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Biomedical subjects

C F Fox

Publications and source records attributed to C F Fox.

At least 55 records · Page 3Linked to original sources
PubMed1980

Effects of cytochalasin B and enucleation on EGF receptor down regulation.

The loss of epidermal growth factor (EGF) binding activity on cultured murine 3T3 cells exposed to EGF (EGF receptor down regulation) was determined in colchicine treated cells, cytochalasin B treated cells, and untreated cells. Neither colchicine nor cytochalasin B altered the affinity of the receptor for EGF, but colchicine decreased maximal EGF binding activity by 20%. The maximal extent of EGF receptor down regulation was similar in colchicine treated cells and cytochalasin B treated cells, but the rate of receptor down regulation was higher in cytochalasin B treated cells. Cytoplasts produced by subjecting cytochalasin B treated cells adhering to the substratum to centrifugal force responded to EGF with nearly normal down regulation kinetics. The results suggest that the cytoskeleton is not obligatorily involved in EGF-induced EGF receptor down regulation.

Animals↗
PubMed1979

Photoaffinity inactivation of the enkephalin receptor.

An arylazide enkephalin derivative, [D-Ala2,Met5]enkephalin-Tyr-N-(2-nitro-4-azidophenyl) ethylenediamine (ETN), has been synthesized. In the dark, it inhibited the binding of [3H]enkephalinamide to enkephalin receptor-rich NG-108 cell membranes with an I50 = 2.2 X 10(-8) M or KI = 7 X 10(-9) M, assuming competitive inhibition. Photolysis of membranes in the presence of ETN caused irreversible inactivation of the enkephalin receptor, but inactivation was prevented by the addition of enkephalin, the half-effective concentration being 3 x 10(-9) M. ETN appears to be an effective photoaffinity label for the enkephalin receptor.

Affinity Labels↗
PubMed1979

Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: the endocytic activation hypothesis.

The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity 125I-EGF, and the fate of the affinity labeled EGF-receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF-induced receptor internalization and EGF-induced stimulation of 3H-thymidine uptake into cellular DNA show that there is a direct correlation between EGF-induced receptor internalization and EGF-induced stimulation of DNA synthesis, but not between EGF binding and EGF-induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF-induced processes requires constant EGF-induced internalization of receptor for a requisite 6-8 h period as an obligatory step in production of "second messenger" in the action of this hormone.

Animals↗
PubMed1979

Hormone-induced modification of EGF receptor proteolysis in the induction of EGF action.

A proposal that EGF action is mediated through enhanced internalization of EGF receptors is modified to account for more recent evidence. EGF receptors turn over at a rapid rate, and the maintenance of a steady state of EGF receptors on the cell surface is provided through a rapid synthesis of EGF receptors, balancing their removal. This rapid turnover of unoccupied receptors may arise through their internalization and proteolysis in the lysosomes, in much the same way as receptors are internalized and degraded when exposed to EGF, which enhances internalization. This provides a dilemmma for the endocytic activation concept, since slight enhancement of receptor internalization gives rise to a strong hormone response. This problem may be solved by the observation that EGF induces a change in its receptor, exposing an otherwise unavailable site for proteolytic cleavage. This hormone-dependent modification of receptors may be the critical step in the induction of responses to EGF and other hormones that are internalized with their receptors. Both platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) are shown to down-regulate EGF receptors, though transiently, placing still more stringent requirements on the specificity by which hormones might act through endocytic activation of their receptors.

Animals↗
PubMed1978

Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril.

The use of the iodinating reagent 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycouril (chloroglycoluril) to selectively label membrane surface proteins was investigated with the following systems: enveloped viruses (Sendai and Newcastle disease viruses), human erythrocytes, and nucleated cells propagated both in suspension (EL-4) and in monolayer culture (BHK-21). Conditions are described for specifically iodinating surface proteins while maintaining full virus integrity or cell viability. Comparison of the chloroglycoluril method with the lactoperoxidase and chloramine-T methods for labeling surface membrane proteins shows that the chloroglycoluril method has a number of advantages: It routinely produces a 3- to 17-fold greater specific radioactivity without sacrificing viral or cellular integrity, it is technically simpler to use, it does not require the addition of extraneous protein to initiate the reaction nor a strong reducing reagent to terminate it. Chloroglycoluril also proved to be an effective substitute for chloramine-T in the nonvectorial labeling of viral and cellular proteins. Membrane protein samples were solubilized with the detergent sodium dodecyl sulfate before iodination or labeled in the presence of high iodide concentrations without prior solubilization. The resulting specific radioactivities generated by the use of chloroglycoluril were equal to or greater than those generated by the chloramine-T method. The effectiveness, simplicity of use, and versatility of chloroglycoluril recommend it as an iodinating reagent for both surface-specific and nonvectorial labeling of membrane systems.

Cell Line↗
PubMed1978

Epidermal growth factor induced membrane changes in 3T3 cells.

Epidermal growth factor (EGF) is a mitogen for Swiss 3T3 cells. Short incubation periods with physiological concentrations of EGF induced increased binding of Swiss 3T3 cells to Con A-coated nylon fibers. This effect was not induced in an EGF non-responsive 33 variant, in the transformed murine XC cells or in Swiss SV3T3 cells. The increase in Con A fiber-binding seems to be specific for EGF, since it was not observed in response to insulin, prostaglandin F2alpha or a higher serum concentration, which also initiate cell devision of confluent quiescent 3T3 cells. EGF also reduced Con A-mediated hemadsorption to 3T3, but had no effect on hemadsorption by the EFG non-responsive 3T3 variant. There was no change in the number of Con A-receptors on 3T3 cells after EGF treatment. Binding to WGA-coated fibers and WGA-mediated hemadsorption were not effected by preincubation with EGF.

Clone Cells↗
PubMed1978

Membrane permeability of bifunctional, amino site-specific, cross-linking reagents.

The membrane permeability of a series of reversible cross-linking reagents which are diazide tartarate derivatives has been compared with that of dimethyl-3,3'-dithiobispropionimidate (DTBP). The diazide tartarate derivatives tested include tartryl-diazide (TDA), tartryl-di(glycylazide) (TDGA), tartryl-di(beta-alanylazide) (TDAA), tartryl-di-(gamma-aminobutyrylazide) (TDBA), tartryl-di(epsilon-aminocaproylazide) (TDCA). TDA, which has the shortest chain length of the diazide tartarate derivatives tested, proved to be readily permeable through the erythrocyte membrane. When added at equal concentration to unsealed ghosts, TDGA was at least as reactive as DTBP in its ability to cross link the internally displayed proteins 1, 2, 4.1, 4.2, and 6. Treatment of resealed ghosts by DTBP produced oligomeric complexes of these proteins plus apparent homooligomeric complexes of hemoglobin. TDGA at the same concentrations did not cross-link any of these components, indicating its membrane-impermeable nature. As the chain length of the homologous series increased from TDGA to TDCA, the cross-linkers became increasingly permeable through the erythrocyte membrane.

Amino Acids↗
PubMed1978

Molecular mechanism of mitogen action: processing of receptor induced by epidermal growth factor.

An affinity labeling technique used previously for identification of a membrane receptor for epidermal growth factor (EGF) was exploited to investigate the physiological fate of receptor after binding of EGF. Incubation of affinity-labeled cells at 37 degrees resulted in a time-dependent loss of radioactivity from the EGF-receptor covalent complex (M(r) 190,000). Ninety percent of the radioactivity lost from the band of M(r) 190,000 during a 1-hr incubation at 37 degrees appeared in three bands of M(r) 62,000, 47,000, and 37,000. The crosslinked EGF-receptor complex (M(r) 190,000) on intact cells was accessible to the action of trypsin at 4 degrees and cofractionated with the plasmalemmal fraction. The proteolytic processing products of receptor were inaccessible to trypsin and banded with the lysosomal fraction upon subcellular fractionation. The rate of internalization and proteolytic processing of radiolabeled receptor was the same as the rate of reduction of binding activity induced by EGF. A study of the relationship between EGF-induced receptor internalization and processing, and stimulation of DNA synthesis, showed that both these processes were half-maximally stimulated at approximately 0.1 nM EGF, a concentration at which only 10% of the receptor sites are occupied. These data indicate that at concentrations of EGF subsaturating for binding but optimal for biological activity, there is a slow, continuous process of receptor internalization and degradation which could be limiting for EGF-induced mitogenesis.

Cell Line↗
PubMed1978

Nearest-neighbor interactions of the major RNA tumor virus glycoprotein on murine cell surfaces.

Formaldehyde-fixed Staphylococcus aureus and monospecific antiserum to gp70, the major envelope glycoprotein of murine leukemia virus, were used to immunoadsorb gp70 from Nonidet P40 extracts prepared from surface-radioiodinated murine cells. The labeled gp70 molecules in these cells were linked to a protein of approximately 15,000 daltons via native disulfide bonding. Prior treatment of cells with the reversible, bifunctional, crosslinking reagent dimethyl-3,3'-dithiobispropionimidate, followed by immunoadsorption and two-dimensional diagonal electrophoresis, revealed apparent homodimers and homotrimers of the 85,000-dalton complex. Identical treatment of purified type C RNA tumor virus from murine cells also revealed homodimeric and homotrimeric species, demonstrating similar self-associating tendencies of this glycoprotein in both intact virus and the plasma membrane of nonproducing murine cells. One cross-linked product consistently detected on the surfaces of murine cells was not present after crosslinking of a representative strain of murine leukemia virus.

Cells, Cultured↗
PubMed1978

Affinity labeling of a cell surface receptor for epidermal growth factor.

The membrane receptor for epidermal growth factor (EGF) on 3T3 cells has been identified and specifically labeled radiochemically using a photoreactive derivative of EGF. Photoreactive 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 1 other major radioactive band which migrated at approximately 190,000 daltons. 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. The crosslinked complex in intact cells is accessible to the action of a macromolecule like trypsin at 4 degrees, suggesting a cell surface location for this complex. Upon incubation of cells at 37 degrees, radioactivity from previously formed EGF-receptor crosslinked complex is converted by cellular action to 3 forms of mol wt less than or equal to 58,000 daltons. These are not accessible to trypsin action upon intact cells.

Affinity Labels↗
PubMed1977

A confirmation of the phase behavior of Escherichia coli cytoplasmic membrane lipids by X-ray diffraction.

The lipid fatty acid composition of the cytoplasmic membranes of Escherichia coli can be varied by growing an unsaturated fatty acid auxotroph in the presence of different fatty acid supplements. Electron spin resonance (ESR) studies of spin-label partitioning into the cytoplasmic membranes of different lipid fatty acid compositions as a function of temperature have been interpreted as indicating a broad order-to-disorder transition in the membrane lipids, the end points of the transition depending upon the fatty acid composition. We have utilized x-ray diffraction to confirm the ESR studies for three different fatty acid supplements (oleic, elaidic, and bromostearic). We found that the characteristic end-point temperatures detected by ESR were indeed the end-point temperatures of a broad order-to-disorder transition of the cytoplasmic membrane lipids. In addition, Patterson functions calculated from lamellar x-ray diffraction from partially oriented cytoplasmic membranes indicate a decrease in average membrane thickness upon fatty acid chain melting.

Cell Membrane↗