Effects of low temperature and lipid modification on the proliferation of cultured mammalian cells.
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Biomedical subjects
Publications and source records attributed to C F Fox.
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A photoreactive derivative of epidermal growth factor (EGF) has been used to identify and specifically label a membrane receptor for EGF on mouse 3T3 cells. Photoactivable EGF, labeled with 125I, was incubated with 3T3 cells and then photolyzed in situ to generate a nitrene capable of reacting with a wide variety of chemical bonds. Analysis of the system by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed, besides the band of EGF, only one other major radioactive band, at a position indicating an apparent molecular weight of 190,000. This band was absent when a nonresponsive and nonbinding variant of 3T3 was used. A direct proportionality between binding activity and crosslinked complex formation was demonstrated using a variety of binding conditions. "Down regulated" cells, in which EGF binding activity was greatly reduced by prolonged incubation with an appropriate concentration of EGF, also had a decrease in covalent complex formation proportional to the decrease in EGF binding activity.
The pulpal responses of three ultraviolet-activated composite resins, Nuva-Fil, Experimental UV No. 1 and Experimental UV No. 2, were tested on adult monkey teeth using silicate and zinc oxide eugenol (ZOE) as positive and negative controls. All materials were placed in Class V cavity preparations in Rhesus monkey teeth using approximately 48 anterior and 63 posterior teeth of both the maxillary and mandibular arches. A total of 111 teeth were utilized and all materials were evaluated at 3 days, 5 and 8 weeks. Following left ventricular perfusion, the teeth were prepared for microscopic evaluation using routine histological procedures. The 3-day pulpal response of all the ultraviolet-activated composites was slight with some disruption and vacuolization in the odontoblastic layer and a slight inflammatory response. At 5 weeks there was a reduction of the inflammatory response and the formation of reparative dentin was noted for all ultraviolet composites. The 8-week pulp response was slight, characterized by a minimal inflammatory response adjacent to the zone of reparative dentin. Generally, ZOE produced the mildest response while silicate produced the most severe response at the three time intervals.
Diagonal electrophoresis, in conjunction with crosslinking by dimethyl-3,3'-dithiobispropionimidate (DTBP), has been used to probe membrane protein topography of Newcastle disease virus (NDV) and cultured mammalian fibroblasts (LM cells). Analysis of DTBP-treated NDV revealed crosslinking between a nucleocapsid protein, VP3, and an externally displayed glycoprotein, VP1, possessing hemagglutination and neuraminidase activities. Similar analysis of LM cells iodinated by the lactoperoxidase method indicated nearest-neighbor interactions of a noncovalent nature involving a major cell surface protein of 72,000 molecular weight. These studies illustrate the feasibility of this methodology for the study of membrane protein disposition in complex systems.
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Three strains of Newcastle disease virus (NDV-HP-16, NDV-L-Kansas, and NDV-N) were propagated in chick embryo fibroblasts, equilibrium labeled with 32Pi, and the composition of phospholipid in the membranous envelope of the virions determined. A phospholipid identifed as monoacylphosphatidylserine was consistently observed in the viral strains which are listed as follows in their order of decreasing abundance of lysophosphatidylserine: NDV-HP 16greater than NDV-L-Kansas greater than NDV-N. The phosphatidylserine concentration in the virion envelopes of these strains decreased in proportion to the increase in the monoacylphosphatidylserine concentration. No other lysophosphatide was observed in significant quantity in virions of these strains. The degree of cell fusion in mouse fibroblast monolayers by each of the viral strains was independent of the lysophosphatidylserine content of the virions. The ability of the viral strains to induce fusion from within, i.e., that occurring in cells that are actively propagating virus was: NDV-L-Kansas greater than NDV-HP-16 greater than NDV-N. The ability of the viral strains to induce fusion from without, i.e., that occurring in response to incubation of cells with large quantities of irradiated virus was: NDV-HP-16 greater than NDV-N greater than NDV-L-Kansas. On the basis of these findings we conclude that there is no direct correlation between the level of lysophosphatide in the virion and its ability to induce cell membrane fusion. A direct correlation was observed, however, between the presence of high monoacylphosphatidylserine content and the ability of a strain to produce lytic infection.
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Lactoperoxidase-catalyzed iodination selectively labels the two glycoproteins (VP1 and VP2) of Newcastle disease virus. The low-molecular-weight, nonglycosylated major viral protein, VP6, was not iodinated in the intact virus but was iodinated in disrupted virions, suggesting a localization on the inner, rather than the outer, envelope surface. Studies on the distribution of virion proteins labeled with 125-I and 3-H-isoleucine between detergent-soluble and detergent-insoluble fractions show that the virion proteins VP4, VP5, and VP6 are solubilized to a much lesser extent than are VP1 and VP2.
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Electron spin resonance analysis of suspensions of animal cell plasma membranes consistently reveals four characteristic temperatures for lateral phase separations in the membrane lipids. Similar analysis of an aqueous dispersion of lipids extracted from these membranes reveals only two characteristic temperatures, indicating that some aspect of lipid organization in membranes is destroyed by the extraction procedure. The characteristic temperatures for surface membranes from two different species of homeothermic animals were nearly identical and were approximately 37 degrees , 31 degrees , 21 degrees , and 15 degrees . A treatment of the physical data revealed that these temperatures could identify independent phase transitions for two hydrocarbon compartments of approximately equal size with lower and upper characteristic temperatures of 21 degrees and 37 degrees , and of 15 degrees and 31 degrees . The analysis of the effects of temperature on a number of physiological parameters indicates that 21 degrees and 37 degrees are likely to define the boundaries for lateral phase separations in the inner monolayer and 15 degrees and 31 degrees the boundaries for lateral phase separations in the outer monolayer.
Attractant-directed motility (chemotaxis) in Escherichia coli has an absolute requirement for a fluid membrane. No such requirement was detected for motility per se.