G1I and I17 K), and cecropin P1 was investigated by monitoring the dynamics of fluorescence dye leakage. A critical peptide concentration was required for dye leakage with the rate of leakage being dependent on peptide concentration above a critical value. A lag time was required for dye leakage for low peptide concentrations that are above the critical value, which decreased at higher peptide concentrations eventually approaching zero. Lag time was found to be in the order I17 K mutant with lower hydrophobicity and higher net charge > G1I with higher hydrophobicity > melittin > cecropin P1. Cecropin P1 exhibited the highest rate of dye leakage followed by melittin, G1I, and I17 K. Size distribution and transmission electron microscopy (TEM) of liposomes exposed to peptides of different concentrations indicated pore formation with accompanied stretching of liposomes at low peptide concentrations for both melittin and cecropin P1. At much higher concentrations, however, size distribution indicated three peaks for both peptides. In both cases, TEM images show that the middle and small peaks are shown to be due to stretched liposome and broken stretched liposome respectively. For melittin, the large peak is due to peptide aggregates as well as aggregates of liposome. For cecropin P1, however, the large peak indicates cecropin P1 aggregates with solubilized lipids thus suggesting carpet mechanism.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Cholesterol; Diffusion; Dimyristoylphosphatidylcholine; Dose-Response Relationship, Drug; Fluoresceins; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Kinetics; Lipid Bilayers; Liposomes; Melitten; Mutation; Peptides; Protein Aggregates; Static Electricity; Structure-Activity Relationship; Thermodynamics

Niosomes are used in studies for drug delivery or gene transfer. However, their physical properties and features relative to liposomes are not well documented. To characterize and more rationally optimize niosome formulations, the properties of these vesicle systems are compared to those of liposomes composed of phosphatidylcholine and phosphatidylethanolamine lipids plus cholesterol. Niosomes are highly stable and only slightly more leaky than liposomes as assayed by calcein leakage; the permeability for ions (KCl) is higher than that of liposomes. Contrary to liposomes, the size of niosomes decreases substantially upon freezing in liquid nitrogen and subsequent thawing, as shown by cryo-EM and dynamic light scattering. The packing of niosomal membranes was determined by laurdan fluorescence and is slightly lower than that of liposomes. We did not succeed in the functional reconstitution of the L-arginine/L-ornithine antiporter ArcD2 in niosomes, which we attribute to the non-ionic nature of the surfactants. The antimicrobial peptides alamethicin and melittin act similarly on niosomes and liposomes composed of unsaturated components, whereas both niosomes and liposomes are unaffected when saturated amphiphiles are used. In conclusion, in terms of stability and permeability for drug-size molecules niosomes are comparable to liposomes and they may offer an excellent, inexpensive alternative for delivery purposes.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Alamethicin; Antimicrobial Cationic Peptides; Arginine; Cholesterol; Cryoelectron Microscopy; Detergents; Drug Delivery Systems; Fluoresceins; Hexoses; Light; Lipids; Liposomes; Melitten; Nitrogen; Ornithine; Osmosis; Permeability; Phosphatidylethanolamines; Polysorbates; Scattering, Radiation; Surface-Active Agents

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.

Topics: Animals; Bee Venoms; Bees; Cell Membrane; Circular Dichroism; Dose-Response Relationship, Drug; Fluoresceins; Lipids; Liposomes; Melitten; Molecular Conformation; Phosphatidylcholines; Protein Structure, Tertiary; Surface Properties

The changes induced by biologically active substances in the permeability to K+ and calcein of liposomes composed of egg phosphatidylcholine and cholesterol were measured simultaneously in order to rapidly screen the sizes of pores formed in a membrane, using different sized markers. The substances examined in the present study were classified into three types based on differences in the rates at which K+ and calcein were released. The first type released only K+, and included gramicidin A. The second type predominantly released K+, preceding the release of calcein, and included amphotericin B and nystatin. The third type, including antimicrobial peptides, such as gramicidin S, alamethicin, and melittin, and several membrane-active drugs, like celecoxib (non-steroidal anti-inflammatory drug), 1-dodecylazacycloheptan-2-one (named azone; skin permeation enhancer), and chlorpromazine (tranquilizer), caused the release of K+ and calcein simultaneously. Thus, the sizes of pores formed in a liposomal membrane increased in the following order: types one, two, and three. We determined the size more precisely by conducting an osmotic protection experiment, measuring the release of calcein in the presence of osmotic protectants of different sizes. The radii of pores formed by the second type, amphotericin B and nystatin, were 0.36 - 0.46 nm, while the radii of pores formed by the third type were much larger, 0.63 - 0.67 nm or more. The permeability changes induced by substances of the third type are discussed in connection with a transient pore formed in a lipid packing mismatch taking place during the phase transition of dipalmitoylphosphatidylcholine liposomes.

Topics: Alamethicin; Amphotericin B; Azepines; Celecoxib; Chlorpromazine; Fluoresceins; Gramicidin; Liposomes; Melitten; Membranes, Artificial; Nystatin; Permeability; Potassium; Pyrazoles; Sulfonamides

Bionanocapsules (BNCs) are hollow nanoscale particles composed of L protein of the hepatitis B virus surface antigen that represent specific affinity for human hepatocytes. BNCs can transfer genes and drugs into human hepatocytes efficiently and specifically. BNC can be expressed in yeast cells. In this study, we developed a new L particle production system using a stably transfected insect cell line. For this purpose, we established a host-vector system using the Trichoplusia ni insect cell line. L particles were efficiently secreted by the overexpression of the L protein, which was fused to the secretion signal peptide. The concentration of L particles was reached approximately 1.7 microg/ml in 5 days during cultivation in a serum-free medium without antibiotic selective pressure. The production of L particles was maintained for at least 75 days. The secretory production of L particles facilitated their easy purification by chromatography. Furthermore, it was demonstrated that purified L particles can transfect only human hepatocytes. Therefore, an insect cell expression system is an attractive tool for the production of BNC.

Topics: Animals; Bees; Cell Line; Cell Line, Tumor; Drosophila; Electroporation; Fluoresceins; Genetic Vectors; Hepatocytes; Humans; Melitten; Moths; Nanocapsules; Promoter Regions, Genetic; Protein Sorting Signals; Recombinant Proteins; Sensitivity and Specificity; Transfection; Viral Envelope Proteins

Peptides derived from the unprocessed N-termini of mouse and bovine prion proteins (mPrPp and bPrPp, respectively), comprising hydrophobic signal sequences followed by charged domains (KKRPKP), function as cell-penetrating peptides (CPPs) with live cells, concomitantly causing toxicity. Using steady-state fluorescence techniques, including calcein leakage and polarization of a membrane probe (diphenylhexatriene, DPH), as well as circular dichroism, we studied the membrane interactions of the peptides with large unilamellar phospholipid vesicles (LUVs), generally with a 30% negative surface charged density, comparing the effects with those of the CPP penetratin (pAntp) and the pore-forming peptide melittin. The prion peptides caused significant calcein leakage from LUVs concomitant with increased membrane ordering. Fluorescence correlation spectroscopy (FCS) studies of either rhodamine-entrapping (REVs) or rhodamine-labeled (RLVs) vesicles, showed that addition of the prion peptides resulted in significant release of rhodamine from the REVs without affecting the overall integrity of the RLVs. The membrane leakage effects due to the peptides had the following order of potency: melittin>mPrPp>bPrPp>pAntp. The membrane perturbation effects of the N-terminal prion peptides suggest that they form transient pores (similar to melittin) causing toxicity in parallel with their cellular trafficking.

Topics: Animals; Biophysics; Carrier Proteins; Cattle; Cell Membrane; Cell-Penetrating Peptides; Circular Dichroism; Diffusion; Diphenylhexatriene; Fluoresceins; Melitten; Mice; Peptides; Prions; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Rhodamines; Spectrometry, Fluorescence; Spectrophotometry; Time Factors

The cytolytic peptides melittin and gramicidin S are naturally occurring agents that provide a comparative model for studies of complement, immunotoxin and cell-mediated membrane permeability. Most attempts to characterize cytolytic peptides have used model membrane systems including phospholipid vesicles or erythrocytes. Membrane vesicles permit the use of self-quenching concentrations of fluorescent permeability markers, while erythrocytes release measurable hemoglobin. Attempts at measuring early membrane permeability changes in nucleated mammalian cells have been limited. To measure the kinetics of mammalian cell membrane permeability changes induced by cytolytic peptides, we developed a 96-well fluorescence cytolysis assay using the cytoplasmic fluorescent dye calcein as the membrane permeability marker. To facilitate rapid assessment of membrane permeability, trypan blue was added to the assay solution to quench (a) released fluorescence and (b) retained intracellular fluorescence. Trypan blue also provided a complementary visual assessment of cell viability. Using this assay, a detailed kinetic analysis demonstrated permeability of the cell membranes within seconds of exposure to the cytolytic peptides. The rapid permeabilization of the cell membranes was confirmed by flow cytometry using the calcium indicator dye fluo-3. The assay also demonstrated a second slower phase of marker release over the next several hours. The fluorescence cytolysis assay was able to reliably detect the biphasic permeability changes associated with the melittin and gramicidin S peptides suggesting the potential utility of this assay in the assessment of other cytolytic agents.

Topics: Animals; Cell Line; Cell Membrane Permeability; Cytotoxins; Fluoresceins; Fluorescent Dyes; Gramicidin; Humans; Mammals; Melitten; Peptides; Sheep; Time Factors; Trypan Blue

A new fluorescence method has been developed to measure simultaneously and independently the release of fluorophores from two vesicle populations. Calcein and sulforhodamine B were used as a probe couple: the leakage of these probes from vesicles can be recorded independently since they can be excited simultaneously at 510 nm, and their individual fluorescence can be isolated by measuring the fluorescence signal at 525 and 590 nm, using a T-shape fluorometer. Controls show that both probes are suitable for the leakage assay based on fluorescence self-quenching, that they do not interact physically or chemically at the concentrations used in the method, and that they leak in a similar fashion from a given vesicle type. This dual-probe technique is applied to examine the specificity of the release relative to the cholesterol content of the vesicles for melittin, a toxin. This new approach shows in a straightforward manner that melittin-induced release for a given population can be modulated by the presence of vesicles with another lipid composition and this competitive release is associated with a preferential distribution of the peptide on the targeted vesicles.

Topics: Cell Membrane Permeability; Cholesterol; Fluoresceins; Fluorescent Dyes; Lipid Bilayers; Liposomes; Melitten; Phosphatidylcholines; Rhodamines; Spectrometry, Fluorescence

Melittin, a bee venom, is a basic amphiphilic peptide, which mainly acts on the lipid matrix of membranes, lysing various cells. To elucidate the molecular mechanism, we investigated its interactions with phospholipid vesicles. The peptide formed a pore with a short lifetime in the membrane, as revealed by the release of an anionic fluorescent dye, calcein, from the liposomes. Our new double-labeling method clarified that the pore size increased with the peptide-to-lipid ratio. Upon the disintegration of the pore, a fraction of the peptides translocated across the bilayer. The pore formation was coupled with the translocation, which was proved by three fluorescence experiments recently developed by our laboratory. A novel model for the melittin pore formation was discussed in comparison with other pore-forming peptides.

Topics: Dextrans; Dithionite; Energy Transfer; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Kinetics; Lipid Bilayers; Liposomes; Melitten; Models, Theoretical; Spectrometry, Fluorescence; Structure-Activity Relationship

Transmembrane osmotic gradients applied on large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles were used to modulate the potency of melittin to induce leakage. Melittin, an amphipathic peptide, changes the permeability of vesicles, as studied using the release of entrapped calcein, a fluorescent marker. A promotion of the ability of melittin to induce leakage was observed when a hyposomotic gradient (i.e., internal salt concentration higher than the external one) was imposed on the vesicles. It is proposed that structural perturbations caused by the osmotic pressure loosen the compactness of the outer leaflet, which facilitates the melittin-induced change in membrane permeability. Additionally, we have shown that this phenomenon is not due to enhanced binding of melittin to the vesicles using intrinsic fluorescence of the melittin tryptophan. Furthermore, we investigated the possibility of using a transmembrane pH gradient to control the lytic activity of melittin. The potency of melittin in inducing release is known to be inhibited by increased negative surface charge density. A transmembrane pH gradient causing an asymmetric distribution of unprotonated palmitic acid in the bilayer is shown to be an efficient way to modulate the lytic activity of melittin, without changing the overall lipid composition of the membrane. We demonstrate that the protective effect of negatively charged lipids is preserved for asymmetric membranes.

Topics: Biophysical Phenomena; Biophysics; Cholesterol; Electrochemistry; Fluoresceins; Hydrogen-Ion Concentration; In Vitro Techniques; Melitten; Membranes, Artificial; Osmolar Concentration; Osmosis; Osmotic Pressure; Permeability; Phosphatidylcholines

The leakage induced by melittin, a membrane-perturbing amphipathic peptide, from large unilamellar 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles was studied using calcein as fluorescent marker. The extent of leakage has been found to be regulated by the melittin/lipid molar ratio. Melittin leads to the complete release of trapped calcein from some vesicles. This all-or-none mechanism leads to the co-existence of two different vesicle populations: the 'empty' and the intact one. Intervesicular migration of melittin was not observed. The results reveal a specific targeting of the lysed vesicles by melittin. The presence of negatively charged lipids (unprotonated palmitic acid or 1-palmitoyl-2-oleoylphosphatidylglycerol) in the neutral POPC matrix inhibits the lytic power of melittin; this inhibition increases with increasing surface charge density. It is proposed that the anchorage of the peptide on the charged surface prevents the formation of defects allowing leakage. A statistical model based on a random distribution of the peptide molecules on the vesicles is proposed to describe the release induced by melittin. It is proposed that about 250 melittin molecules per vesicle are required to affect the bilayer permeability and to empty a vesicle of its content. This large number suggests that leakage is more likely due to collective membrane perturbation by the peptide rather than to the formation of a well-defined pore.

Topics: Electrochemistry; Fluoresceins; Liposomes; Melitten; Models, Statistical; Palmitic Acid; Palmitic Acids; Phosphatidylcholines; Phosphatidylglycerols; Spectrometry, Fluorescence; Structure-Activity Relationship

Synthetic calmodulin-binding (CaM-binding) peptides (CBPs) representing CaM-binding domains of Ca2+/CaM-dependent enzymes have been reported to interfere with the activity of the melanocyte-stimulating hormone (MSH) receptor function in melanoma cells [Gerst, J. E., & Salomon, Y. (1988) J. Biol. Chem. 263, 7073-7078]. We postulated that membrane lipids may play an important role in the mode of action of CBPs on cells. We therefore tested the ability of CBPs to interact with membrane bilayers. Using artificial phospholipid vesicles, or M2R melanoma cells and cell membranes derived therefrom, as models, we report here that synthetic peptides representing the CaM-binding domains of skeletal muscle myosin light chain kinase (M5) and the human erythrocyte calcium pump (C28W), as well as other CBPs, interact with lipid bilayers and cell membranes. Significant interactions of CBPs with the lipid bilayer were detected in both model systems. M5 and C28W were found to partition into the lipid bilayer of melanoma cell membranes and soybean lecithin vesicles, and surface partition constants obtained (for the liposome model) were in the range 10(3)-10(4) M-1. In addition, C28W and its N-modified NBD derivative were found to inhibit [125I]iodo-[Nle4,D-Phe7]alpha MSH binding to cultured M2R melanoma cells. These and other CBPs were also found to induce the release of cations and calcein from liposomes, suggesting that the interaction of CBPs with the lipid bilayer increases membrane permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: alpha-MSH; Amino Acid Sequence; Animals; Binding Sites; Calcium-Transporting ATPases; Calmodulin; Calmodulin-Binding Proteins; Cell Membrane; Cell Membrane Permeability; Erythrocytes; Fish Venoms; Fluoresceins; Humans; Lipid Bilayers; Melanoma, Experimental; Melitten; Mice; Molecular Sequence Data; Muscles; Myosin-Light-Chain Kinase; Peptides; Tumor Cells, Cultured

The effects of the lytic peptides, melittin and delta-haemolysin, are compared in vesicles of gel-phase dipalmitoylphosphatidylcholine (DPPC), using calcein as trapped marker. At low concentration, both toxins cause vesicles to lose contents in 5 mM phosphate buffer near neutral pH, with melittin being the more active. As phosphate concentration is increased, the kinetics of melittin-induced leakage change from a slow, sustained loss to a rapid 'burst' of leakage when melittin is present mainly as tetramer in solution, under conditions where it is reported to lose haemolytic activity towards erythrocytes. At low phosphate concentration, the leakage induced by delta-haemolysin is preceded by a lag phase, though fluorescence measurements show that binding of toxin is rapid. At higher phosphate concentration, the toxin binds rapidly to vesicles, but causes no leakage of entrapped calcein. Steady-state fluorescence spectra show no obvious differences in tryptophan emission for delta-haemolysin bound to lipid in high- or low-phosphate buffer. Spin-label fluorescence-quenching studies show that the single tryptophan residue of delta-haemolysin is buried within the lipid bilayer at all phosphate concentrations used. In gel-phase DPPC, delta-haemolysin shows no tendency to cause vesicle aggregation over several hours, as judged by light scattering, though a slow non-linear effect is seen above the lipid phase transition temperature. These effects are contrasted with those of melittin under similar conditions.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Bacterial Proteins; Bee Venoms; Cell Membrane; Fluoresceins; Hemolysin Proteins; Hemolysis; Hydrogen-Ion Concentration; Kinetics; Lipid Bilayers; Melitten; Phosphatidylcholines; Scattering, Radiation; Spectrometry, Fluorescence