Pre-formed vesicles

Measurements of the quantities of glycolipids inserted into the membrane have also been reported by a technique based on the use of C-labeled lipid anchors. In this method, the carbohydrate (a-o-Man) was covalently coupled to the anchor at the surface of a pre-formed vesicle. Indeed, the liposome structure was shown to remain intact in the treatment. Nevertheless, the measurement of the incorporated mannose was performed after separation of bound and unbound material by centrifugation. The yields of coupling were shown to increase with the increase of the initial mannose/ C-anchor ratio, but non covalent insertions were displayed at high initial mannose concentrations. Therefore, the aforementioned method was not as accurate as could have been expected for the use of radioactive materials [142]. Radiolabeled phospholipids were also used for such determinations thus the amounts of glycosphingolipids incorporated into liposomes were quantified by the use of H-phospholipids whereas the amounts of glycolipids were determined by a sphingosine assay [143]. 

Figure 10.11 The use of ferritin as a label for the mechanism of growth of vesicles (adapted from Berclaz et al, 2001a b). Schematic representation of the possible vesicle formation and transformation processes when oleate, and oleic acid, are added to pre-formed vesicles which have been labelled, (a) The situation if only de novo vesicle formation occurs, (b) Growth in size of the pre-formed and labeled vesicles which may lead to division, either yielding vesicles that all contain marker molecules (case i, a statistical redistribution of the ferritin molecules) or also yielding vesicles that do not contain markers (case ii). Compare all this with Figure 10.9.

Consider the experiment illustrated in Figure 10.19, which shows oleate vesicle formation when an aliquot of concentrated surfactant is added to water compared to the situation in which the same amount of surfactant is added to a solution containing pre-formed vesicles. In the second case, the formation of vesicles is remarkably accelerated, as if in the presence of a strong catalytic effect whereas over one hour is needed to reach the turbidity plateau for oleate addition to water, the plateau is reached in less than ten minutes, curve (b), in the second case. 

We have seen experiments of growth of vesicles, whereby fresh surfactant binds to the surface of pre-formed vesicles. How would you devise an experiment, so that the growth rate is determined - or affected - by the vesicle content ... 

Tetanus is a disease caused by the release of neurotoxins from the anaerobic, spore-forming rod Clostridium tetani. The clostridial protein, tetanus toxin, possesses a protease activity which selectively degrades the pre-synaptic vesicle protein synaptobrevin, resulting in a block of glycine and y-aminobutyric acid (GABA) release from presynaptic terminals. Consistent with the loss of neurogenic motor inhibition, symptoms of tetanus include muscular rigidity and hyperreflexia. The clinical course is characterized by increased muscle tone and spasms, which first affect the masseter muscle and the muscles of the throat, neck and shoulders. Death occurs by respiratory failure or heart failure. 

The time course of an actual experiment is shown in Figure 7.17, which shows the hydrolysis of oleic anhydride catalyzed by spontaneously formed oleate vesicles. Note the sigmoid behavior, typical of an autocatalytic process. The lag phase is due to the preliminary formation of vesicles, and in fact the length of the lag phase is shortened when already formed vesicles are pre-added, as shown in the hg-ure. Some mechanistic details of these processes will be discussed in Chapter 10. In this work, an analysis of the number and size distribution of vesicles at the beginning and the end of the reaction was also performed by electron microscopy. 

In contrast to this is the addition of oleate surfactant - in the form of micelles or free monomer - to oleate or to POPC vesicles. In this case, the ratio of the two competitive rates is such that a considerable binding of the added fresh surfactant to the pre-existing vesicles takes place. The efficient uptake of oleate molecules by POPC liposomes (Lonchin et al., 1999) as well as to oleate vesicles (Blochiger et al., 1998) is well documented in the literature. 

Figure 10.10 Transmission electron micrograph of ferritin entrapped in POPC liposomes (palmitoyloleoylphosphatidylcholine). Cryo-TEM micrographs of (a) ferritin-containing POPC liposomes prepared using the reverse-phase evaporation method, followed by a sizing down by extrusion through polycarbonate membranes with 100 nm pore diameters ([POPC] = 6.1 mM) and (b) the vesicle suspension obtained after addition of oleate to pre-formed POPC liposomes ([POPC] = 3 mM, [oleic acid - - oleate] = 3 mM). (Adapted from Berclaz et al, 2001a, b.)...

Figure 10.12 Number-weighted size distributions as obtained by cryo-TEM (adapted from Berclaz et al, 2001a, b). (a) Distribution for the pre-formed POPC vesicles ([POPC] = 1.9 mM). (b) Distribution for the vesicle suspension obtained upon addition of oleate to pre-formed ferritin-containing POPC vesicles ([POPC] = 0.2 mM [oleic acid -I- oleate] = 5 mM). Empty ( ) and ferritin-containing ( ) vesicles are represented individually in the histogram, (c) Direct comparison of the number-weighted size distribution of the pre-formed POPC vesicles, which contained at least one ferritin molecule ( ) with the number-weighted size distribution of the ferritin-containing vesicles obtained after oleate addition to pre-formed POPC vesicles ( ). Note that the total of all ferritin-containing vesicles was set to 100%.

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.)... 

Other than accelerating the formation of spherical nanoparticles of silica from metastable silicic acid solutions, the silaffins appear to have no structure-directing activity. If they are responsible for silica formation in the living diatom, as seems quite likely, control of the higher order architecture of the resulting silica apparently must be determined by the pre-formed shape of the silica deposition vesicle (the envelope within which the silica grows) serving as a complex three-dimensional mold. 

DNA, coding for the GFP, was introduced in liposomes composed by eggPC, together with the whole T T machinery (PURESYSTEM). GFP was synthesized inside giant vesicles prepared by centrifugation of a pre-formed w/o emulsion. 

Templates made of surfactants are very effective in order to control the size, shape, and polydispersity of nanosized metal particles. Surfactant micelles may enclose metal ions to form amphiphilic microreactors (Figure 11a). Water-in-oil reverse micelles (Figure 11b) or larger vesicles may function in similar ways. On the addition of reducing agents such as hydrazine nanosized metal particles are formed. The size and the shape of the products are pre-imprinted by the constrained environment in which they are grown. 

FIGURE 1—9- Neurotransmitter synthesis in a neuropeptidergic neuron. Neurotransmitter synthesis occurs only in the cell body because the complex machinery for neuropeptide synthesis is not transported into the axon terminal. Synthesis of a specific neuropeptide begins with the transcription of the pre-propeptide gene in the cell nucleus into primary RNA, which can be rearranged or edited to create different versions of RNA, known as alternative splice variants or pre-propeptide RNA. Next, RNA is translated into a pre-propeptide, which enters the endoplasmic reticulum, where its peptide tail is clipped off by an enzyme called a signal peptidase to form the propeptide, the direct precursor of the neuropeptide neurotransmitter. Finally, the propeptide enters synaptic vesicles, where it is converted into the neuropeptide itself. Synaptic vesicles loaded with neuropeptide neurotransmitters are transported down to the axon terminals, where there is no reuptake pump for neuropeptides. The action of peptides is terminated by catabolic peptidases, which cut the peptide neurotransmitter into inactive metabolites. 

FIGURE 5—68. Substance P neurons and neurokinin 1 receptors, part 1. For neurons utilizing substance P, synthesis starts with the gene called pre-protachykinin A (PPT-A). This gene is transcribed into RNA, which is then edited to form three alternative mRNA splice variants, alpha, beta, and gamma. The actions of the mRNA version called alpha-PPT-A mRNA are shown here. This mRNA is then transcribed into a protein called alpha-PPT-A, which is substance P s grandparent. It is converted in the endoplasmic reticulum into the parent of substance P, called protachykinin A (alpha-PT-A). Finally, this protein is clipped even shorter by another enzyme, called a converting enzyme, in the synaptic vesicle and forms substance P itself. 

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