Search PubMed⌕ Search

Biomedical subjects

H S Wiley

Publications and source records attributed to H S Wiley.

At least 73 records · Page 4Linked to original sources
PubMed1984

Epidermal growth factor rapidly induces a redistribution of transferrin receptor pools in human fibroblasts.

Treatment of human fibroblasts with epidermal growth factor (EGF) results in a rapid increase (less than 5 min) in the ability of the cells to bind 125I-labeled transferrin to surface receptors. Scatchard analyses of EGF-treated cells indicate that this increase was due to an increase in the number of transferrin receptors at the cell surface rather than to alterations in ligand-receptor affinity. The EGF-induced increase in transferrin receptors was transient, reaching a peak by 5 min and then declining back to near basal levels by 45 min. Increases in transferrin receptor number were observed when approximately equal to 1% of the EGF receptors were occupied and were maximal at 16% occupancy. EGF treatment accelerated the rate at which previously internalized 125I-labeled transferrin-receptor complexes were returned to the cell surface. The kinetics and magnitude of the loss of intracellular transferrin receptors was sufficient to account for the increase in surface transferrin receptors. We conclude from these studies that one of the earliest effects of EGF treatment is the induced translocation of an intracellular compartment to the cell surface. This intracellular compartment contains transferrin receptors and may be part of the pathway involved in the normal recycling of cell surface proteins.

Cells, Cultured↗
PubMed1983

Photoaffinity labeling of specific alpha-thrombin binding sites on Chinese hamster lung cells.

Binding sites for alpha-thrombin on cultured Chinese hamster lung cells were identified using photoactivatable cross-linking conjugates of diisopropylphosphorofluoridate-inactivated alpha-thrombin. A series of photoaffinity reagents was synthesized that permitted systematic variation of the extent of thrombin modification and of steric factors affecting the ability of the photoaffinity reagent to contact thrombin receptors. The reagents were synthesized with a tritium label to accurately determine the number of photoaffinity molecules linked to each thrombin. Also, they were synthesized with different length spacer arms between the photoreactive cross-linking group (a nitroarylazide) and the end which linked to alpha-thrombin (a succinimide ester). By calculating the percentage of the thrombin surface that would be accessed by modifying it with a fixed molar excess of each reagent, it was possible to select the photoaffinity reagents that would be most effective for cross-linking 125I-labeled diisopropylphosphorofluoridate-inactivated alpha-thrombin to its cellular binding sites. The validity of this selection procedure was confirmed in experiments in which an Mr = 150,000 cellular component was labeled. This component had the properties of a specific binding site for thrombin since labeling was readily competed for by nonlabeled alpha-thrombin. The cross-linking achieved was due to the photoactivatable reagent since no detectable cross-linked complex was formed in the absence of photoactivation or with 125I-labeled diisopropylphosphorofluoridate-inactivated alpha-thrombin that was not conjugated with the photoaffinity reagent.

Affinity Labels↗
PubMed1983

The oocyte as an endocytic cell.

Oocytes of Xenopus laevis grow primarily by sequestering vitellogenin (VTG) selectively from the maternal bloodstream. Morphological observations have demonstrated that an endocytic system is responsible for VTG uptake. Binding studies indicate the presence of 2-28 X 10(10) surface VTG receptors per oocyte. These are continuously internalized into endosomes whether or not they are occupied by VTG, and other macromolecules may become trapped in the process. VTG-containing endosomes give rise to dense transitional yolk bodies; these fuse with yolk platelets only after the cleavage of vitellogenin. In the absence of VTG, endosomes appear to fuse directly with yolk platelets. From these observations it is postulated that receptor occupancy can act as a transmembrane signal which directs the postendocytic compartmentation of proteins. Yolk platelet proteins do not undergo subsequent turnover, whereas adventitiously incorporated protein is gradually lost from the oocyte by a dual mechanism which may involve both lysosomal proteolysis and secretion from the oocyte as a consequence of membrane recycling. Although these observations may not apply to all growing oocytes, the X. laevis oocyte nevertheless appears to be a particularly attractive experimental system for studies of endocytic compartmentation and membrane receptor recycling.

Animals↗
PubMed1982

Epidermal growth factor stimulates fluid phase endocytosis in human fibroblasts through a signal generated at the cell surface.

We have investigated the stimulation of fluid phase endocytosis by epidermal growth factor (EGF) in normal human fibroblasts using 125I-labeled polyvinylpyrrolidone (125I-PVP) as a fluid phase marker. We found that EGF initially induced a threefold increase in the rate of 125I-PVP uptake. This initial burst of fluid uptake terminated within 10 min. Thereafter, the rate of fluid uptake in EGF-treated cells was approximately 40% higher than in control cells. To identify the cellular site of EGF action in stimulating fluid phase endocytosis, we examined the kinetics of the induction of this response as well as the kinetics of cell surface binding and internalization of 125I-EGF. Although there was no detectable lag between binding of EGF to the cell surface and its internalization, the kinetics of the two processes were quite different. Significantly, the kinetics of induction of 125I-PVP uptake matched the kinetics of binding of 125I-EGF to its cell surface receptors, indicating that the signal for the increase in fluid phase endocytosis is generated at the cell surface. To determine if EGF-stimulated fluid phase endocytosis was related to EGF-stimulated endocytosis of its own receptor, we compared the EGF dose dependency and time course of the two processes. Although the stimulated endocytosis of the EGF receptor was not saturable with respect to the concentration of EGF used, the stimulation of fluid phase endocytosis was half maximal at an EGF concentration of 1 ng/ml and saturated at a concentration of 5 ng/ml. Also, the stimulation of fluid phase endocytosis was sevenfold greater initially after adding EGF than after a 30-min continuous incubation with the hormone, whereas the enhanced clearance of the EGF receptor did not change during this time period. We conclude that the EGF-stimulated increase in fluid phase endocytosis is not directly coupled to EGF-stimulated endocytosis of its own receptor but instead to a separate signal generated at the cell surface.

Cells, Cultured↗
PubMed1980

Differential postendocytotic compartmentation in Xenopus oocytes is mediated by a specifically bound ligand.

Xenopus laevis oocytes were used as a model system to study the intracellular fate of proteins incorporated by endocytosis. We found that the intracellular stability and compartmentation of proteins incorporated by receptor-mediated endocytosis differed substantially from that of proteins incorporated by nonspecific endocytosis. After its uptake, the specifically sequestered yolk precursor protein vitellogenin was converted to the yolk proteins lipovitellin and phosvitin which were stable with time (up to 13 days in culture). In contrast, nonspecifically incorporated albumin (125I-labeled or 3H-labeled bovine serum albumin) was rapidly degraded. To determine whether the differential stability of these proteins was related to their entry into different postendocytotic compartments, we examined the intracellular transfer pathways taken by these proteins. The transfer of vitellogenin from coated vesicles to the yolk platelets was found to involve a secondary compartment formed by the fusion of the incoming endosomes. This compartment, termed transitional yolk bodies (TYB), underwent a progressive condensation until it attained its terminal density (1.21 g/cm3) after approximately 1.5 hr. The fusion of the TYB with the yolk platelets then occurred coincidentally with the time at which vitellogenin was proteolytically processed into the yolk proteins within the TYB. When the proteolytic cleavage of vitellogenin was blocked there was no fusion of the two compartments. In contrast, we found that albumin incorporated in the absence of vitellogenin was directly transferred from endosomes to yolk platelets without the formation of, or fusion with a secondary compartment. However, when oocytes were exposed simultaneously to albumin and vitellogenin both proteins followed identical routes of compartmentation (that of vitellogenin) with no evidence of direct transfer of either protein to the yolk platelets. These results suggest that the incorporation of a specifically bound ligand can result in the formation of a unique intracellular compartment. Moreover, since yolk platelets were able to fuse only with vesicles lacking occupied receptors (in the case of albumin alone) or with a compartment in which the specific ligand had been proteolytically cleaved and presumably released from its receptor (in the case of VTG), we suggest that occupied receptors can act as a transmembrane signal which directs the postendocytotic compartmentation of proteins.

Animals↗
PubMed1980

Growth of anuran oocytes in serum-supplemented medium.

Appropriately-sized oocytes from Xenopus laevis can be grown in vitro in vitellogenin-containing serum for up to 2 weeks. The source of serum appears to be unimportant. Rates of oocyte growth are somewhat better than those achieved in the presence of vitellogenin alone. Rana pipiens oocytes grow about twice as fast as X. laevis oocytes under identical conditions. The oocyte culture conditions described appear to be applicable to amphibian oocytes in general.

Animals↗
PubMed1980

Proteins iodinated by the chloramine-T method appear to be degraded at an abnormally rapid rate after endocytosis.

Proteins labeled with either (3)H by reductive methylation or (125)I by the chloramine-T method were incubated with Xenopus laevis oocytes; the incorporation and acid precipitability of the proteins were then studied. The uptake rates of both specifically incorporated (vitellogenin) and nonspecifically incorporated proteins (bovine serum albumin and X. laevis serum proteins lacking albumin) were not influenced by the method of labeling. However, (125)I-labeled proteins were apparently degraded at rates far exceeding their (3)H-labeled counterparts, based on the generation of acid-soluble radioactivity. Thus, after a 3-hr incubation, 3-5 times more (125)I-labeled bovine serum albumin and X. laevis serum proteins lacking albumin were degraded than the corresponding (3)H-labeled proteins (95% compared to 30% and 75% compared to 15%, respectively), whereas after a 24-hr incubation, the degradation of (125)I-labeled vitellogenin was 15 times greater than that of [(3)H]vitellogenin labeled in vivo (60% compared to 4%). Moreover, examination of the relative amounts of (3)H- compared to (125)I-labeled bovine serum albumin deposited into the exogenously derived yolk platelet compartment of the oocyte revealed 7 times more acid-precipitable (3)H-labeled protein, indicating that the observed discrepancies were not due to reincorporation of the (3)H-labeled ligands. Passage of dissolved oocytes previously exposed to (125)I-labeled bovine serum albumin (chloramine-T method) over a column of Bio-Gel P-10 revealed some breakdown of bovine serum albumin to intermediate molecular weight components and the presence of a large amount ( approximately 90%) of labeled low molecular weight compounds, which analysis showed to be 72% free iodine. The evolution of either iodotyrosine or free iodine would nevertheless be perceived as protein degradation by most analytical procedures (e.g., acid precipitation or autoradiography). We conclude, therefore, that apparent degradation rates observed for endocytotically incorporated proteins may vary depending on the method used to label the protein and caution should be exercised when interpreting results obtained with labeled, particularly chloramine-T labeled, proteins.

Animals↗
PubMed1979

The origin of yolk-DNA in Xenopus laevis.

Xenopus laevis serum and plasma was found to contain an average of 25 microgram DNA/ml. Isolated X. laevis oocytes incubated in medium containing 25 microgram DNA/ml labeled with either 125I, 32P or 14C and from three different sources (bovine, E. coli and X. laevis), incorporated the label at an average rate of 0.11 ng.mm-2.hr-1. Sucrose gradient fractionation of oocytes revealed that 40-75% of the acid-precipitable label incorporated was associated with the yolk platelets. Additional incubations of oocytes in unlabeled medium demonstrated that the DNA incorporated into the yolk platelets was undergoing turnover; only 20% of the yolk-associated DNA was still present after a one-week incubation. Our data suggest that yolk-DNA arises by the adventitious uptake of DNA present in the maternal serum by vitellogenic oocytes.

Animals↗