alamethicin and fluorexon

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 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

Alamethicin is a well-studied channel-forming peptide that has a prototypical amphipathic helix structure. It permeabilizes both microbial and mammalian cell membranes, causing loss of membrane polarization and leakage of endogenous contents. Antimicrobial peptide-lipid systems have been studied quite extensively and have led to significant advancements in membrane biophysics. These studies have been performed on lipid bilayers that are generally charged or zwitterionic and restricted to a thickness range of 3-5 nm. Bilayers of amphiphilic diblock copolymers are a relatively new class of membranes that can have significantly different physicochemical properties compared with those of lipid membranes. In particular, they can be made uncharged, nonzwitterionic, and much thicker than their lipid counterparts. In an effort to extend studies of membrane-protein interactions to these synthetic membranes, we have characterized the interactions of alamethicin and several other membrane-active peptides with diblock copolymer bilayers. We find that although alamethicin is too small to span the bilayer, the peptide interacts with, and ruptures, thick polymer membranes.

Topics: Alamethicin; Circular Dichroism; Fluoresceins; Hydrophobic and Hydrophilic Interactions; Spectrometry, Fluorescence; Water

Amoebapores, the pore-forming polypeptides of the protozoan parasite Entamoeba histolytica, and NK-lysin, an effector molecule of porcine NK (natural killer) and cytotoxic T cells, belong to the same protein family, the saposin-like proteins. As both types of protein are implicated in the killing of microbes in vivo, it appears that phylogenetically diverse organisms such as amoebae and mammals use similar effector molecules to fulfil a comparable task. However, structural features have led to the assumption that the proteins display their activities according to different modes of action. To address this question, we analysed the antibacterial, cytotoxic and pore-forming activities of these proteins in parallel and in comprehensive detail. Interestingly, the comparison of activities revealed significant differences. Whereas NK-lysin, recombinantly expressed, is efficient at a broad range of pH values, the amoebapores exhibited a pronounced pH dependence of all their activities, with markedly decreased activity at pH values above 6. Moreover, increasing salinity affects amoebapores more drastically than NK-lysin. All of the proteins compared were found to be potently active against Gram-positive bacteria, but only NK-lysin was equally efficient against Gram-negative bacteria. However, the amoebapores displayed five times higher pore-forming activity than NK-lysin, which is in accordance with the more hydrophobic character of the amoebapores compared with the essentially cationic NK-lysin.

Topics: Alamethicin; Animals; Anti-Bacterial Agents; Bacillus subtilis; Cell Division; Dose-Response Relationship, Drug; Entamoeba histolytica; Escherichia coli; Fluoresceins; Humans; Hydrogen-Ion Concentration; Ion Channels; Jurkat Cells; Leukocytes, Mononuclear; Liposomes; Protein Isoforms; Proteolipids; Protozoan Proteins; Swine

Interactions of hypelcin A, an alpha-aminoisobutyric acid containing antibiotic peptide, with phosphatidylcholine vesicles were investigated to obtain information on its bioactive mechanism. The peptide induced the leakage of a fluorescent dye, calcein, entrapped in sonicated vesicles. The leakage rate depended on both the peptide and the lipid concentrations. Analysis of this dependency indicated that the leakage was due to the monomeric peptide and that the membrane-perturbing activity of the monomer was higher for solid distearoylphosphatidylcholine vesicles than for fluid egg yolk phosphatidylcholine vesicles. Hypelcin A also affected the gel to liquid-crystalline phase transition of dipalmitoylphosphatidylcholine multilamellar vesicles. The transition was broadened with a reduced transition enthalpy, suggesting the peptide strongly binds the surrounding lipids to perturb the bilayer lipid packing. A circular dichroism study revealed that the helical content of hypelcin A increases upon membrane binding. We concluded that the monomeric peptide with an increased helical content, complexed with the lipids, perturbs the lipid organization and induces the increased permeability.

Topics: Alamethicin; Amino Acid Sequence; Aminoisobutyric Acids; Anti-Bacterial Agents; Circular Dichroism; Fluoresceins; Kinetics; Lipid Bilayers; Molecular Sequence Data; Permeability; Phosphatidylcholines; Protein Conformation; Temperature