Intralipid 10%: physicochemical characterization.

OBJECTIVES: Parenteral fat emulsions contain two populations of particles: artificial chylomicrons rich in triacylglycerols (TAG), and liposomes (bilayer of phospholipids [PL] enveloping an aqueous phase). Centrifugation permits isolating the liposomes in the infranatant called mesophase. The aim of the present work was to better characterize this mesophase chemically and to view the particles it contains by electron microscopy. METHODS: Electron microscopy (Philips 410) was performed after cryofracture on native 10% Intralipid, mesophase (centrifugation for 1 h at 27 000 g), and a liposome-enriched fraction (ring of density 1.010-1.030 g/l obtained after centrifuging mesophase in a KBr density gradient at 100 000 g for 24 h). The TAG and protein content of the mesophase was analyzed and the proteins partially characterized by immunodetection (Western-blot). RESULTS: This electron microscope study of 10% Intralipid gives evidence for the coexistence of artificial chylomicrons (mean diameter, 260 nm) and liposomes (43 nm), the latter being smaller than expected and containing 8% w/w TAG after purification. The solubilization of TAG in PL bilayers (reported to be < or = 3.1% w/w) might have been increased in parenteral emulsions by the manufacturing process or/and the high TAG/PL ratio. Minute amounts of proteins have also been detected and partially characterized using a specific antibody raised against the human 7 kDa Anionic Polypeptide Factor (APF), known to strongly interact with PL in bile. CONCLUSIONS: This work has shown that the size (mean diameter, 43 nm) of the liposomes present in 10% Intralipid is smaller than that usually assumed. Traces of hydrophobic proteins in the emulsion may account for certain allergic reactions sometimes observed in infused patients.

Self-assembly of regular hollow icosahedra in salt-free catanionic solutions.

Self-assembled structures having a regular hollow icosahedral form (such as those observed for proteins of virus capsids) can occur as a result of biomineralization processes, but are extremely rare in mineral crystallites. Compact icosahedra made from a boron oxide have been reported, but equivalent structures made of synthetic organic components such as surfactants have not hitherto been observed. It is, however, well known that lipids, as well as mixtures of anionic and cationic single chain surfactants, can readily form bilayers that can adopt a variety of distinct geometric forms: they can fold into soft vesicles or random bilayers (the so-called sponge phase) or form ordered stacks of flat or undulating membranes. Here we show that in salt-free mixtures of anionic and cationic surfactants, such bilayers can self-assemble into hollow aggregates with a regular icosahedral shape. These aggregates are stabilized by the presence of pores located at the vertices of the icosahedra. The resulting structures have a size of about one micrometre and mass of about 1010 daltons, making them larger than any known icosahedral protein assembly or virus capsid. We expect the combination of wall rigidity and holes at vertices of these icosahedral aggregates to be of practical value for controlled drug or DNA release.

Selective adhesion, lipid exchange and membrane-fusion processes between vesicles of various sizes bearing complementary molecular recognition groups.

Equimolar mixtures of large unilamellar vesicles (LUVs) obtained from mixtures of egg lecithin and lipids containing complementary hydrogen bonding head groups (barbituric acid (BAR) and 2,4,6-triaminopyrimidine (TAP)) were shown to aggregate and fuse. These events have been studied in detail using electron microscopy and dynamic light scattering, and by fluorimetry using membrane or water-soluble fluorescence probes. It was shown that aggregation was followed by two competitive processes: a) lipid mixing leading to redispersion of the vesicles; b) fusion events generating much larger vesicles. In order to better understand the nature of the interaction, the effects of ionic strength and surface concentration of recognition lipids on the aggregation process were investigated by dynamic light scattering. Additionally, it was possible to inhibit the aggregation kinetics through addition of a soluble barbituric acid competitor. The study was extended to giant unilamellar vesicles (GUVs) to investigate the size effect and visualise the phenomena in situ. The interactions between complementary LUVs and GUVs or GUVs and GUVs were studied by optical microscopy using dual fluorescent labelling of both vesicle populations. A selective adhesion of LUVs onto GUVs was observed by electron and optical microscopies, whereas no aggregation took place in case of a GUV/GUV mixture. Furthermore, a fusion assay of GUV and LUV using the difference of size between GUV and LUV and calceine self-quenching showed that no mixing between the aqueous pools occured.

VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals.

Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995). This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesicles from rapidly frozen nerve endings and synaptosomes. VAMP was found associated with synaptic vesicles, as also shown by conventional electron microscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gold labeling of synaptic vesicles. Comparison of gold particle densities suggests that the presynaptic plasma membrane contains 3 VAMP molecules per molecule of syntaxin. After biotinylation of intact synaptosomes, the synaptosomal plasma membrane was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles. VAMP was present in both membranes whereas syntaxin and SNAP 25 were highly enriched in the synaptosomal plasma membrane. This membrane has a low content of classical synaptic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine transporter). The VAMP to syntaxin stoichiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2, in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synaptic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of synaptic vesicles, a process which could, as far as SNAREs are concerned, very much resemble homotypic fusion.

Visualization of in vitro protein-rejecting properties of PEGylated stealth polycyanoacrylate nanoparticles.

The in vitro protein-rejecting properties of PEG-coated polyalkylcyanoacrylate (PACA) nanoparticles were for the first time visualized after freeze-fracture of the nanoparticles pre-incubated with fibrinogen as a model blood protein. The reduced protein association to the nanoparticles was evidenced also by two-dimensional PAGE after incubation of the nanoparticles with human plasma. In vivo experiments showed the 'stealth' long-circulating properties of the PEGylated nanoparticles after intravenous administration to mice. Thus, the images obtained after nanoparticle-protein incubation were predictive of the behavior observed in vivo. In conclusion, freeze-fracture analysis represents a novel and original qualitative approach to investigate the interactions between proteins and particulate systems.

Pegylated nanoparticles from a novel methoxypolyethylene glycol cyanoacrylate-hexadecyl cyanoacrylate amphiphilic copolymer.

PURPOSE: The aim of this work was to develop PEGylated poly(alkylcyanoacrylate) nanoparticles from a novel methoxypolyethyleneglycol cyanoacrylate-co-hexadecyl cyanoacrylate copolymer. METHODS: PEGylated and non-PEGylated nanoparticles were formed by nanoprecipitation or by emulsion/solvent evaporation. Nanoparticles size, zeta potential and surface hydrophobicity were investigated. Surface chemical composition was determined by X-ray photoelectron spectroscopy. Nanoparticle morphology was investigated by transmission electron microscopy after freeze-fracture. Nanoparticles cytotoxicity was assayed in vitro, onto mouse peritoneal macrophages. Cell viability was determined through cell mitochondrial activity, by a tetrazolium-based colorimetric method (MTT test). Finally, the degradation of PEGylated and non-PEGylated poly(hexadecyl cyanoacrylate) nanoparticles was followed spectrophotometrically during incubation of nanoparticles in fetal calf serum. RESULTS: Monodisperse nanoparticles with a mean diameter ranging between 100 and 200 nm were obtained using nanoprecipitation or emulsion/solvent evaporation as preparation procedures. A complete physico-chemical characterization, including surface chemical analysis, allowed to confirm the formation of PEG-coated nanoparticles. The PEGylation of the cyanoacrylate polymer showed reduced cytotoxicity towards mouse peritoneal macrophages. Furthermore, the presence of the PEG segment increased the degradability of the poly(hexadecyl cyanoacrylate) polymer in presence of calf serum. CONCLUSIONS: We succeeded to prepare PEGylated nanoparticles from a novel poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecyl cyanoacrylate) by two different techniques. Physico-chemical characterization showed the formation of a PEG coating layer. Low cytotoxicity and enhanced degradation were also shown.

Calmodulin is essential for assembling links necessary for exocytotic membrane fusion in Paramecium.

Calmodulin has long been suspected to be involved in calcium-regulated exocytosis but its precise site(s) of action has not yet been identified. In Paramecium, a genetic approach to the problem is possible as in vivo-selected mutations in the calmodulin gene that prevent the activation of some channels have been characterized. Three of these calmodulin mutants were examined for exocytotic capacity and the mutant cam1 was found to be defective for exocytosis at 35 degrees C. The loss of exocytotic capacity in cam1 cells can be restored by transformation with the wild-type calmodulin gene, demonstrating that its exocytotic lesion is indeed due to the mutation in the calmodulin gene. The cam1 mutant displays abnormal exocytotic sites at the non-permissive temperature: it lacks the links ('rosettes' of intramembranous particles in the plasma membrane and the fibrous 'connecting material') which normally connect plasma and trichocyst membranes. Upon shift of cam1 cells from the permissive to a non-permissive temperature, performed sites remain functional. These results demonstrate that calmodulin is necessary for the assembly of these links at the exocytotic site. These results do not, however, exclude the possibility of calmodulin also being involved in Ca(2+)-dependent steps of the stimulus-exocytosis coupling.

Calcium-induced acetylcholine release and intramembrane particle occurrence in proteoliposomes equipped with mediatophore.

Proteoliposomes obtained from the mediatophore, a purified Torpedo electric organ nerve terminals protein, and endogenous lipids were used for a study of calcium-induced release of acetylcholine and freeze-fracture electron microscopy. Large intramembrane particles were induced by the influx of calcium into proteoliposomes, as previously observed for synaptosomes or stimulated electric organ nerve terminals. The involvement of mediatophore in a calcium dependent acetylcholine translocation seems therefore to be related to the occurrence of a category of intramembrane particles in the course of the release process.

Fine structure of the chorion of Manduca sexta and Sesamía nonagrioides as revealed by scanning electron microscopy and freeze-fracturing.

The fine structure of Manduca sexta and Sesamia nonagrioides chorion was investigated by scanning electron microscopy and freeze-fracturing. In both species the mature chorion exhibits a complex ultrastructure on its outer surface, with a large number of aeropyles forming polygonal arrays. The micropyle is surrounded by a rosette of approximately 80 follicular cell imprints. Scanning electron microscopy of vertically ripped sections reveals that both chorions consist of two main layers: a trabecular layer closest to the oocyte and a lamellar layer. The technique of freeze-fracturing, utilizing single-sided and rotary shadowing, clearly shows that fibrils, approximately 3-4 nm in diameter, constitute chorionic lamellae in both species. The fibrils appear to have a 'beaded' structure, with a 2-3 nm axial periodicity. Freeze-fracturing also provides a direct visualization of the helicoidal arrangement of these fibrils for the formation of chorion supramolecular architecture.

Effect of cetiedil on acetylcholine release and intramembrane particles in cholinergic synaptosomes.

The release of acetylcholine (ACh) from instantly frozen Torpedo electric organ synaptosomes in the course of stimulation is systematically associated with an increase in the number of large intramembrane particles counted on freeze-fracture replicas. The drug cetiedil, which is a potent inhibitor of ACh release, also blocks the increase in the number of large particles. The blockage was studied either after ionophore A 23187 or Glycera neurotoxin action in the presence of calcium.

Reconstitution of a functional synaptosomal membrane possessing the protein constituents involved in acetylcholine translocation.

Reconstitution of a functional presynaptic membrane possessing calcium-dependent acetylcholine release properties has been achieved. The proteoliposomal membrane obtained gains its acetylcholine-releasing capabilities from presynaptic membrane proteins. At the peak of acetylcholine release, intramembrane particles became more numerous in one of the proteoliposomal membrane faces. This phenomenon resembles the intramembrane particle rearrangements found in stimulated synaptosomes. No visible structures capable of releasing acetylcholine as a result of the calcium influx were found inside the proteoliposomes. This supports the view that the release of free cytosolic acetylcholine from stimulated nerve terminals can be directly attributed to presynaptic membrane proteins. These proteins were extracted in a functional form from the synaptosomal membrane.

Freeze-fracture electron microscopic analysis of solutions of biological molecules.

Freeze-fracture electron microscopy was used to study the morphology of proteins in solution. The size of the particles appearing on the fractured surface, replicated with tungstentantalum, were measured in a direction perpendicular to the shadowing angle. The distributions of the measured particle sizes could be correlated with the known shape and dimensions for each protein. It is concluded that freeze-fracture electron microscopy is a useful technique to study the morphology of biological molecules in solution, particularly hydrophobic proteins which may be difficult to study by other microscopic techniques.

Rearrangement of intramembrane particles as a possible mechanism for the release of acetylcholine.

1. A chemiluminescent procedure for measuring acetylcholine (ACh) has recently been described. The procedure is based on the hydrolysis of ACh by acetylcholinesterase and on the oxidation of choline to betaine and H2O2 by choline oxidase. The H2O2 generated reacts with luminol in presence of peroxidase to produce a light emission. This method is sensitive in the pmol/ml range. 2. On isolated synaptosomes from electric organ, it is possible to obtain an estimate of the cytoplasmic ACh compartment by measuring the light emission after a single freezing and thawing cycle. The vesicular pool which resists several freezing and thawing cycles is then estimated by opening the compartment with a detergent. Increasing the intensity of stimulation of synaptosomes with different agents depletes the ACh content down to the vesicular pool. 3. The release of ACh is not associated with any change in the number of synaptic vesicles as seen in cryofractured synaptosomes. The only ultrastructural change detected common to all stimulations was a decreased density of P face intramembrane particles smaller than 11 nm and an increased density of E face 8 to 18 nm particles. The very significant particle changes were more intense for the conditions releasing more ACh. It is suggested that these particles are involved in the release of ACh from the cytoplasm. An attempt to directly correlate the release of ACh with intramembrane particle changes is discussed.

[Changes in the number and distribution of intramembranous particles of electric organ synaptosomes of Torpedo during synaptic activity]. / Modification du nombre et de la distribution des particules intramembranaires de synaptosomes d'organe électrique de Torpille lors de l'activité synaptique.

Cholinergic synaptosomes were depolarized with KCl or treated with a venom extracted from the annelid Glycera convoluta. This venom was shown to increase considerably the frequency of the miniature endplate potentials at neuromuscular junctions. The synaptosomes were frozen and fractured in the absence of any fixative or cryoprotectant. Synaptic activity decreased the number of small (6 to 8 nm) particles in the P faces of the presynatic membrane, while the large particles (above 8 nm) increased on both P and E faces. It is suggested that these modifications are related to ionic flux or more directly to the release of transmitter.

Effect of succinylphosphatidylcholine on phosphatidylcholine vesicles: structural studies by gel chromatography, electron microscopy and nuclear magnetic resonance.

The effect of an aqueous dispersion of succinylphosphatidylcholine on an aqueous suspension of phosphatidylcholine vesicles was studied by gel chromatography, freeze-fracture electron microscopy and proton nuclear magnetic resonance with Mn2+ (broadening paramagnetic reagent). Total phospholipid concentrations were in the range 10--20 mM. Succinylphosphatidylcholine is in micellar form and behaves as a detergent. The structures obtained depend on the molar percentage of succinylphosphatidylcholine. Above a succinylphosphatidylcholine molar percentage of 60%, mixed micelles are formed, assumed to be essentially spherical. Below a succinylphosphatidylcholine molar percentage of 30%, principally mixed vesicles are observed, with an external diameter of 215--240 A, and an almost constant internal volume. Between 30 and 60% of succinlyphosphatidylcholine, a mixture of these structures is obtained; rod-shaped profiles are also observed in electron microscopy, which may correspond to sections of leaky vesicles or to a new kind of cylindrical micelle.