n-n--4-xylylenebis(pyridinium) and 8-amino-1-3-6-naphthalenetrisulfonic-acid

Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non-human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis, and localized aggressive periodontitis. Aggregatibacter actinomycetemcomitans secretes a repeats-in-toxin protein, leukotoxin, which exclusively kills lymphocyte function-associated antigen-1-bearing cells. The toxin's pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell's plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and destabilization. In this study, we tested this hypothesis by analysing lipid-induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin-membrane association, we observed an increase in leukotoxin α-helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans.

Topics: Aggregatibacter actinomycetemcomitans; Bacterial Toxins; Cell Death; Cell Membrane; Circular Dichroism; Cytotoxins; Ethanolamines; Exotoxins; Fluorescent Dyes; Humans; Jurkat Cells; Liposomes; Lymphocyte Function-Associated Antigen-1; Lymphocytes; Lysophosphatidylcholines; Naphthalenes; Phosphatidylcholines; Protein Binding; Protein Structure, Secondary; Pyridinium Compounds; Surface Plasmon Resonance

Plantaricin149a (Pln149a) is a cationic antimicrobial peptide, which was suggested to cause membrane destabilization via the carpet mechanism. The mode of action proposed to this antimicrobial peptide describes the induction of an amphipathic α-helix from Ala7 to Lys20, while the N-terminus residues remain in a coil conformation after binding. To better investigate this assumption, the purpose of this study was to determine the contributions of the Tyr1 in Pln149a in the binding to model membranes to promote its destabilization. The Tyr to Ser substitution increased the dissociation constant (KD) of the antimicrobial peptide from the liposomes (approximately three-fold higher), and decreased the enthalpy of binding to anionic vesicles from -17.2 kcal/mol to -10.2 kcal/mol. The peptide adsorption/incorporation into the negatively charged lipid vesicles was less effective with the Tyr1 substitution and peptide Pln149a perturbed the liposome integrity more than the analog, Pln149S. Taken together, the peptide-lipid interactions that govern the Pln149a antimicrobial activity are found not only in the amphipathic helix, but also in the N-terminus residues, which take part in enthalpic contributions due to the allocation at a lipid-aqueous interface.

Topics: Amino Acid Sequence; Anti-Infective Agents; Bacteriocins; Calorimetry; Circular Dichroism; Hemolysis; Humans; Liposomes; Microbial Sensitivity Tests; Molecular Sequence Data; Naphthalenes; Peptides; Phosphatidylglycerols; Phospholipids; Pyridinium Compounds; Spectrometry, Fluorescence; Structure-Activity Relationship; Tryptophan; Tyrosine

To develop mucoadhesive liposomes by anchoring the polymer chitosan-thioglycolic acid (chitosan-TGA) to the liposomal surface to target intestinal mucosal membranes.. Liposomes consisting of phosphatidylcholine (POPC) and a maleimide-functionalized lipid were incubated with chitosan-TGA, leading to the formation of a thioether bond between free SH-groups of the polymer and maleimide groups of the liposome. Uncoated and newly generated thiomer-coated liposomes were characterized according to their size, zeta potential, and morphology using photon correlation spectroscopy and transmission electron microscopy. The release behavior of calcitonin and the fluorophore/quencher-couple ANTS/DPX (8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis- pyridinium bromide) from coated and uncoated liposomes, was investigated over 24 hours in simulated gastric and intestinal fluids. To test the mucoadhesive properties of thiomer-coated and uncoated liposomes in-vitro, we used freshly excised porcine small intestine.. Liposomes showed a concentration-dependent increase in size - from approximately 167 nm for uncoated liposomes to 439 nm for the highest thiomer concentration used in this study. Likewise, their zeta potentials gradually increased from about -38 mV to +20 mV, clearly indicating an effective coupling of chitosan-TGA to the surface of liposomes. As a result of mucoadhesion tests, we found an almost two-fold increase in the mucoadhesion of coupled liposomes relative to uncoupled ones. With fluorescence microscopy, we saw a tight adherence of coated particles to the intestinal mucus.. Taken together, our current results indicate that thiomer-coated liposomes possess a high potential to be used as an oral drug-delivery system.

Topics: Adhesiveness; Animals; Cell Line, Tumor; Cell Survival; Chitosan; Histocytochemistry; In Vitro Techniques; Intestinal Mucosa; Intestine, Small; Liposomes; Maleimides; Microscopy, Fluorescence; Naphthalenes; Particle Size; Phosphatidylcholines; Pyridinium Compounds; Sulfhydryl Compounds; Swine; Thioglycolates

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis(1). Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)(1). After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues(2). In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)(3-6). Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation(7,8). In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members(7,9). As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane. Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization(10). LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)(10). This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for ki

Topics: Animals; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Humans; Liposomes; Naphthalenes; Proto-Oncogene Proteins c-bcl-2; Pyridinium Compounds; Xenopus

We describe fluorescence assays for membrane fusion involving the fusion of liposomes with each other and with cultured cells, fluorescence methods to assess liposome uptake by cells and the intracellular delivery of liposome contents, and assays to evaluate liposome membrane permeability. The Tb/DPA and ANTS/DPX assays monitor the intermixing of aqueous contents of liposomes. The NBD-PE/Rhodamine-PE assay follows the intermixing of liposomal lipids. A variation of this method is suitable for detecting the mixing of the inner monolayers of liposomes. The lipid-mixing assay is also used to study the fusion of cationic liposomes and lipoplexes with cultured cells. The intracellular delivery of liposome contents are monitored, via fluorescence microscopy or flow cytometry, by measuring the release of calcein from the liposome interior, and normalized to cell-associated liposomes quantitated with Rhodamine-PE in the membrane of the same liposomes. The release of liposome contents is monitored by the increase in fluorescence of encapsulated carboxyfluorescein, calcein, or ANTS/DPX, or by the decrease in fluorescence of encapsulated Tb/DPA.

Topics: Animals; Cell Line; Cell Membrane Permeability; Flow Cytometry; Fluoresceins; Humans; Liposomes; Membrane Fusion; Microscopy, Fluorescence; Naphthalenes; Permeability; Picolinic Acids; Pyridinium Compounds; Terbium

We have been examining the mechanism and kinetics of the interactions of a selected set of peptides with phospholipid membranes in a quantitative manner. This set was chosen to cover a broad range of physical-chemical properties and cell specificities. Mastoparan (masL) and mastoparan X (masX) are two similar peptides from the venoms of the wasps Vespula lewisii and Vespa xanthoptera, respectively, and were chosen to complete the set. The rate constants for masX association with and dissociation from membranes are reported here for the first time. The kinetics of dye efflux induced by both mastoparans from phospholipid vesicles were also examined and quantitatively analyzed. We find that masL and masX follow the same graded kinetic model that we previously proposed for the cell-penetrating peptide transportan 10 (tp10), but with different parameters. This comparison is relevant because tp10 is derived from masL by addition of a mostly nonpolar segment of seven residues at the N-terminus. Tp10 is more active than the mastoparans toward phosphatidylcholine vesicles, but the mastoparans are more sensitive to the effect of anionic lipids. Furthermore, the Gibbs free energies of binding and insertion of the peptides calculated using the Wimley-White transfer scales are in good agreement with the values derived from our experimental data and are useful for understanding peptide behavior.

Topics: Amino Acid Sequence; Animals; Cell Membrane Permeability; Fluoresceins; Fluorescent Dyes; Galanin; Humans; Intercellular Signaling Peptides and Proteins; Lipid Bilayers; Membrane Lipids; Models, Molecular; Molecular Sequence Data; Naphthalenes; Peptides; Phospholipids; Protein Isoforms; Pyridinium Compounds; Recombinant Fusion Proteins; Thermodynamics; Wasp Venoms; Wasps

To better understand the influence of phospholipid acyl-chain composition on the formation of pores by cytotoxic amphipathic helices in biological membranes, the leakage of aqueous contents induced by the synthetic peptide GALA (WEAALAEALAE ALAEHLAEALAEALEALAA) from large unilamellar phospholipid vesicles of various compositions has been studied. Peptide-mediated leakage was examined at pH 5.0 from vesicles made of phosphatidylcholine (PC) and phosphatidylglycerol (PG) with the following acyl-chain compositions: 1-palmitoyl-2-oleoyl (PO), 1,2-dioleoyl (DO), 1, 2-dielaidoyl (DE), and 1,2-dipetroselinoyl (DPe). A mathematical model predicts and simulates the final extents of GALA-mediated leakage of 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and p-xylene-bis-pyridinium bromide (DPX) from 1-palmitoyl-2-oleoyl-phosphatidylcholine/1-palmitoyl-2-oleoyl-phospha tidylglycerol (POPC/POPG) and 1, 2-dielaidoyl-sn-glycero-3-phosphocholine/1, 2-dielaidoyl-phosphatidylglycerol (DEPC/DEPG) liposomes at pH 5.0 as a function of peptide concentration in the bilayer, by considering that GALA pores responsible for this leakage have a minimum size of 10 +/- 2 monomers and are formed by quasiirreversible aggregation of the peptide. With the phospholipid acyl-chain compositions tested, GALA-induced ANTS/DPX leakage follows the rank order POPC/POPG approximately DEPC/DEPG > DPePC/DPePG > DOPC/DOPG. Results from binding experiments reveal that this reduced leakage from DOPC/DOPG vesicles cannot be explained by a reduced binding affinity of the peptide to these membranes. As shown by monitoring the leakage of a fluorescent dextran, an increase in the minimum pore size also does not explain the reduction in ANTS/DPX leakage. The data suggest that surface-associated GALA monomers or aggregates are stabilized in bilayers composed of phospholipids containing a cis unsaturation per acyl chain (DO and DPe), while transbilayer peptide insertion is reduced. GALA-induced ANTS/DPX leakage is also decreased when the vesicles contain phosphatidylethanolamine (PE). This lends further support to the suggestion that factors stabilizing the surface state of the peptide reduce its insertion and subsequent pore formation in the bilayer.

Topics: 4-Chloro-7-nitrobenzofurazan; Amino Acid Sequence; Animals; Dextrans; Fluorescent Dyes; Ion Channels; Kinetics; Liposomes; Molecular Sequence Data; Naphthalenes; Peptides; Permeability; Phospholipids; Pyridinium Compounds; Xanthenes

The potential biomedical utility of the photoinduced destabilization of liposomes depends in part on the use of green to near infrared light with its inherent therapeutic advantages. The polymerization of bilayers can be sensitized to green light by associating selected amphiphilic cyanine dyes, i.e. the cationic 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), or the corresponding anionic disulfonated DiI (DiI-DS), with the lipid bilayer. The DiI sensitization of the polymerization of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine/1,2-bis[10-(2', 4'-hexadienoyloxy)-decanoyl]-sn-glycero-3-phosphocholine liposomes caused liposome destabilization with release of encapsulated aqueous markers. In separate experiments, similar photosensitive liposomes were endocytosed by cultured HeLa cells. Exposure of the cells and liposomes to 550 nm light caused a net movement of the liposome-encapsulated 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) from low pH compartment(s) to higher pH compartment(s). This suggests that photolysis of DiI-labelled liposomes results in delivery of the contents of the endocytosed liposomes to the cytoplasm. The release of HPTS into the cytoplasm appears to require the photoactivated fusion of the labelled liposomes with the endosomal membrane. These studies aid in the design of visible light sensitive liposomes for the delivery of liposome-encapsulated reagents to the cytoplasm.

Topics: Arylsulfonates; Carbocyanines; Color; Cytoplasm; Drug Carriers; Endocytosis; Endosomes; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Light; Liposomes; Membrane Fusion; Naphthalenes; Phosphatidylcholines; Phosphatidylethanolamines; Photolysis; Pyridinium Compounds; Sulfonic Acids; Temperature; Ultraviolet Rays

The incorporation of a reduced amide bond, psi(CH(2)NH), into peptide results in an increase in the net positive charge and the perturbation of alpha-helical structure. By using this characteristic of the reduced amide bond, we designed and synthesized novel pseudopeptides containing reduced amide bonds, which had a great selectivity between bacterial and mammalian cells. A structure-activity relationship study on pseudopeptides indicated that the decrease in alpha-helicity and the increase in net positive charge in the backbone, caused by the incorporation of a reduced amide bond into the peptide, both contributed to an improvement in the selectivity between lipid membranes with various surface charges. However, activity results in vitro indicated that a perturbation of alpha-helical structure rather than an increase in net positive charge in the backbone is more important in the selectivity between bacterial and mammalian cells. The present result revealed that the backbone of membrane-active peptides were important not only in maintaining the secondary structure for the interactions with lipid membranes but also in direct interactions with lipid membranes. The present study showed the unique function of a reduced amide bond in cytolytic peptides and a direction for developing novel anti-bacterial agents from cytolytic peptides that act on the lipid membrane of micro-organisms.

Topics: Amides; Amino Acid Sequence; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Bacteria; Candida albicans; Cell Membrane; Chromatography, High Pressure Liquid; Circular Dichroism; Coloring Agents; Erythrocytes; Fluoresceins; Hemolysis; Liposomes; Mice; Microbial Sensitivity Tests; Naphthalenes; Oxidation-Reduction; Peptides; Phospholipids; Protein Structure, Secondary; Pyridinium Compounds; Sodium Dodecyl Sulfate; Static Electricity; Structure-Activity Relationship; Substrate Specificity; Trifluoroethanol

We prepared large unilamellar vesicles (LUVs) with three different stratum corneum lipid compositions: constant amounts of ceramides (55 wt %) and fatty acids (15%) with varying amounts of cholesterol sulfate (0-15%) and cholesterol (15-30%). One of the compositions served as a model for normal stratum corneum, while the second one served as a model for recessive X-linked ichthyosis stratum corneum. The third composition consisted of no cholesterol sulfate. Intervesicle lipid interactions in these LUVs were monitored by fluorescence methods for content leakage, and contents mixing at pH 9, in the absence and presence of Ca2+, and at pH 6. Since the content leakage and contents mixing assays were originally developed for phospholipid vesicles, we characterized the probe binding and the probe quenching properties for stratum corneum LUV systems, and modified the assays slightly accordingly. The time-dependent fluorescence intensity changes in the probe-containing LUVs at pH 9 and 6 and in response to the addition of calcium were monitored. Our results demonstrated that all three types of LUVs were relatively stable at pH 9. Addition of Ca2+ or decreasing the pH to 6 activated intervesicle lipid mixing followed by vesicle fusion and lysis. We found that the LUVs with no cholesterol sulfate and 30% cholesterol exhibited a more extensive Ca2+- or low-pH-activated intervesicle lipid interaction than LUVs with either 5% cholesterol sulfate and 25% cholesterol or 15% cholesterol sulfate and 15% cholesterol. These results suggest that fusogenic agents such as Ca2+ and H+ act to neutralize the fatty acids in the lipid bilayer of stratum corneum vesicles. The inclusion of 5-15% cholesterol sulfate helps to prevent the collapse of fused vesicles into other structures.

Topics: Animals; Binding Sites; Calcium; Cattle; Chromatography, Thin Layer; Fluorescence Polarization; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Ichthyosis; Lipid Bilayers; Lipid Metabolism; Lipids; Mice; Models, Chemical; Naphthalenes; Pyridinium Compounds; Skin; Spectrometry, Fluorescence

The ability of the shark antimicrobial aminosterol squalamine to induce the leakage of polar fluorescent dyes from large unilamellar phospholipid vesicles (LUVs) has been measured. Micromolar squalamine causes leakage of carboxyfluorescein (CF) from vesicles prepared from the anionic phospholipids phosphatidylglycerol (PG), phosphatidylserine (PS), and cardiolipin. Binding analyses based on the leakage data show that squalamine has its highest affinity to phosphatidylglycerol membranes, followed by phosphatidylserine and cardiolipin membranes. Squalamine will also induce the leakage of CF from phosphatidylcholine (PC) LUVs at low phospholipid concentrations. At high phospholipid concentrations, the leakage of CF from PC LUVs deviates from a simple dose-response relationship, and it appears that some of the squalamine can no longer cause leakage. Fluorescent dye leakage generated by squalamine is graded, suggesting the formation of a discrete membrane pore rather than a generalized disruption of vesicular membranes. By using fluorescently labeled dextrans of different molecular weight, material with molecular weight /=10,000 is retained. Negative stain electron microscopy of squalamine-treated LUVs shows that squalamine decreases the average vesicular size in a concentration-dependent manner. Squalamine decreases the size of vesicles containing anionic phospholipid at a lower squalamine/lipid molar ratio than pure PC LUVs. In a centrifugation assay, squalamine solubilizes phospholipid, but only at significantly higher squalamine/phospholipid ratios than required for either dye leakage or vesicle size reduction. Squalamine solubilizes PC at lower squalamine/phospholipid ratios than PG. We suggest that squalamine complexes with phospholipid to form a discrete structure within the bilayers of LUVs, resulting in the transient leakage of small encapsulated molecules. At higher squalamine/phospholipid ratios, these structures release from the bilayers and aggregate to form either new vesicles or squalamine/phospholipid mixed micelles.

Topics: Animals; Anti-Bacterial Agents; Cattle; Cholestanols; Detergents; Dogfish; Fluoresceins; Liposomes; Membrane Lipids; Micelles; Microscopy, Electron; Naphthalenes; Permeability; Phosphatidylcholines; Phospholipids; Pyridinium Compounds

Tryptophan, proline, and basic amino acids have all been implicated as being important in the assembly and structure of membrane proteins. Indolicidin, an antimicrobial 13-residue peptide-amide isolated from the cytoplasmic granules of bovine neutrophils, is highly enriched in these amino acids: five tryptophans, three prolines, three basic residues, and no acidic residues. Consistent with the likely importance of these amino acids in membrane protein assembly, indolicidin is known to be highly membrane-active and is believed to act by disruption of cell membranes. We have, therefore, examined the interactions of native indolicidin with large unilamellar vesicles (LUV) formed from palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylglycerol (POPG), in order to use it as a model system for studying membrane protein insertion and for evaluating the relative contributions of hydrophobic and electrostatic forces in peptide-bilayer interactions. Equilibrium dialysis measurements indicate that indolicidin binds strongly, but reversibly, to both neutral POPC and anionic POPG vesicles with free energies of transfer of -8.8 +/- 0.2 and -11.5 +/- 0.4 kcal/mol, respectively. The extremely strong partitioning into POPG vesicles necessitated the development of a new equilibrium dialysis method that is described in detail. Tryptophan fluorescence measurements show that indolicidin is located in the bilayer interface and that indole fluorescence is affected by the type of lipid used to form the LUVs. Circular dichroism (CD) measurements reveal unordered conformations in aqueous and bulk organic solutions and a somewhat more ordered, but not alpha-helical, conformation in SDS micelles and lipid bilayers. Fluorescence requenching measurements (Ladokhin et al. 1995. Biophys. J. 69:1964-1971) on vesicles loaded with the fluorophore/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX), show that indolicidin induces membrane permeabilization. For anionic POPG, leakage is graded with a high preference for the release of cationic DPX over anionic ANTS. For neutral POPC vesicles no such preference is observed. Leakage induction is more effective with POPG vesicles than with POPC vesicles, as judged by three quantitative measures that are developed in the Appendix.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Kinetics; Lipid Bilayers; Liposomes; Naphthalenes; Octoxynol; Peptides; Permeability; Phosphatidylcholines; Phosphatidylglycerols; Pyridinium Compounds; Spectrometry, Fluorescence; Spectrophotometry; Tryptophan

Cecropin A is a naturally occurring, linear, cationic, 37-residue antimicrobial peptide. The precise mechanism by which it kills bacteria is not known, but its site of action is believed to be the cell membrane. To investigate the nature of its membrane activity, we examined the ability of cecropin A to alter membrane permeability in synthetic lipid vesicles and in Gram-negative bacteria. Cecropin A exerted distinctly different types of membrane activity depending on its concentration. In synthetic lipid vesicles, cecropin A dissipated transmembrane electrochemical ion gradients at relatively low concentrations, but much higher concentrations were required to release an encapsulated fluorescent probe. Cecropin A dissipated ion gradients whether or not the vesicle membranes contained anionic lipid, although the presence of anionic lipid dramatically increased peptide binding, and modestly increased the release of an encapsulated probe. Cholesterol did not prevent the dissipation of ion gradients by low concentrations of peptide, but it did inhibit release of the encapsulated probe by high concentrations of peptide. At the highest concentrations examined, cecropin A remained monomeric in solution, and did not aggregate, lyse, or otherwise alter vesicle size. In Gram-negative bacteria, cecropin A was potently bactericidal at concentrations which dissipated ion gradients in lipid vesicles, but much higher concentrations were required to cause the release of cytoplasmic contents. These findings point to the conclusion that cecropin A kills bacteria by dissipating transmembrane electrochemical ion gradients. They weigh against theories comparing the antimicrobial activity of cecropin A to the release of encapsulated probes from lipid vesicles, and against roles for cholesterol or anionic lipid headgroups in the selectivity of peptide action against bacteria.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Centrifugation, Isopycnic; Dose-Response Relationship, Drug; Fluoresceins; Fluorescence Polarization; Gram-Negative Bacteria; Membrane Potentials; Membranes; Molecular Sequence Data; Naphthalenes; Peptides; Permeability; Protein Binding; Pyridinium Compounds; Scattering, Radiation

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide ('DPX') to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 microM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

Topics: Calcium; Cell Membrane; Cytoplasmic Granules; Humans; Membrane Fusion; Naphthalenes; Neutrophils; Phospholipids; Pyridinium Compounds

Agents such as antimicrobial peptides and toxins can permeabilize membrane vesicles to cause leakage of entrapped contents in either a graded or an all-or-none fashion. Determination of which mode of leakage is induced is an important step in understanding the molecular mechanism of membrane permeabilization. Wimley et al. (1994, Protein Sci. 3:1362-1378) have developed a fluorescence method for distinguishing the two modes that makes use of the dye/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) without the usual need for the physical separation of vesicles from released contents. Their "requenching" method establishes the mode of release through the fluorescence changes that occur when DPX is added externally to a solution of vesicles that have released some fraction of their contents. However, the requenching method as originally stated ignored the possibility of preferential release of dye or quencher. Here we extend the theory of the method to take into account preferential release and the effects of graded leakage. The ratio of the rates of release of the cationic quencher DPX and anionic dye 8-aminonapthalene-1,3,6 trisulfonic acid can be estimated by means of the theory. For graded leakage, we show that the release of the markers does not coincide with the fluorescence changes observed in the standard leakage assay. This is true for self-quenching dyes as well and means that 1) the amount of released material will be overestimated and 2) the kinetics will be nonexponential and have artificially high apparent rates. We show how the extended requenching analysis allows the results of leakage experiments to be corrected for artifacts that result from graded and preferential leakage. Experimental evidence is presented for the existence of peptide-induced preferential graded leakage of DPX from both neutral and anionic vesicles.

Topics: Amino Acid Sequence; Animals; Biophysical Phenomena; Biophysics; Cell Membrane Permeability; In Vitro Techniques; Kinetics; Lipid Bilayers; Liposomes; Membrane Lipids; Models, Chemical; Molecular Sequence Data; Naphthalenes; Oligopeptides; Pyridinium Compounds; Spectrometry, Fluorescence

We have investigated the effects of the Ca(2+)-requiring enzyme phospholipase C on the stability of sonicated vesicles made with different molar ratios of cholesterol to lecithin. Vesicle aggregation is detected by following turbidity with time. Upon the addition of phospholipase C and after a short lag period, the turbidity of a vesicle dispersion increases continuously with time. The rate of increase of turbidity increases with both the enzyme-to-vesicle ratio and the cholesterol content of the vesicles. Vesicle fusion and leakage of contents are monitored by a contents-mixing fusion assay using 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylylenebis(pyridinium bromide) (DPX) as the fluorescence probes [Ellens, H., Bentz, J. & Szoka, F.C. (1985) Biochemistry 24, 3099-3106]. The results clearly show that phospholipase C induces vesicle fusion. The rate of vesicle fusion correlates with the enzyme-to-vesicle ratio but not with the cholesterol content of the membrane. Negligible aggregation and fusion of vesicles occurs when the experiment is repeated with buffer free of Ca2+. The membrane-destabilizing diacylglycerol, a product of lecithin hydrolysis by phospholipase C, is speculated to play a major role in driving the observed vesicle aggregation and fusion. The kinetics of vesicle aggregation and vesicle fusion can be predicted by linking Michaelis-Menten enzyme kinetics to a mass-action model.

Topics: Cholesterol; Fluorescent Dyes; Kinetics; Lipid Bilayers; Membrane Fusion; Naphthalenes; Phosphatidylcholines; Pyridinium Compounds; Type C Phospholipases

Topics: Calcium; Chromatography, Gel; Indicators and Reagents; Kinetics; Liposomes; Membrane Fusion; Models, Biological; Naphthalenes; Picolinic Acids; Pyridinium Compounds; Spectrometry, Fluorescence; Terbium

The effects on membranes of pardaxin, an amphipathic polypeptide, purified from the gland secretion of the Red Sea Moses sole flatfish Pardachirus marmoratus are dose-dependent and range from formation of voltage-gated, cation-selective pores to lysis. We have now investigated the interactions of pardaxin with small unilamellar liposomes. Light scattering showed that pardaxin (10(-7)-10(-9) M) mediated the aggregation of liposomes composed of phosphatidylserine but not of phosphatidylcholine. Aggregation of phosphatidylserine vesicles was impaired by vesicle depolarization. Furthermore, pardaxin-mediated aggregation between fluorescent-labeled PS vesicles was accompanied by leakage of the vesicle contents, and not by fusogenic process within the aggregates. We suggest that pardaxin is a unique polypeptide, that induces vesicle aggregation and membrane destabilization, but not membrane fusion; the mechanism of the aggregation activity of pardaxin is related to its amphipathic properties.

Topics: Cell Membrane Permeability; Fish Venoms; Fluorescent Dyes; Gramicidin; Liposomes; Membrane Fusion; Membrane Potentials; Naphthalenes; Phosphatidylserines; Pyridinium Compounds; Sodium Chloride; Spectrometry, Fluorescence

The kinetics of electrically induced fusion of human erythrocyte ghosts were monitored by the Tb/DPA and ANTS/DPX fluorescence fusion assays. Ghosts were aligned by dielectrophoresis using a 3-MHz 350-V/cm alternating field and were fused by single 15- or 50-microseconds electric field pulses of amplitude 2.5-5.0 kV/cm. Fusion was detected immediately after the pulse. The peak fluorescence change due to fusion was always obtained within 7 s of pulse application, and was highest for a 5.0 kV/cm 15-microseconds pulse. Probe leakage was measured separately and became apparent only 2-3 s after the initiation of fusion. Increasing pulse amplitudes produced higher fusion yields but produced more leakage from the fusion products. 50-microseconds pulses produced less fusion, resulting from a disruption of the dielectrophoretic alignment by fluid turbulence immediately after pulse application. Probe leakage was observed only when pulse application was preceded by dielectrophoresis, suggesting that close membrane positioning allows for additional membrane destabilization caused by the high field pulse. The fluorescence kinetics are interpreted using a simplified model depicting three major types of events: (a) fusion without observable leakage, (b) fusion followed by probe leakage, and (c) contact-related leakage from ghosts which do not undergo contents mixing.

Topics: Cell Fusion; Electric Conductivity; Electrophoresis; Erythrocyte Membrane; Humans; Kinetics; Naphthalenes; Picolinic Acids; Pyridinium Compounds

Liposomes composed of oleic acid and phosphatidylethanolamine (3:7 mole ratio) aggregate, become destabilized, and fuse below pH 6.5 in 150 mM NaCl. Fusion is monitored by (i) the intermixing of internal aqueous contents of liposomes, utilizing the quenching of aminonaphthalene-3,6,8-trisulfonic acid (ANTS) by N,N'-p-xylylenebis(pyridinium bromide) (DPX) encapsulated in two separate populations of vesicles, (ii) a resonance energy transfer assay for the dilution of fluorescent phospholipids from labeled to unlabeled liposomes, (iii) irreversible changes in turbidity, and (iv) quick-freezing freeze-fracture electron microscopy. Destabilization is followed by the fluorescence increase caused by the leakage of coencapsulated ANTS/DPX or of calcein. Ca2+ and Mg2+ also induce fusion of these vesicles at 3 and 4 mM, respectively. The threshold for fusion is at a higher pH in the presence of low (subfusogenic) concentrations of these divalent cations. Vesicles composed of phosphatidylserine/phosphatidylethanolamine or of oleic acid/phosphatidylcholine (3:7 mole ratio) do not aggregate, destabilize, or fuse in the pH range 7-4, indicating that phosphatidylserine and phosphatidylcholine cannot be substituted for oleic acid and phosphatidylethanolamine, respectively, for proton-induced membrane fusion. Freeze-fracture replicas of oleic acid/phosphatidylethanolamine liposomes frozen within 1 s of stimulation with pH 5.3 display larger vesicles and vesicles undergoing fusion, with membrane ridges and areas of bilayer continuity between them. The construction of pH-sensitive liposomes is useful as a model for studying the molecular requirements for proton-induced membrane fusion in biological systems and for the cytoplasmic delivery of macromolecules.

Topics: Calcium; Chemical Phenomena; Chemistry; Energy Transfer; Fluoresceins; Freeze Fracturing; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Liposomes; Magnesium; Microscopy, Electron; Naphthalenes; Oleic Acid; Oleic Acids; Phosphatidylethanolamines; Pyridinium Compounds; Spectrometry, Fluorescence

A new liposome fusion assay has been developed that monitors the mixing of aqueous contents at neutral and low pH. With this assay we have investigated the ability of H+ to induce membrane destabilization and fusion. The assay involves the fluorophore 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and its quencher N,N'-p-xylylenebis(pyridinium bromide) (DPX). ANTS is encapsulated in one population of liposomes and DPX in another, and fusion results in the quenching of ANTS fluorescence. The results obtained with the ANTS/DPX assay at neutral pH give kinetics for the Ca2+-induced fusion of phosphatidylserine large unilamellar vesicles (PS LUV) that are very similar to those obtained with the Tb3+/dipicolinic acid (DPA) assay [Wilschut, J., & Papahadjopoulos, D. (1979) Nature (London) 281, 690-692]. ANTS fluorescence is relatively insensitive to pH between 7.5 and 4.0. Below pH 4.0 the assay can be used semiquantitatively by correcting for quenching of ANTS due to protonation. For PS LUV it was found that, at pH 2.0, H+ by itself causes mixing of aqueous contents, which makes H+ unique among the monovalent cations. We have shown previously that H+ causes a contact-induced leakage from liposomes composed of phosphatidylethanolamine and the charged cholesteryl ester cholesteryl hemisuccinate (CHEMS) at pH 5.0 or below, where CHEMS becomes protonated. Here we show that H+ causes lipid mixing in this pH range but not mixing of aqueous contents. This result affirms the necessity of using both aqueous space and lipid bilayer assays to comprehend the fusion event between two liposomes.

Topics: Animals; Calcium; Cattle; Drug Stability; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Liposomes; Magnesium; Naphthalenes; Phosphatidylethanolamines; Phosphatidylserines; Pyridinium Compounds; Spectrometry, Fluorescence