Extruded vesicles

Zhang, F. and E.S. Rowe. 1994. Calorimetric studies of the interactions of cytochrome c with dioleoylphosphatidylglycerol extruded vesicles ionic strength effects. Biochim Biophys Acta 1193 219-225. 

Figure 10.20 (a) Matrix effect for oleate addition to pre-formed POPC liposomes. In this case, mixed oleate/POPC vesicles are finally formed. Note the extraordinary similarity between the size distribution of the pre-formed liposomes and the final mixed ones. By contrast, the size distribution of the control (no pre-existing liposomes) is very broad, (i) Sodium oleate added to POPC liposomes, radius = 44.13, P-index = 0.06 (ii) POPC liposomes, radius = 49.63, P-index = 0.05 (iii) sodium oleate in buffer, radius = 199.43, P-index = 0.26. (b) matrix effect for the addition of fresh oleate to pre-existing extruded oleate vesicles. In this case, the average radius of the final vesicles is c. 10% greater than the pre-added ones, and again the difference with respect to the control experiment (no pre-added extruded vesicles) is striking, (i) Oleate vesicles extruded 100 nm, radius = 59.77, P-index = 0.06 (ii) oleate added to oleate vesicles, extended 100 nm, radius = 64.82, P-index 0.09 (iii) sodium oleate in buffer, radius = 285.88, P-index = 0.260. (Modified from Rasi et al, 2003.)... 

Kolchens, S., Ramaswami, V., Birgenheier, J., Nett, L., and O Brien, D. F. (1993), Quasielastic light scattering determination of the size distribution of extruded vesicles, Chem. Phys. Lipids, 65,1-10. 

Figure 3. Freeze-fracture electron microscopy analysis of the vesicle size distribution in the case of the spontaneous vesiculation of oleic acid/oleate. (A) Vesicles formed from the hydrolysis of 25 mM oleic anhydride (overall concentration) at 30 °C, yielding 50 mM oleic acid/oleate. (B) Vesicles extruded throughout 50 nm diameter filters. (C) Vesicles formed upon hydrolyzing 20 mM oleic anhydride (same conditions as in A) in the presence of pre-added extruded vesicles B—all in 0.2 M bicine buffer pH 8.5. For details see ref. 8.

Liposomes should be stored at 4°C and used within 1 mo. Extruded vesicles should not be frozen. Vesicles will naturally begin to coalesce within 6 mo to 1 yr. 

DNA polymerase has been entrapped within POPC extruded vesicles, together with a template DNA, primers, and dNTPs. Thermal cycles allow the polymerase chain reaction occurs within vesicles. 

Ribosomes were entrapped inside POPC vesicles together with poly(U), acting as template mRNA, t-RNA , Phe-tRNA-synthetase, Phe, and all low mw compounds required for the translation. Poly(Phe) is produced accordingly inside extruded vesicles. 

Massenburg, D., and Lentz, B., 1994, Polyfethylene glycol)-induced fusion and rapture of dipalmitoylphosphatidylcholine large, unilamellar extruded vesicles. Biochemistry 32 9172-9180. 

Figure 9,5 Zeta potential versus concentration of the charged lipid DOPG in DOPC vesicles. Eight samples, each containing 10mg of lipid (of different DOPC DOPG ratios) were dried down, rehydrated with 1 ml of 0.1 mM NaCl and then extruded through 200 nm filters in a Lipex Biomembranes extruder 11 times. The samples were then diluted with 1 ml of 0.1 mM NaCl. 1.3 ml of each sample was placed in a different cuvette. The zeta potential of the extruded vesicles was measured 10 times for each sample using a Brookhaven Zeta PlusPCS machine.

Finally, the actions of the so-called 5-HT releasing agent , if-fenfluramine, which is well known for its anorectic effects, should be mentioned here. This compound inhibits 5-HT uptake but its metabolite, if-norfenfluramine, increases 5-HT release as do high doses of (i-amphetamine. It is important to realise that this 5-HT release is independent of nerve impulses and the action of such compounds rests on their effects on the 5-HT transporters on the storage vesicles and terminal membrane. Once these drugs have been taken up into 5-HT neurons by the transporter, they cause 5-HT to leak out of its storage vesicles and, ultimately, to be extruded from the neuron by retrotransport (see below and Chapter 4 for further details). 

Reverse-phase evaporation in a nitrogen atmosphere was used to prepare lipids. A lipid film previously formed was redissolved in diethyl ether and an aqueous phase containing the dyebath components added to the phospholipid solution. The resulting two-phase system was sonicated at 70 W and 5 °C for 3 minutes to obtain an emulsion. The solvent was removed at 20 °C by rotary evaporation under vacuum, the material forming a viscous gel and then an aqueous solution. The vesicle suspension was extruded through a polycarbonate membrane to obtain a uniform size distribution (400 nm). 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

Figure 10.15 Intensity-weighted size distribution of POPC in 0.2 M bicine buffer solution, pH 8.5 0.5 ml solution of 1 mM 30 nm extruded POPC vesicles mixed with 0.5 ml solution of 1 mM 200 nm extruded POPC vesicles. The measuring angle is 90°. (From Cheng and Luisi, 2003.)...

Figure 10.16 Effect of Ca + on the turbidity change upon mixing oleate vesicle solutions 0.25 ml 1 mM 60 nm radius extruded oleic acid vesicles + 0.25 ml 1 mM 200 nm radius extruded oleic acid vesicles + 1.5 ml bicine buffer. Calcium ion concentration (a) 0 mM (b) 1 irtM (c) 2.5 mM (d) 5 mM (e) added excess EDTA to (d). (Adapted from Cheng and Luisi, 2003.)...

Experiments have been set up in this direction (Cheng and Luisi, 2003) utilizing extruded oleate vesicles having radii of 31.6 and 64.1 nm. For the same surfactant concentration, the total surface areas are the same for the two families of vesicles (neglecting the differences arising in aggregation number due to the differences... 

Diatoms are unicellular, photosynthetic microalgae that are abundant in the world s oceans and fresh waters. It is estimated that several tens of thousands of different species exist sizes typically range from ca 5 to 400 pm, and most contain an outer wall of amorphous hydrated silica. These outer walls (named frustules ) are intricately shaped and fenestrated in species-specific (genetically inherited) patterns5,6. The intricacy of these structures in many cases exceeds our present capability for nanoscale structural control. In this respect, the diatoms resemble another group of armored unicellular microalgae, the coccolithophorids, that produce intricately structured shells of calcium carbonate. The silica wall of each diatom is formed in sections by polycondensation of silicic acid or as-yet unidentified derivatives (see below) within a membrane-enclosed silica deposition vesicle 1,7,8. In this vesicle, the silica is coated with specific proteins that act like a coat of varnish to protect the silica from dissolution (see below). The silica is then extruded through the cell membrane and cell wall (lipid- and polysaccharide-based boundary layers, respectively) to the periphery of the cell. 

Liposome Preparation Techniques In most cases, liposomes are named by the preparation method used for their formation, Such as sonicated, dehydrated-rehy-drated vesicle (DRV), reverse-phase evaporation (REV), one step, and extruded. Several reviews have summarized available liposome preparation methods [91,124, 125], Liposome formation happens spontaneously when phospholipids are dispersed in water. However, the preparation of drug-encapsulating liposomes with high drug encapsulation and specific size and lamellarity is not always an easy task. The most important methods are highlighted below. 

In humanS/ P-gp is expressed on trophoblastic cells throughout pregnancy (82/ 84-86). It has been located on apical surfaces of endodermal cells of the mouse yolk sac (87)/ in the vesicles of the brush border membrane of the human syncytiotrophoblast that directly faces maternal blood/ but not within maternal vascular endothelium (83/ 84/ 86-95). Actively transporting molecules in a basolateral-to-apical direction/ the role of P-gp within the placenta is similar to its function at other sites it extrudes drugs from the placenta back into maternal circulation/ thereby protecting the developing fetus from potential toxic factors within the maternal circulation (95). 

In other coccoliths species the intracellular vesicles may be mineralized and completed after the scale has been extruded onto the cell surface but still covered by an organic membrane. In the genus Chyrsotila, nonmotile cells produce a mucilaginous sheath in which spherulitic aragonite crystallites form, which eventually encapsulate the cell (Green and Course, 1983). There are... 

After sonication the samples are filtered by syringe filters with the pore size of 0.45 pm to separate large vesicles that may blockade the extruder membrane (see Note 3). 

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