Effect of PEGylated lipid and Lecinol S-10 on physico-chemical properties and encapsulation efficiency of palmitoleate–palmitoleic acid vesicles

A vesicle is a microscopic particle composed of a lipid bilayer membrane that separates the inner aqueous compartment from the outer aqueous environment. Palmitoleate–palmitoleic acid vesicles were prepared and their physico-chemical properties were investigated. Moreover, mixed vesicles composed of palmitoleic acid and PEGylated lipid and/or a mixture of phospholipids were also prepared. The stabilizing effects of these double-chain lipids on the formation of palmitoleate–palmitoleic acid vesicles were studied. Stability of the vesicle suspension was examined using particle size and zeta potential at 30?°C. The magnitude of the zeta potential was relatively lower in the vesicle suspension with the presence of phospholipid. Although some of the mixed vesicles that were formed were not very stable, they displayed potential for encapsulating the active ingredient calcein and the encapsulation efficiencies of calcein were encouraging. The palmitoleate–palmitoleic acid–DPPE-PEG2000 vesicle showed the most promising stability and encapsulation efficiency.     

Spherical reconstruction: a method for structure determination of membrane proteins from cryo-EM images

We propose a new method for single-particle reconstruction, which should be generally applicable to structure determination for membrane proteins. After reconstitution into a small spherical vesicle, a membrane protein takes a particular orientation relative to the membrane normal, and its position in the projected image of the vesicle directly defines two of its three Euler angles of orientation. The spherical constraint imposed by the vesicle effectively reduces the dimensionality of the alignment search from 5 to 3 and simplifies the detection of the particle. Projection images of particles in vesicles collectively take all possible orientations and therefore cover the whole Fourier space. Analysis of images of vesicles in ice showed that the vesicle density is well described by a simple model for membrane electron scattering density. In fitting this model we found that osmotically swollen vesicles remain nearly spherical through the freezing process. These results satisfy the basic experimental requirements for spherical reconstruction. A computer simulation of particles in vesicles showed that this method provides good estimates of the two Euler angles and thus may improve single-particle reconstruction and extend it to smaller membrane proteins.     

Effect of Hb-encapsulation with vesicles on H2O2 reaction and lipid peroxidation

We encapsulated a purified and concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane to form Hb vesicles (particle diameter, ca. 250 nm) for the development of artificial oxygen carriers. Reaction of Hb inside the vesicle with hydrogen peroxide (H(2)O(2)) is one of the important safety issues to be clarified and compared with a free Hb solution. During the reaction of the Hb solution with H(2)O(2), metHb (Fe(III)) and ferrylHb (Fe(IV)=O) are produced, and H(2)O(2) is decomposed by the catalase-like reaction of Hb. The aggregation of discolored Hb products due to heme degradation is accompanied by the release of iron (ferric ion). On the other hand, the concentrated Hb within the Hb vesicle reacts with H(2)O(2) that permeated through the bilayer membrane, and the same products as the Hb solution are formed inside the vesicle. However, there is no turbidity change, no particle diameter change of the Hb vesicles, and no peroxidation of lipids comprising the vesicles after the reaction with H(2)O(2). Furthermore, no free iron is detected outside the vesicle, though ferric ion is released from the denatured Hb inside the vesicle, indicating the barrier effect of the bilayer membrane against the permeation of ferric ion. When vesicles composed of egg york lecithin (EYL) as unsaturated lipids are added to the mixture of Hb and H(2)O(2), the lipid peroxidation is caused by ferrylHb and hydroxyl radical generated from reaction of the ferric iron with H(2)O(2), whereas no lipid peroxidation is observed in the case of the Hb vesicle dispersion because the saturated lipid membrane of the Hb vesicle should prevent the interaction of the ferrylHb or ferric iron with the EYL.     

Absence of subtransition in racemic dipalmitoylphosphatidylcholine vesicles

dl-Dipalmitoylphosphatidylcholine multilamellar vesicle suspensions were examined by the method of differential scanning calorimetry. A lack of the subtransition at 18°C was established. Such a subtransition is characteristic for l-dipalmitoylphosphatidylcholine suspensions. This lack is supposed to be the result of the impossibility of the racemic phospholipid mixture to form the low-temperature crystal structure L_c.     

Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 2. 1,6-Diphenyl-1,3,5-hexatriene in lipid bilayers

The application of a new spectroscopic tool [Knutson, J. R., Davenport, L., & Brand, L. (1986) Biochemistry (preceding paper in this issue)] for studying rotational microheterogeneity of probe location in lipid bilayer systems is described. Anisotropy decay associated spectra are derived from experimentally obtained polarized emission components. "Early" difference spectra (IV - IH) contain contributions from both fast and slow rotors, while "late" difference spectra predominantly reflect the emission from slowly rotating fluorophores. Anisotropy decay associated spectra have been used to resolve the emission spectra of 1,6-diphenyl-1,3,5-hexatriene (DPH) imbedded within a known rotationally heterogeneous mixture of two vesicle types (L-alpha-dimyristoyllecithin and L-alpha-dipalmitoyllecithin). At 29 degrees C, diphenylhexatriene within pure dimyristoyllecithin vesicles rotates rapidly, with a small r infinity, while diphenylhexatriene in dipalmitoyllecithin vesicles exhibits a large r infinity. Spectra for diphenylhexatriene imbedded in the two vesicle types show small but significant spectral differences. A spectrum of a mixture of the two vesicle types with DPH lies between these characteristic component spectra. The spectrum extracted for "immobilized" probes in the mixture correctly overlays the dipalmitoyllecithin spectrum. Further studies have shown that diphenylhexatriene exhibits more than one emission anisotropy decay associated spectrum in vesicles of a single lipid type, when that lipid is near its phase transition temperature. Diphenylhexatriene apparently inhabits more than one rotational environment even in these "homogeneous" vesicle preparations.     

Fusion of isolated cardiac sarcolemmal and sarcoplasmic reticulum vesicles. An approach to the mechanism

A procedure for the fusion of isolated cardiac sarcolemmal and sarcoplasmic reticulum vesicles is described. When the mixture of vesicles was incubated in a medium containing CaCl₂ and ATP, membrane fusion rather than vesicle aggregation or molecular exchange was demonstrated. This was achieved either by studying changes in vesicle density using sucrose gradients, fluorescence quenching using fluorescamine labeled sarcoplasmic reticulum, or by separation of the different vesicle sizes using gel-filtration. Although extensive fusion was observed when inside-out sarcolemmal vesicles were used, right-side-out vesicles showed no capacity to fuse with sarcoplasmic reticulum vesicles. The relationship between fusion and other aspects of cardiac sarcolemmal function was discussed.     

Apolipoprotein and lipid distribution between vesicles and HDL-like particles formed during lipolysis of human very low density lipoproteins by perfused rat heart

A study was undertaken to determine the relative association of lipid and apolipoproteins among lipoproteins produced during lipolysis of very low density lipoproteins (VLDL) in perfused rat heart. Human VLDL was perfused through beating rat hearts along with various combinations of albumin (0.5%), HDL2, the infranatant of d greater than 1.08 g/ml of serum, and labeled sucrose. The products were resolved by gel filtration, ultracentrifugation, and hydroxylapatite chromatography. The composition of the lipoprotein products was assessed by analysis of total lipid profiles by gas-liquid chromatography and immunoassay of apolipoproteins. A vesicle particle, which trapped and retained 1-2% of medium sucrose, co-isolated with VLDL and VLDL remnants by gel filtration chromatography but primarily with the low density lipoprotein (LDL) fraction when isolated by ultracentrifugation. The vesicle was resolved from apoB-containing LDL lipolysis products by hydroxylapatite chromatography of the lipoproteins. The vesicle lipoprotein contained unesterified cholesterol (34%), phosphatidylcholine and sphingomyelin (50%), cholesteryl ester (6%), triacylglycerol (5%), and apolipoprotein (5%). The apolipoprotein consisted of apoC-II (7%), apoC-III (93%), and trace amounts of apoE (1%). When viewed by electron microscopy the vesicles appeared as rouleaux structures with a diameter of 453 A, and a periodicity of 51.7 A. The mass represented by the vesicle particle in terms of the initial amount in VLDL was: cholesterol (5%), phosphatidylcholine and sphingomyelin (3%), apoC-II (0.5%), apoC-III (2.2%). The majority of the apoC and E released from apoB-containing lipoproteins was associated with neutral-lipid core lipoproteins proteins which possessed size characteristics of HDL. The vesicles were also formed in the presence of HDL and serum and were not disrupted by serum HDL. It is concluded that lipolysis of VLDL in vitro results in the production of VLDL remnants and LDL apoB-containing lipoproteins, as well as HDL-like lipoproteins. A vesicular lipoprotein which has many characteristics of lipoprotein X found in cholestasis, lecithin: cholesterol acyltransferase deficiency, and during Intralipid infusion is also formed. The majority of apolipoprotein C and E released from apoB-containing lipoproteins is associated with the HDL-like lipoprotein. It is suggested that the formation and stability of the vesicle lipoprotein may be related to the high ratio of cholesterol/phospholipid in this particle.     

Purification of the vesamicol receptor.

The vesamicol receptor (VR) present in cholinergic synaptic vesicles isolated from the electric organ of Torpedo was solubilized in cholate detergent and stabilized with glycerol and a phospholipid mixture. The receptor was purified in 7% yield by hydroxylapatite, wheat germ lectin affinity, DEAE anion-exchange, and size exclusion chromatographies based on a [3H]vesamicol binding assay. A final specific binding of 4400 pmol/mg of protein was obtained. The cholate-solubilized VR complex exhibited variable aggregation states with particle molecular masses of 210-3500 kDa in different experiments. The purified VR exhibited very heterogeneous electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with very diffuse protein staining at about 240 kDa. No "classical" polypeptide or glycopeptide band was detected. One form of the SV1 epitope, which is characteristic of cholinergic synaptic vesicle proteoglycan, copurified precisely with the VR. The SV2 epitope, which is found in most neuronal and endocrine secretory vesicles, also closely purified with the VR. Substantially purified VR retained both enantioselectivity for (-)-vesamicol and a linked AcCh-binding site. This confirms the allosteric model for the VR in the AcCh transporter. The physicochemical properties of the VR and copurification of it with the SV1 epitope strongly suggest that the VR is associated with cholinergic vesicle proteoglycan. A second proteoglycan that is not associated with the VR but which carries the SV1 and SV2 epitopes also was observed.     

Steroid glucuronides as male pheromones in the reproduction of the African catfish Clarias gariepinus--a brief review

After ovulation, female African catfish are strongly attracted by the odor of male conspecifics. This attraction depends on the presence of the seminal vesicle, a part of the male reproductive organs. Removal of the seminal vesicle illustrates this fact. A low dose of seminal vesicle fluid, added to the water, appears to be highly attractive for catfish which have ovulated. Fractionation of the fluid and testing of the different fractions shows that steroid glucuronides could be responsible for the attraction. These steroid glucuronides can be identified with gas chromatographic-mass spectrometric analysis. A mixture of glucuronides, prepared to resemble the composition of the seminal vesicle fluid, evokes a dose-dependent attraction. The most potent odorant, observed by measuring electrical responses from the olfactory epithelium and from the olfactory tract appears to be 3,17-dihydroxy-5β-pregnan-20-one-3-glucuronide.     

Cross-correlation laser scattering.

The dependence of phospholipid vesicle size on lipid composition is investigated by photon correlation spectroscopy. For each lipid composition prolonged ultracentrifugation was used to isolate a nearly uniform population of minimum-sized vesicles. The residual size variations in the samples were sufficient to cause polydispersity that made comparisons between samples difficult. Analyses of the data by the method of cumulants and by a method for approximating the particle size distributions directly are presented. The latter method made possible unambiguous comparisons that revealed small but systematic dependences of vesicle size on composition in vesicles containing mixtures of egg phosphatidylcholine and phosphatidylethanolamine, egg phosphatidylcholine and beef brain sphingomyelin, and in single lipid vesicles of egg phosphatidylcholine, dioleylphosphatidylcholine, and beef brain sphingomyelin. These size dependences are quantified within the resolution limits of the technique and their implications are discussed.     

In vitro stimulatory effect of anti-apoptotic seminal vesicle protein 4 on purified peroxidase enzymes

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 mum native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme-electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.     

Steroid glucuronides as male pheromones in the reproduction of the African catfish Clarias gariepinus—a brief review

After ovulation, female African catfish are strongly attracted by the odor of male conspecifics. This attraction depends on the presence of the seminal vesicle, a part of the male reproductive organs. Removal of the seminal vesicle illustrates this fact. A low dose of seminal vesicle fluid, added to the water, appears to be highly attractive for catfish which have ovulated. Fractionation of the fluid and testing of the different fractions shows that steroid glucuronides could be responsible for the attraction. These steroid glucuronides can be identified with gas chromatographic-mass spectrometric analysis. A mixture of glucuronides, prepared to resemble the composition of the seminal vesicle fluid, evokes a dose-dependent attraction. The most potent odorant, observed by measuring electrical responses from the olfactory epithelium and from the olfactory tract appears to be 3α,17α-dihydroxy-5β-pregnan-20-one-3α-glucuronide.     

Calcium influx and mitochondrial alterations at synapses exposed to snake neurotoxins or their phospholipid hydrolysis products

Snake presynaptic phospholipase A2 neurotoxins (SPANs) bind to the presynaptic membrane and hydrolyze phosphatidylcholine with generation of lysophosphatidylcholine (LysoPC) and fatty acid (FA). The LysoPC+FA mixture promotes membrane fusion, inducing the exocytosis of the ready-to-release synaptic vesicles. However, also the reserve pool of synaptic vesicles disappears from nerve terminals intoxicated with SPAN or LysoPC+FA. Here, we show that LysoPC+FA and SPANs cause a large influx of extracellular calcium into swollen nerve terminals, which accounts for the extensive synaptic vesicle release. This is paralleled by the change of morphology and the collapse of membrane potential of mitochondria within nerve bulges. These results complete the picture of events occurring at nerve terminals intoxicated by SPANs and define the LysoPC+FA lipid mixture as a novel and effective agonist of synaptic vesicle release.     

Interaction of short-chain lecithin with long-chain phospholipids: characterization of vesicles that form spontaneously

Stable unilamellar vesicles formed spontaneously upon mixing aqueous suspensions of long-chain phospholipid (synthetic, saturated, and naturally occurring phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) with small amounts of short-chain lecithin (fatty acid chain lengths of 6-8 carbons) have been characterized by using NMR spectroscopy, negative staining electron microscopy, differential scanning calorimetry, and Fourier transform infrared (FTIR) spectroscopy. This method of vesicle preparation can produce bilayer vesicles spanning the size range 100 to greater than 1000 A. The combination of short-chain lecithin and long-chain lecithin in its gel state at room temperature produces relatively small unilamellar vesicles, while using long-chain lecithin in its liquid-crystalline state produces large unilamellar vesicles. The length of the short-chain lecithin does not affect the size distribution of the vesicles as much as the ratio of short-chain to long-chain components. In general, additional short-chain decreases the average vesicle size. Incorporation of cholesterol can affect vesicle size, with the solubility limit of cholesterol in short-chain lecithin micelles governing any size change. If the amount of cholesterol is below the solubility limit of micellar short-chain lecithin, then the addition of cholesterol to the vesicle bilayer has no effect on the vesicle size; if more cholesterol is added, particle growth is observed. Vesicles formed with a saturated long-chain lecithin and short-chain species exhibit similar phase transition behavior and enthalpy values to small unilamellar vesicles of the pure long-chain lecithin prepared by sonication. As the size of the short-chain/long-chain vesicles decreases, the phase transition temperature decreases to temperatures observed for sonicated unilamellar vesicles. FTIR spectroscopy confirms that the incorporation of the short-chain lipid in the vesicle bilayer does not drastically alter the gauche bond conformation of the long-chain lipids (i.e., their transness in the gel state and the presence of multiple gauche bonds in the liquid-crystalline state).     

Phospholipid exchange between bilayer membranes.

The mode of interaction of aqueous dispersions of phospholipid vesicles is investigated. The vesicles (average diameter 950 A) are prepared from total lipid extracts of Escherichia coli composed of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. One type of vesicle contains trans-delta 9-octadecenoate, the other type trans-delta 9-hexadecenoate as predominant acyl chain component. The vesicles show order in equilibrium disorder transitions at transition temperatures, Tt = 42 degrees C and Tt = 29 degrees C, respectively. A mixture of these vesicles is incubated at 45 degrees C and lipid transfer is studied as a function of time using the phase transition as an indicator. The system reveals the following properties: Lipids are transferred between the two vesicle types giving rise to a vesicle population where both lipid components are homogeneously mixed. Lipid transfer is asymmetric, i.e. trans-delta 9-hexadecenoate-containing lipid molecules appear more rapidly in the trans-delta 9-octadecenoate-containing vesicles than vice versa. At a given molar ratio of the two types of vesicles the rate of lipid transfer is independent of the total vesicle concentration. It is concluded that lipid exchange through the water phase by way of single molecules or micelles is the mode of communication of these negatively charged lipid vesicles.     

Vesicle size distributions measured by flow field-flow fractionation coupled with multiangle light scattering.

The separation method, flow field-flow fractionation (flow FFF), is coupled on-line with multiangle laser light scattering (MALLS) for simultaneous measurement of the size and concentration of vesicles eluting continuously from the fractionator. These size and concentration data, gathered as a function of elution time, may be used to construct both number- and mass-weighted vesicle size distributions. Unlike most competing, noninvasive methods, this flow FFF/MALLS technique enables measurement of vesicle size distributions without a separate refractive index detector, calibration using particle size standards, or prior assumptions about the shape of the size distribution. Experimentally measured size distributions of vesicles formed by extrusion and detergent removal are non-Gaussian and are fit well by the Weibull distribution. Flow FFF/MALLS reveals that both the extrusion and detergent dialysis vesicle formation methods can yield nearly size monodisperse populations with standard deviations of approximately 8% about the mean diameter. In contrast to the rather low resolution of dynamic light scattering in analyzing bimodal systems, flow FFF/MALLS is shown to resolve vesicle subpopulations that differ by much less than a factor of two in mean size.     

Dynamics of phospholipid aggregation in ethanol-water solutions

The kinetics of liposome and vesicle formation of a synthetic lecithin has been studied by light scattering techniques. It is shown, that by evaporation of alcohol from a lipid-alcohol-water mixture, the aggregates formed undergo several changes in shape. A hypothesis is presented, visualising the formation of liposomes or vesicles from monomers, which is consistent with the experimental observations.     

Automatic cryo-EM particle selection for membrane proteins in spherical liposomes

Random spherically constrained (RSC) single particle reconstruction is a method to obtain structures of membrane proteins embedded in lipid vesicles (liposomes). As in all single-particle cryo-EM methods, structure determination is greatly aided by reliable detection of protein “particles” in micrographs. After fitting and subtraction of the membrane density from a micrograph, normalized cross-correlation (NCC) and estimates of the particle signal amplitude are used to detect particles, using as references the projections of a 3D model. At each pixel position, the NCC is computed with only those references that are allowed by the geometric constraint of the particle’s embedding in the spherical vesicle membrane. We describe an efficient algorithm for computing this position-dependent correlation, and demonstrate its application to selection of membrane-protein particles, GluA2 glutamate receptors, which present very different views from different projection directions.     

Structure of Catecholamine Secretory Vesicles from PC12 Cells

Catecholamine secretory organelles were partially purified from PC12 cells. Measurement of the sedimentation coefficient (540S in 0.32 M sucrose), density in an isoosmotic gradient (1.139 g/cm), and density in an isoosmotic gradient using D2O as a solvent (1.205 g/cm3) have allowed us to calculate the molecular weight (1.17 X 10(9) daltons), radius (74 nm), and water content (62% vol/vol) of the secretory vesicle. The vesicle appears to contain ATP, but the molar ratio of 3,4-dihydroxyphenylethylamine (dopamine) to ATP in the particles is high (16.5) and the ATP was frequently asymmetrically distributed in the vesicle fraction. The particle behaves like a true secretory particle in that the dopamine content of the particle is increased by pargyline, diminished by depolarization, and abolished by reserpine. Sequential purification of PC12 lysates on controlled pore glass columns and isoosmotic Ficoll gradients produced a 20-30-fold purification, but this enrichment is not sufficient to produce a homogeneous population of vesicles. An 82,000-dalton protein copurifies with secretory granules and appears to be the major secreted protein. At this stage of purification this single protein makes up about 30% of the protein in the vesicle-containing fractions and so the vesicles must be approaching homogeneity.     

Coupling field theory with continuum mechanics: a simulation of domain formation in giant unilamellar vesicles

Domain formation is modeled on the surface of giant unilamellar vesicles using a Landau field theory model for phase coexistence coupled to elastic deformation mechanics (e.g., membrane curvature). Smooth particle applied mechanics, a form of smoothed particle continuum mechanics, is used to solve either the time-dependent Landau-Ginzburg or Cahn-Hilliard free-energy models for the composition dynamics. At the same time, the underlying elastic membrane is modeled using smooth particle applied mechanics, resulting in a unified computational scheme capable of treating the response of the composition fields to arbitrary deformations of the vesicle and vice versa. The results indicate that curvature coupling, along with the field theory model for composition free energy, gives domain formations that are correlated with surface defects on the vesicle. In the case that external deformations are included, the domain structures are seen to respond to such deformations. The present simulation capability provides a significant step forward toward the simulation of realistic cellular membrane processes.