1-2-oleoylphosphatidylcholine and 6-carboxyfluorescein

Liposomes are applicable to fabrication of colloidal carriers of drugs and proteins. Physicochemical stimuli-triggered leakage from liposomes offers a wide variety of applications in biochemical and biomedical fields. In this work, effects of bubbles on the characteristics of PEGylated liposomes encapsulating 5(6)-carboxyfluorescein were examined. The liposomes were composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-10 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with poly(ethylene glycol) (DSPE-PEG). The mean molecular mass M

Topics: Drug Carriers; Fluoresceins; Lipid Bilayers; Liposomes; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols

Entry of cell-penetrating peptides (CPPs) into living cells by translocating across plasma membranes is an important physiological phenomenon. To elucidate the mechanism of the translocation of CPPs across lipid bilayers, it is essential to reveal its elementary processes. For this purpose, here, we have developed a new method for the continuous, quantitative detection of the entry of CPPs into giant unilamellar vesicles (GUVs), where we investigate the interaction of fluorescent probe-labeled CPPs with single GUVs containing large unilamellar vesicles (LUVs) and fluorescent probes in their lumens using confocal microscopy. Using this method, we investigated the interaction of carboxyfluorescein (CF)-labeled transportan 10 (CF-TP10) with single GUVs comprised of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) containing LUVs of the same membrane and Alexa Fluor 647 hydrazide (AF647) in their lumens. At low concentrations of CF-TP10, first the fluorescence intensity (FI) of the GUV membrane increased with time, and then after some lag time the FI of the GUV lumen due to CF-TP10 increased continuously with time without leakage of AF647. At higher concentrations of CF-TP10, after the FI of the GUV lumen due to CF-TP10 increased significantly, leakage of AF647 started. These results indicate that CF-TP10 entered the GUV lumen by translocating across the GUV membrane and then bound to the LUVs there without pore formation and that CF-TP10 concentration in the lumen increased with time. The rate of entry of CF-TP10 into GUV lumen increased with CF-TP10 concentration. We discussed the kinetics of entry of CF-TP10 into single GUVs.

Topics: Carbocyanines; Cell-Penetrating Peptides; Fluoresceins; Fluorescent Dyes; Microscopy, Confocal; Phosphatidylcholines; Phosphatidylglycerols; Recombinant Fusion Proteins; Unilamellar Liposomes

The presence of large hydrophobic aromatic residues in cell-penetrating peptides or proteins has been demonstrated to be advantageous for their cell penetration. This phenomenon has also been observed when AuNPs were modified with peptides containing aromatic amino acids. However, it is still not clear how the presence of hydrophobic and aromatic groups on the surface of anionic AuNPs affects their interaction with lipid bilayers. Here, we studied the interaction of a range of anionic amphiphilic AuNPs coated by different combinations of hydrophobic and anionic ligands with four different types of synthetic lipid vesicles. Our results demonstrated the important role of the surface aromatic or bulky groups, relative to the hydrocarbon chains, in the interaction of anionic AuNPs with lipid bilayers. Hydrophobic interaction itself arising from the insertion of aromatic/bulky ligands on the surface of AuNPs into lipid bilayers is sufficiently strong to cause overt disruption of lipid vesicles and cell membranes. Moreover, by comparing the results obtained from AuNPs coated with aromatic ligands and cyclohexyl ligands lacking aromaticity respectively, we demonstrated that the bulkiness of the terminal groups in hydrophobic ligands instead of the aromatic character might be more important to the interaction of AuNPs with lipid bilayers. Finally, we further correlated the observation on model liposomes with that on cell membranes, demonstrating that AuNPs that are more disruptive to the more negatively charged liposomes are also substantially more disruptive to cell membranes. In addition, our results revealed that certain cellular membrane domains that are more susceptible to disruption caused by hydrophobic interactions with nanoparticle surfaces might determine the threshold of AuNP-mediated cytotoxicity.

Topics: Dynamic Light Scattering; Fatty Acids; Fluoresceins; Gold; Hydrophobic and Hydrophilic Interactions; Ligands; Lipid Bilayers; Liposomes; Metal Nanoparticles; Methylamines; Oleic Acids; Phosphatidylcholines; Sulfhydryl Compounds; Surface Properties; Surface-Active Agents; Tryptamines

The cell-penetrating peptide, transportan 10 (TP10), can translocate across the plasma membrane of living cells and thus can be used for the intracellular delivery of biological cargo such as proteins. However, the mechanisms underlying its translocation and the delivery of large cargo remain unclear. In this report we investigated the entry of TP10 into a single giant unilamellar vesicle (GUV) and the TP10-induced leakage of fluorescent probes using the single GUV method. GUVs of 20% dioleoylphosphatidylglycerol (DOPG)/80% dioleoylphosphatidylcholine (DOPC) were prepared, and they contained a water-soluble fluorescent dye, Alexa Fluor 647 hydrazide (AF647), and smaller vesicles composed of 20% DOPG/80% DOPC. The interaction of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) with these loaded GUVs was investigated using confocal microscopy. The fluorescence intensity of the GUV membrane increased with time to a saturated value, then the fluorescence intensity due to the membranes of the smaller vesicles inside the GUV increased prior to leakage of AF647. This result indicates that CF-TP10 entered the GUV from the outside by translocating across the lipid membrane before CF-TP10-induced pore formation. The rate constant of TP10-induced pore formation in lipid membranes increased with an increase in TP10 concentration. Large molecules such as Texas Red Dextran 40,000, and vesicles with a diameter of 1-2 μm, permeated through the TP10-induced pores or local rupture in the lipid membrane. These results provide the first direct experimental evidence that TP10 can deliver large cargo through lipid membranes, without the need for special transport mechanisms such as those found in cells.

Topics: Cell-Penetrating Peptides; Fluoresceins; Fluorescence; Fluorescent Dyes; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylglycerols; Recombinant Fusion Proteins; Unilamellar Liposomes

Membrane fusion is broadly envisioned as a two- or three-step process proceeding from contacting bilayers through one or two semistable, nonlamellar lipidic intermediate structures to a fusion pore. A true fusion event requires mixing of contents between compartments and is monitored by the movement of soluble molecules between trapped compartments. We have used poly(ethylene glycol) (PEG) to rapidly generate an ensemble aggregated state A that proceeds sequentially through intermediates (I₁ and/or I₂) to a final fusion pore state (FP) with rate constants k₁, k₂, and k₃. Movement of moderately sized solutes (e.g., Tb³⁺/dipicolinic acid) has been used to detect pores assigned to intermediate states as well as to the final state (FP). Analysis of ensemble kinetic data has required that mixing of contents occurs with defined probabilities (αi) in each ensemble state, although it is unclear whether pores that form in different states are different. We introduce here a simple new assay that employs fluorescence resonance energy transfer (FRET) between a 6-carboxyfluorescein (donor) and tetramethylrhodamine (acceptor), which are covalently attached to complementary sequences of 10 bp oligonucleotides. Complementary sequences of fluorophore-labeled oligonucleotides were incorporated in vesicles separately, and the level of FRET increased in a simple exponential fashion during PEG-mediated fusion. The resulting rate constant corresponded closely to the slow rate constant of FP formation (k₃) derived from small molecule assays. Additionally, the total extent of oligonucleotide mixing corresponded to the fraction of content mixing that occurred in state FP in the small molecule assay. The results show that both large "final pores" and small (presumably transient) pores can form between vesicles throughout the fusion process. The implications of this result for the mechanism of membrane fusion are discussed.

Topics: Cell Membrane Structures; Fluoresceins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Indicators and Reagents; Kinetics; Membrane Fusion; Oligonucleotides; Phosphatidylcholines; Phosphatidylethanolamines; Picolinic Acids; Polyethylene Glycols; Porosity; Rhodamines; Solubility; Sphingomyelins; Surface Properties; Terbium

In order to enhance the membrane disruption of antimicrobial peptides both targeting and multivalent presentation approaches were explored. The antimicrobial peptides anoplin and temporin L were conjugated via click chemistry to vancomycin and to di- and tetravalent dendrimers. The vancomycin unit led to enhanced membrane disruption of large unilamellar vesicles (LUVs) displaying the vancomycin target lipid II, but only for temporin L and not for anoplin. The multivalent presentation led to enhanced LUV membrane disruption in the case of anoplin but not for temporin L.

Topics: Antimicrobial Cationic Peptides; Biophysics; Chromatography, High Pressure Liquid; Drug Design; Fluoresceins; Humans; Lipids; Models, Chemical; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Proteins; Vancomycin; Wasp Venoms

The interaction of hemoglobin with phospholipid bilayer vesicles (liposomes) has been analyzed in several studies to better understand membrane-protein interactions. However, not much is known on hemoglobin interactions with the aminophospholipids, predominantly localized in the inner leaflet of erythrocytes, e.g., phosphatidylserine (PS), phosphatidylethanolamine (PE) in membranes containing phosphatidylcholine (PC). Effects of cholesterol, largely abundant in erythrocytes, have also not been studied in great details in earlier studies. This work therefore describes the study of the interactions of different hemoglobin variants HbA, HbE and HbF and the globin subunits of HbA with the two aminophospholipids in the presence and absence of cholesterol. Absorption measurements indicate preferential oxidative interaction of HbE and alpha-globin subunit with unilamellar vesicles containing PE and PS compared to normal HbA. Cholesterol was found to stabilize such oxidative interactions in membranes containing both the aminophospholipids. HbE and alpha-globin subunits were also found to induce greater leakage of membrane entrapped carboxyfluorescein (CF) using fluorescence measurements. HbE was found to induce fusion of membrane vesicles containing cholesterol and PE when observed under electron microscope. Taken together, these findings might be helpful in understanding the oxidative stress-related mechanism(s) involved in the premature destruction of erythrocytes in peripheral blood, implicated in the hemoglobin disorder, HbE/beta-thalassemia.

Topics: Cholesterol; Fluoresceins; Glycerophospholipids; Hemoglobin A; Hemoglobins; Humans; Membranes, Artificial; Microscopy, Electron, Transmission; Mutant Proteins; Oxidation-Reduction; Phosphatidylcholines; Protein Binding; Protein Subunits; Spectrum Analysis; Time Factors; Unilamellar Liposomes

The increasing resistance of human pathogens to conventional antibiotics presents a growing threat to the chemotherapeutic management of infectious diseases. The lanthionine antibiotics, still unused as therapeutic agents, have recently attracted significant scientific interest as models for targeting and management of bacterial infections. We investigated the action of one member of this class, subtilin, which permeabilizes lipid membranes in a lipid II-dependent manner and binds bactoprenyl pyrophosphate, akin to nisin. The role the C and N termini play in target recognition was investigated in vivo and in vitro by using the natural N-terminally succinylated subtilin as well as enzymatically truncated subtilin variants. Fluorescence dequenching experiments show that subtilin induces leakage in membranes in a lipid II-dependent manner and that N-succinylated subtilin is roughly 75-fold less active. Solid-state nuclear magnetic resonance was used to show that subtilin forms complexes with membrane isoprenyl pyrophosphates. Activity assays in vivo show that the N terminus of subtilin plays a critical role in its activity. Succinylation of the N terminus resulted in a 20-fold decrease in its activity, whereas deletion of N-terminal Trp abolished activity altogether.

Topics: Alanine; Anti-Bacterial Agents; Bacteriocins; Cell Membrane; Coated Vesicles; Diphosphates; Fluoresceins; Lactococcus lactis; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Succinic Acid; Sulfides; Tryptophan; Uridine Diphosphate N-Acetylmuramic Acid

This report describes the design, synthesis, and biochemical evaluation of alkene- and alkane-bridged AB(C)-ring mimics of the lantibiotic nisin. Nisin belongs to a class of natural antimicrobial peptides, and has a unique mode of action: its AB(C)-ring system binds to the pyrophosphate moiety of lipid II. This mode of action was the rationale for the design of smaller nisin-derived peptides to obtain novel potential antibiotics. As a conformational constraint the thioether bridge was mimicked by an alkene- or alkane isostere. The peptides of the linear individual ring precursors were synthesized on solid support or in solution, and cyclized by ring-closing metathesis in solution with overall yields of between 36 and 89 %. The individual alkene-bridged macrocycles were assembled in solution by using carbodiimide-based synthesis protocols for the corresponding AB(C)-ring mimics. These compounds were tested for their binding affinity toward lipid II by evaluation of their potency to inhibit nisin-induced carboxyfluorescein release from large unilamellar vesicles. It was found that these AB(C)-ring mimics were not able to induce membrane leakage; however, they acted by inhibiting nisin-induced carboxyfluorescein release; this indicates their affinity toward lipid II. These results imply that an alkene or alkane moiety is a suitable thioether bridge mimic.

Topics: Alkanes; Alkenes; Anti-Bacterial Agents; Catalysis; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Computers, Molecular; Cyclization; Drug Design; Fluoresceins; Models, Molecular; Molecular Mimicry; Nisin; Peptide Fragments; Phosphatidylcholines; Polyisoprenyl Phosphate Oligosaccharides; Stereoisomerism; Unilamellar Liposomes; Uridine Diphosphate N-Acetylmuramic Acid

Changes in the peptide chain of amphiphilic heptapeptides known to form ion-conducting pores in bilayers dramatically alter transport efficacy and the aggregation number of pore formation.

Topics: Biological Transport; Dihydroxyphenylalanine; Fluoresceins; Glycine; Ions; Lipid Bilayers; Liposomes; Oligopeptides; Phosphatidylcholines; Porosity; Surface-Active Agents; Time Factors

Previously, it was shown that the tetrameric potassium channel KcsA when present in a lipid bilayer can be dissociated by trifluoroethanol [van den Brink-van der Laan, E., et al. (2004) Biochemistry 43, 4240-4250]. Here, we demonstrate that this is a general property of small alcohols. We found that small alcohols dissociate the KcsA tetramer, at a concentration that depends on their membrane affinity. Importantly, the efficiency of the alcohol-induced tetramer dissociation was found to correlate with the efficiency of both alcohol-induced bilayer leakage and acyl chain disordering. Our data suggest that the ability of small alcohols to induce KcsA tetramer dissociation and to function as anesthetics depends on their effect on the membrane lateral pressure.

Topics: Alcohols; Bacterial Proteins; Cell Membrane; Fluoresceins; Fluorescent Dyes; Lipid Bilayers; Models, Molecular; Molecular Weight; Phosphatidylcholines; Potassium Channels; Pressure; Protein Structure, Quaternary; Protein Subunits; Statistics as Topic

Gene 9 minor coat protein from bacteriophage M13 is known to be located in the inner membrane after phage infection of Escherichia coli. The way of insertion of this small protein (32 amino acids) into membranes is still unknown. Here we show that the protein is able to insert in monolayers. The limiting surface pressure of 35 mN/m for 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol lipid systems indicates that this spontaneous insertion can also occur in vivo. By carboxyfluorescein leakage experiments of vesicles it is demonstrated that protein monomers, or at least small aggregates, are more effective in releasing carboxyfluorescein than highly aggregated protein. The final orientation of the protein in the bilayer after insertion was addressed by proteinase K digestion, thereby making use of the unique C-terminal location of the antigenic binding site. After insertion the C-terminus is still available for the enzymatic digestion, while the N-terminus is not. This leads to the overall conclusion that the protein is able to insert spontaneously into membranes without the need of any machinery or transmembrane gradient, with the positively charged C-terminus remaining on the outside. The orientation after insertion of gene 9 protein is in agreement with the 'positive inside rule'.

Topics: Bacteriophage M13; Binding Sites; Binding Sites, Antibody; Blotting, Western; Capsid; Capsid Proteins; Endopeptidase K; Escherichia coli; Fluoresceins; Lipid Bilayers; Phosphatidylcholines; Phosphatidylglycerols; Pressure

Liposomes are commonly used as models for chilling and freezing damage, with leakage of water-soluble contents from the aqueous interior as the most frequently used measurement of damage. In order to achieve an understanding of the mechanism of the leakage, we have conducted a study of the factors that influence the leakage from liposomes during phase transitions. While such investigations have appeared sporadically in the literature, a detailed study has not been undertaken previously, despite the fact that liposomes are widely used as models for stress injury. Thus, we suggest that these findings will be of general interest in the cryobiology community. We now report that the following variables affected leakage from liposomes during chilling: (i) increasing the rate of cooling and warming resulted in decreased leakage; (ii) maximal leakage occurred at the measured phase transition temperature; (iii) addition of defect-forming additives such as a second phospholipid or a surfactant increased leakage from the liposomes during the phase transition but not above or below that temperature; (iv) small unilamellar vesicles leaked much more rapidly than large unilamellar vesicles; and (v) increasing the pH of the external buffer decreased leakage of carboxyfluorescein, an effect that is probably particular to ionizable solutes.

Topics: Fluoresceins; Fluorescent Dyes; Freezing; Hydrogen-Ion Concentration; In Vitro Techniques; Lipid Bilayers; Liposomes; Particle Size; Permeability; Phosphatidylcholines; Phospholipids; Thermodynamics

Temperature-dependent internal content release from liposomes was examined using di-oleoylphosphatidylcholine (DOPC)/cholesterol liposomes with encapsulated Pluronic F127 molecules. The interaction of Pluronic F127 with the lipid bilayer at elevated temperature causes the release of encapsulated contents. Content release was measured using fluorescent markers of two different sizes: small, carboxyfluorescein (CF), and large, bovine serum albumin-conjugated fluorescein iso-thiocyanate (BSA-FITC). Release of CF was studied using fluorescence de-quenching, while that of BSA-FITC was studied using fluorescence emission quenching due to fluorescence resonance energy transfer (FRET). Temperature-controlled complete internal content release was achieved at a precise temperature by controlling the concentration of the encapsulated Pluronic. Increasing cholesterol % in the liposome composition resulted in a sharper transition with temperature in content release. The onset temperature of content release increased with decrease in Pluronic concentration. For the same Pluronic concentration, the onset temperature also depended on the size of the encapsulated marker and was higher for larger markers. We have established that onset of content release is determined by the critical micellar temperature (CMT) of the Pluronic. Temperature-sensitive liposomes, made stealth using di-stearoyl(polyethylene glycol 5000) phosphatidylethanolamine (DSPEG5000PE) in conjunction with Pluronic F127, had similar temperature sensitivity and efficiency in content release compared to the non-stealth liposomes.

Topics: Cholesterol; Fluoresceins; Liposomes; Molecular Weight; Phosphatidylcholines; Phosphatidylethanolamines; Poloxamer; Polyethylene Glycols; Temperature

We have investigated, both experimentally and theoretically, the efflux of carboxyfluorescein (a self-quenching fluorescent dye) from vesicles of different sizes and lipid species (POPC, DOPC) after having added the bee venom peptide melittin. This comprises quantitative analyses regarding the extent of lipid-associated peptide, the mode as well as the temporal progress of dye release and the possible leakage mechanism. Our results indicate a graded efflux characterized by a single-pore retention factor reflecting the formation of pores whose lifetimes are rather small (millisecond range). The observed fluorescence signal arising from the dequenching of effluent dye has been converted to the number of pore openings over the course of time. All the resulting curves exhibit a pronounced slowing down of the pore formation rate revealing two distinct relaxation steps at about 20 and 200 s, respectively, being largely independent of vesicle type and peptide to lipid ratio. The pore formation rate itself increases in proportion to the amount of membrane bound peptide. We give a quantitative account of our experimental findings based on a novel reaction scheme applicable to any of our various liposome systems. It implies that the pore formation rate is controlled by a passage through two intermediate monomeric peptide states. These states are thought to become well populated in the initial stage of lipid bilayer perturbation, but would practically die out after some time owing to a restabilization of the membrane system.

Topics: Binding Sites; Energy Transfer; Fluoresceins; Fluorescent Dyes; In Vitro Techniques; Kinetics; Liposomes; Melitten; Phosphatidylcholines; Spectrometry, Fluorescence

The interaction of nisin Z and a nisin Z mutant carrying a negative charge in the C-terminus ([Glu-32]-nisin Z) with anionic lipids was characterized in model membrane systems, and bacterial membrane systems. We focused on three possible steps in the mode of action of nisin, i.e., binding, insertion, and pore formation of nisin Z. Increasing amounts of anionic lipids in both model and natural membranes were found to strongly enhance the interaction of nisin Z with the membranes at all stages. The results reveal a good correlation between the anionic lipid dependency of the three stages of interaction, of which the increased binding is probably the major determinant for antimicrobial activity. Maximal nisin Z activity could be observed for negatively charged lipid concentrations exceeding 50-60%, both in model membrane systems as well as in bacterial membrane systems. We propose that the amount of negatively charged lipids of the bacterial target membrane is a major determinant for the sensitivity of the organism for nisin. Nisin Z induced leakage of the anionic carboxyfluorescein was more efficient as compared to the leakage of the potassium cation. This lead to the conclusion that an anion-selective pore is formed. In contrast to the results obtained for nisin Z, the binding of [Glu-32]-nisin Z to vesicles remained low even in the presence of high amounts of negatively charged lipids. The insertion and pore-forming ability of [Glu-32]-nisin Z were also decreased. These results demonstrate that the C-terminus of nisin is responsible for the initial interaction of nisin, i.e., binding to the target membrane.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Cell Membrane; Fluoresceins; Glucose; Lipid Bilayers; Membranes, Artificial; Molecular Sequence Data; Nisin; Phosphatidylcholines; Phosphatidylglycerols; Potassium

We investigated the interaction of the peptide melittin with differently sized vesicles consisting of various lipid compositions. This system was characterized by dynamic light scattering to estimate the size of vesicles. For SUV we obtained a radius of 12 nm, for LUV 53 nm. The pore forming process of melittin in vesicles was investigated by efflux of encapsulated fluorescent dyes at a self-quenching concentration. The influence of the following parameters on efflux and pore formation was estimated: lipid composition (POPC and DOPC), vesicle size (SUV and LUV) and size of the encapsulated dye (carboxyfluorescein and FITC-dextran). We found that under similar conditions vesicles of DOPC give always less leakage than vesicles of POPC independent of the fluorescent dye. For SUV and LUV we have obtained a different leakage behaviour at identical surface concentrations of melittin (if the same partition coefficient is assumed). From efflux measurements with different dyes we concluded that 6-20 molecules of melittin are necessary to form a pore. The possibility that not pore formation but fusion is the mechanism of melittin induced efflux was disproved by fusion experiments using a resonance energy transfer assay.

Topics: Amino Acid Sequence; Cell Membrane; Dextrans; Fluorescein-5-isothiocyanate; Fluoresceins; Light; Liposomes; Melitten; Membrane Fusion; Membrane Lipids; Membranes, Artificial; Molecular Sequence Data; Phosphatidylcholines; Scattering, Radiation

The effect of ethanol on stability of intact yeast cells has been investigated. Several strains with differences in trehalose metabolism were examined for their ability to survive in the presence of 10% (v/v) ethanol. A positive correlation was observed between cell viability and trehalose concentration. When leakage of electrolytes from the cells was recorded by observing changes in conductivity of the medium, we found that ethanol increases leakage, but the presence of trehalose reverses that effect. Similar studies were done with liposomes of similar composition to those seen in intact cells in log and stationary phases. In the presence of ethanol, carboxyfluorescein trapped in the liposomes leaked to the medium. When trehalose was added inside, outside or on both sides of the membrane, the ethanol-induced leakage was strongly inhibited. More leakage was observed in liposomes in gel phase state than in liquid-crystalline phase, suggesting that the thermotropic behavior of the lipids in the plasma membrane, together with trehalose, plays a role in enhancing ethanol tolerance.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cell Membrane Permeability; Cell Survival; Dose-Response Relationship, Drug; Ethanol; Fluoresceins; Liposomes; Phosphatidylcholines; Saccharomyces cerevisiae; Trehalose

The interaction of seminalplasmin (SPLN), a 47-residue antibacterial peptide, and its 13-residue fragment (SPF), which has antibacterial and hemolytic activities, with model membranes has been investigated. The fluorescence characteristics of the single Trp residue in these peptides indicate strong binding to lipid vesicles. SPLN binds more strongly to dioleoylphosphatidylglycerol vesicles compared to dioleoylphosphatidylcholine and phosphatidylserine vesicles. Localization studies using fluorescence quenchers like NO3-, I-, and acrylamide indicate that the Trp residues in both of the peptides are located away from the head group region and are associated with the hydrophobic core. Both peptides cause release of carboxyfluorescein from zwitterionic as well as anionic vesicles. The biological activities of SPLN and SPF have been rationalized in terms of lipid-peptide interactions. It is proposed that the specificity in biological activity arises due to differences in the manner in which the peptides associate with the bacterial and red blood cell surfaces.

Topics: Acrylamide; Acrylamides; Amino Acid Sequence; Animals; Cattle; Circular Dichroism; Fluoresceins; Iodides; Liposomes; Molecular Sequence Data; Nitrates; Peptide Fragments; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Proteins; Seminal Vesicle Secretory Proteins; Spectrometry, Fluorescence

The fluorescent dye carboxyfluorescein (CF), entrapped in liposomes and administered by intraduodenal injection to thoracic duct-cannulated rats, was not detected in thoracic duct lymph at any time after dosing. However, the dye was present in blood, and in higher concentration in portal than in systemic blood. Similarly, free CF given intraperitoneally (i.p.) appeared to be absorbed into portal blood and was quickly cleared from the circulation, with less than 1% of the injected dye being recovered in thoracic duct lymph within 24 h after dosing. In contrast, 27% of the injected CF was recovered in thoracic duct lymph of rats receiving CF/liposomes i.p. These findings indicate that liposomal entrapment effectively limits passage of CF into the splanchnic blood vessels while enhancing the lymphatic uptake of the dye from the peritoneal cavity.

Topics: Animals; Coloring Agents; Drug Stability; Fluoresceins; Injections, Intraperitoneal; Intestinal Absorption; Intubation, Gastrointestinal; Liposomes; Lymphatic System; Male; Phosphatidylcholines; Rats