Membrane components, methods

Another 3-D cornea model, comprising rabbit primary cultures of epithelial and stromal cells as well as mouse immortalized endothelial cells, was described in 1994 by Zieske and coworkers [70], They showed the influence of endothelial cells on the formation of a tightly packed, multilayered epithelium as well as the expression of laminin, type VII collagen, a6 integrin, keratin K3, and a-enolase. Furthermore, their findings suggested that the formation of an in vivo-like epithelium requires the cultivation of the 3-D corneal construct under AIC conditions. By contrast, LCC methods of cultivating corneal equivalents in the absence of endothelial cells failed to promote the expression of differentiation markers and basement membrane components. 

Rates of lateral diffusion of membrane components have also been determined using optical methods. The early experiments of Frye and Ediden16 demonstrated lateral motion of fluorescent-labeled surface antigens in heterokaryons of mouse and human cells. They observed intermixing of fluorescent-labeled antibodies against mouse cell and human cell antigens. Optical methods may also be characterized as either transient or steady state. The use of fluorescence correlation spectroscopy as... 

The usefulness of the gold-catalyzed aldol reaction was demonstrated by application of the method to the asymmetric synthesis of the important membrane components D-erythro and ffereo-sphingosines, and their stereoisomers (Scheme 8B1.2) [13], and MeBmt, an unusual amino acid in the immunosuppressive undecapeptide cyclosporine (Scheme 8B1.3) [14]. 

Cholesterol-rich domains of biological membranes have been isolated on the basis of their insolubility in 1% Triton at 4° C (36). This method has come into question because of the possibility that the Triton itself causes rearrangement of membrane components (37, 38). As indicated above, this fraction should not be referred to as a raft. However, the cholesterol-rich domain in the form of caveolae can be isolated without use of detergent (39, 40), providing stronger evidence that this cholesterol-rich domain exists in a biological membrane before extraction. 

Ribeiro-Neto FAP, Mattera R, Hildebrandt JD et al. (1985) ADP-ribosylation of membrane components by pertussis and cholera toxin. In Methods Enzymol. 109 566-572... 

Porosities of membrane components vary widely and values are reported ranging from 20 to 60%. Commonly, values of 30-40% are used. Pore sizes range from macropores (>500 nm) via mesopores (20-500 nm) to micropores (<2 nm). A great problem is the lack of reliable measurement methods to measure the porosity and pore size distribution of supported membranes (see Chapter 4). 

Because of the stability problems with conventional liposomes, scientists have sought many methods to stabilize them. One important development is the sterically stabilized liposomes (SSLs), which are sometimes called stealth liposomes. 25-28 Synthetic polymers are used for steric stabilization. Another approach involves cross-linking membrane components covalently or by the polymerization of polymerizable lipids.29,30 A third approach utilizes unusually stable archaebacterial membrane lipids mimics.31... 

Membrane proteins can thus be separated (and visualized) following disruption of protein interactions with other protein or lipid membrane components, by gel electrophoresis. One useful method is the layering of an SDS extract solution onto a polyacrylamide gel followed by application of an electrical field. The resulting differences in electrophoretic mobility then separates the individual proteins based on their mass (not charge). This is called SDS-polyacrylamide-gel electrophoresis (SDS-PAGE). 

The lateral mobility of proteins and lipids in natural and artificial lipid bilayer membranes was determined by different methods. For long-range mobility, fluorescence recovery after photobleaching (13-15) and electrophoresis of membrane components (16) were employed. We employed the electrophoresis method for determination of the eletrophoretic and diffusional mobilities of PSI in the plane of hypotonically inflated, spherical thylakoid vesicles. To monitor the redistribution of PSI particles, we made use of the spatial characteristics of the contribution of PSI particles to electrophotoluminescence (EPL) (17, 18). The contribution of PSII to EPL was eliminated by heat treatment of the chloroplasts (19). The EPL originates from the PSI particles at the hemisphere of the vesicles at which the induced electrical field destabilizes the photoinduced charge separation (18). The electrophoretic and diffusional mobilities were measured in vesicular suspensions to avoid immobilization for microscopic visualization (20). The photosynthetic membranes are devoid of cytoskeletal elements that might interfere with the lateral mobility. 

The estimated arnounts show how difficult it is to measure such an amount of carbon accurately. Combe et al. (1999) reported that this amount adsorbed could not be measured by mass balance (total amount adsorbed about 10 pg on a 28.7 10 trF membrane section (Clark (1999)). This amount is very close to the estimated monolayer adsorption value. The organic carbon was desorbed from the membranes with NaOH and then measured with UV. Despite the results being very close to the calculated monolayer value, this method is subject to errors from (i) the contamination of the NaOH with membrane components, (ii) the erratic concentration detetmination using UV (the selective adsorption of UV absorbing compounds could lead to an overestimation), and (ill) possibly the modification of UV absorbance characteristics of the organics due to the extreme pH variation. 

In order to reduce such interferences, successful efforts have been made to isolate the cell membranes, or even their transport-active constituents. One way to achieve this is by preparation of isolated membranes which have a natural tendency to form closed and homogenous vesicles [31,32]. Another approach is by reconstituted systems , i.e. to isolate membrane components involved in specific transport processes, and to incorporate them in artificial lipid membranes, usually liposomes [33,34]. Vesicles have been successfully prepared from various cells and tissues and tested for transport activities. Whenever membranous material has been isolated from other cellular components it tends to form vesicles spontaneously, sometimes with an uniform orientation, right-side-out or inside-out vesicles, respectively. For mixtures of vesicles of the two orientations, methods were developed to separate the two polarities. Furthermore, one can separate vesicles from different cell types or even from different regions of the cell, e.g. brush-border membranes form basal lateral ones [35,36]. 

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