Wiley
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Regulation of dome formation in differentiated epithelial cell cultures Abstract Rat mammary (Rama 25) and dog kidney (MDCK) epithelial cell cultures formed ‘domes’ of cells due to fluid accumulation in focal regions between the culture dish and the cell monolayer. Addition of ouabain caused collapse of domes, suggesting that transport functions were required for maintenance of domes. Dome formation in both epithelial cell lines was stimulated by a broad spectrum of known inducers of erythroid differentiation in Friend erythroleukemia cells. Among these inducers were: (1) polar solvents such as dimethylsulfoxide, dimethylformamide, and hexamethylene bisacetamide; (2) purines such as hypoxanthine, inosine, and adenosine; (3) low‐molecular‐weight fatty acids such as n‐butyrate; and (4) conditions expected to elevate levels of cyclic AMP. In the latter group were activators of adenylate cyclase such as cholera toxin and prostaglandin E1 ; cyclic AMP phosphodiesterase inhibitors such as theophylline and 1‐methyl‐3‐isobutylxanthine; and analogs of cyclic AMP. Induction of domes occurred 15–30 h after addition of inducer to the culture medium. Induction by chemicals was serum‐dependent and required protein synthesis but not DNA synthesis. Induced dome formation was reversible after removal of inducer, requiring the contiuous presence of inducer. Reversal was also observed after either removal of serum or addition of inhibitors of protein synthesis. These results suggest the hypothesis that domes arise in these epithelial cultures by a process that is similar to cell differentiation and is influenced by cyclic AMP.
Wiley - Tập 12 Số 2 - Trang 259-272 - 1979
Molecular interactions in the bacterial phosphoenolpyruvate‐phosphotransferase system (PTS)
Wiley - Tập 2 Số 5-6 - Trang 695-714 - 1974
Reconstituted low density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes to cells Abstract Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3‐pyrenemethyl‐23, 24‐dinor ‐5‐cholen‐22‐oate‐3β‐yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane‐extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor‐mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence‐activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.
Wiley - Tập 10 Số 4 - Trang 467-478 - 1979
Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents Abstract Treatment of isolated human erythrocyte membranes with Triton X‐100 at ionic strength ⋍0.04 preferentially released all the glycerolipid and glycoprotein species. At low ionic strength, certain nonglycosylated polypeptides were also selectively solubilized. The liberated polypeptides were free of lipids, but some behaved as if associated into specific oligomeric complexes. Each detergent‐insoluble ghost residue appeared by electron microscopy to be a filamentous reticulum with adherent lipoid sheets and vesicles. The residues contained most of the membrane sphingolipids and the nonglycosylated proteins. The polypeptide elution profile obtained with nonionic detergents is therefore nearly reciprocal to that previously seen with a variety of agents which perturb proteins. These data afford further evidence that the externally‐oriented glycoproteins penetrate the membrane core where they are anchored hydrophobically, whereas the nonglycosylated polypeptides are, in general, bound by polar associations at the inner membrane surface. The filamentous meshwork of inner surface polypeptides may constitute a discrete, self‐associated continuum which provides rather than derives structural support from the membrance.
Wiley - Tập 1 Số 3 - Trang 233-248 - 1973
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