External bilayer diffusion

The most intriguing aspect of the self-spreading lipid bilayer is that any molecule in the bilayer can be transported without any external bias. The unique characteristic of the spreading layer offers the chance to manipulate molecules without applying any external biases. This concept leads to a completely non-biased molecular manipulation system in a microfluidic device. For this purpose, the use of nano-space, which occasionally offers the possibility of controlling molecular diffusion dynamics, would be a promising approach. 

Fig.3 A migrating zone of solute molecules (spots) interacting with lipid bilayers (rings) in a chromatographic or electrophoretic separation system. The free solute molecules move (arrows) relative to the liposomes or vesicles in a flow of eluent or in an electric field. The solute molecules may either partition into the membranes and diffuse between the external and internal aqueous compartments of the structures as depicted, or interact with the external surface of the membranes and stay outside.

Is it possible to use simple bilayer vesicles (liposomes) to test the involvement of other modes of motion than lateral diffusion of lipids (e.g., motions across the bilayer that would be important in the transmission of signals from the cell interior to the external cell surface) ... 

In the fourth subtechnique, flow FFF (F/FFF), an external field, as such, is not used. Its place is taken by a slow transverse flow of the carrier liquid. In the usual case carrier permeates into the channel through the top wall (a layer of porous frit), moves slowly across the thin channel space, and seeps out of a membrane-frit bilayer constituting the bottom (accumulation) wall. This slow transverse flow is superimposed on the much faster down-channel flow. We emphasized in Section 7.4 that flow provides a transport mechanism much like that of an external field hence the substitution of transverse flow for a transverse (perpendicular) field is feasible. However this transverse flow—crossflow as we call it—is not by itself selective (see Section 7.4) different particle types are all transported toward the accumulation wall at the same rate. Nonetheless the thickness of the steady-state layer of particles formed at the accumulation wall is variable, determined by a combination of the crossflow transport which forms the layer and by diffusion which breaks it down. Since diffusion coefficients vary from species to species, exponential distributions of different thicknesses are formed, leading to normal FFF separation. 

Plasmid/cationic carrier complexes have been proposed to internalize into the endosome and initiate the destabilization of endosomal membranes. This destabilization would induce diffusion of anionic lipids from the external layer of the endosomal membrane into the complexes and form charge neutralized ion pairs with the cationic lipids. Destabilization and/or fusion of the complex with the plasma membrane would permit the same anionic lipids to diffuse to the surface, as would fusion with the endosomal membrane. Release of the condensed DNA from the cationic lipid in the endosome is likely to generate a mechanical or osmotic stress that raptures the endosomal bilayer and releases DNA into the cytoplasm. In contrast, DNA release from complexes on the cell surface might be unable to stress the membrane to a degree sufficient to rapture. 

The effect of US on content release is attributed to the rarefaction phase of the sound wave. Thus, when the negative US wave impinges upon the liposomes, the air pocket expands and stresses the bounding monolayers and also those in the adjacent bilayer. If the pressure drop is large enough, then the stress exceeds the elastic limit of the weakest surface and, at some point, either the bilayer or the monolayer rends. When the integrity of the vesicle is lost, some or all contents are released. If the air in the pocket is expanded faster than it is diffused to the external aqueous phase, then, the monolayer... 

The diffusion of small molecules into the skin from the external world is limited by the stratum comeum (SC). There is now considerable evidence that a major pathway for such diffusion through the stratum comeum itself consists of the intercellular spaces and, in particular, the lipid component of the intercellular spaces. Such lipids are arranged in lamellae that may well be bilayers and that in other respects also resemble biological membranes. In addition, there are other components within the intercellular spaces (e.g., proteins and an aqueous phase) that, although not well understood, mean that the intercellular diffusion path is a heterogeneous material. Within such material, lipids appear to play a very important role (Potts and Guy, 1992), and... 

Membranes are structurally and functionally asymmetric, as exemplified by the restriction of sugar residues to the external surface of mammalian plasma membranes. Membranes are dynamic structures in which proteins and lipids diffuse rapidly in the plane of the membrane (lateral diffusion), unless restricted by special interactions. In contrast, the rotation of lipids from one face of a membrane to the other (transverse diffusion, or flip-flop) is usually very slow. Proteins do not rotate across bilayers hence, membrane asymmetry can be preserved. The degree of fluidity of a... 

V +] or when sensitizers that did not bind to the PC membrane at all were present in the external medium. Additional studies using C-radiolabeled (C7)2V + demonstrated that transmembrane diffusion of the viologen did occur on relevant time-scales, and a full kinetic analysis of apparent transmembrane reduction of vi-ologens across PC liposomal membranes provided direct evidence for the proposed viologen-mediated transmembrane redox pathway [109], Thus, in retrospect, it is clear that these reactions did not occur by electron tunneling across the bilayer. 

Fig. 2.19 Diagram of the plasma membrane showing its integral proteins (fluid mosaic model) (adapted from S.J. Singer et af, 1972 and H. Knufermann, 1976). 1 external aqueous milieu, 2 internal aqueous milieu, 3 fracture plane of the apolar membrane layer, 4 externally orientated intrinsic protein (ectoprotein), 5 internally orientated intrinsic protein (endoprotein), 6 external extrinsic protein, 7 internal intrinsic protein, 8, 9 membrane-penetrating proteins with hydrophobic interactions in the inside of the membrane (P = polar region), 10 membrane pervaded by glycoprotein with sugar residues (, 11 lateral diffusion (A) and flip-flop (B), 12 hydrophilic region (A) and hydrophobic region (B) of the bilayer membrane...

The cell plasma membrane consists of a variety of proteins associated with the lipid bilayer and they perform multitasks in cell function. The control of transport of ions and molecules across membrane is accomplished through specialized function of membrane proteins. These proteins are distributed in membrane on the outer surface, some on the inner surface, and some others are transmembrane proteins with external and cytoplasmic domains. The majority of the transmembrane proteins are the ion channels or signaling proteins. Generally, hpid to protein ratio is 60 40 but this ratio is found variable in different cells and types of membranes. Membrane proteins impart the dynamic structure and selectivity to membrane function. Both proteins and hpids show motional and diffusion properties within the bUayer structure. 

The relationship between the intra- and extravesicular pH in the above experiments has been studied by NMR spectroscopy 91). Vesicles containing a mixture of NaH2P04 and NaNOs were studied to determine whether the presence of the diffusable NO3 ion would permit the influx of hydroxide as observed for intravesicular Ag20 formation. The intravesicular phosphate resonance, initially at -14.98 ppm, did not shift significantly until above pHout H O, after which it shifted steadily downfield due to OH influx (Fig. 23). Similar experiments undertaken with phosphate in the absence of nitrate showed that a pH gradient of 6 units could be maintained across the bilayer membrane at an external pH of 12.5 (Fig. 24). 

In Ch. 24 Aoki uses FT-IR reflection spectroscopy to monitor the transfer of protons and of water molecules from a layer of H2O ice to a layer of D2O ice as a function of time and external pressure. The H/D mutual diffusion coefficient measured at 400 K shows a monotonic decrease by two orders of magnitude as the pressure increases from 8 to 63 GPa. In order to separate molecular from protonic diffusion experiments were also carried out on H2 0 ice bilayer. 

The mechanism by which phospholipid inserts into the outer membrane is unclear. Pulse-chase experiments indicate that newly synthesised PE is first located in the inner leaflet of the outer membrane and later rotates ( flip-flops ) through the lipid bilayer to become part of the external lipid leaflet. Attempts to visualise discrete sites of PE insertion into the outer membrane have failed. This is not surprising since the lateral diffusion time... 

Figure 6.7 Change in the fluorescence intensity of pyranine entrapped in unilamellar vesicles of asolectin following a rapid decrease in external pH. The downward arrow indicates the addition of HCl which lowered the external pH to 6.2. (a) asolectin vesicles alone the rapid drop of fluorescence intensity (direct diffusion of protons through the vesicle bilayers) is followed by a very slow one (b) asolectin vesicles doped with valinomycin the rapid change is not affected but the slow change is much accelerated (c) asolectin vesicles + nonionic surfactant Triton XlOO monophasic variation of intensity that reflects the lysis of the vesicles by the added Triton XlOO. Reproduced from Reference 80 with permission of the American Chemical Society.

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