Cell-surface, isolation

Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

When liposomes are formed, they can be made to entrap certain compounds inside themselves, eg, drugs and isolated genes. There is interest in using liposomes to distribute drugs to certain tissues, and if components (eg, antibodies to certain cell surface molecules) could be incorporated into liposomes so that they would be targeted to specific tissues or tumors, the therapeutic impact would be considerable. DNA entrapped inside liposomes appears to be less sensitive to... 

Valenzuela-Femandez A, Palanche T, Amara A, et al. Optimal inhibition of X4 HIV isolates by the CXC chemokine stromal cell-derived factor 1 alpha requires interaction with cell surface heparan sulfate proteoglycans. J Biol Chem 2001 276(28) 26550-26558. 

The glycocalyx and the mucus layer make up the structure of the unstirred water layer (UWL) [73]. The thickness of the UWL is estimated to be 30-100 pm in vivo, consistent with very efficient stirring effects [74]. In isolated tissue (in the absence of stirring), the mucus layer is 300-700 pm thick [73]. The pH in the unstirred water layer is 5.2-6.2, and might be regulated independently of the luminal pH (Section 2.3). The mucus layer may play a role in regulating the epithelial cell surface pH [73]. 

Most receptors (a notable exception being the steroid receptors that influence DNA transcription) are located on the cell surface, and purified cell membranes are thus an obvious choice of preparation. When a tissue is homogenized, however, any membrane fraction isolated may well contain membranes from intracellular organelles in addition to cell membranes from all the cell types present in the tissue. Thus, brain membranes will contain membranes not only from neurons but... 

The corrector activity of 15 was not limited to recombinant systems as it was confirmed in cultured HBE cells isolated from F508del homozygous patients. After 48-h incubation with 6.7 pM 15, a 2-fold increase in forskolin-stimulated chloride current (compared to DMSO-treated cells) or 14% of the response obtained in non-CF-HBE was observed. It was also reported that 14 and 15 increase the cell surface density of other trafficking-deficient proteins, including G601S-hERG. The authors speculate that this class of compounds may act on the folding pathway shared by both misfolded proteins rather than on CFTR itself [16]. 

Reported applications of BSOCOES include studying the polypeptide antigens on lymphocyte cell surfaces (Zarling et al., 1980), crosslinking labeled (3-endorphin to its opioid receptors (Howard et al., 1985), and isolation and characterization of calcitonin receptors in rat kidney (Bouizar et al., 1986). 

The isolated cells may be lysed using standard mechanical or detergent methods and the biotinylated cell-surface proteins analyzed or isolated using (strept)avidin reagents. 

The biotinylated glycans on the cell surfaces subsequently may be probed with (strept)avidin reagents to detect the azido-sialic acid modifications. Alternatively, the cells may be lysed and the glycoproteins isolated using an immobilized (strept)avidin or monomeric avidin affinity resin. 

IL-2-stimulated cytotoxic T cells appear even more efficacious than LAK cells in promoting tumour regression. The approach adopted here entails removal of a tumour biopsy, followed by isolation of T-lymphocytes present within the tumour. These tumour-infiltrating lymphocytes (TILs) are cytotoxic T-lymphocytes that apparently display a cell surface receptor which specifically binds the tumour antigen in question. They are thus tumour-specific cells. Further activation of these TILs by in vitro culturing in the presence of IL-2, followed by reintroduction into the patient along with IL-2, promoted partial/full tumour regression in well over 50 per cent of treated patients. 

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