Biomedical subjects

J L Cooper

Publications and source records attributed to J L Cooper.

28 records · Page 2Linked to original sources

PubMed1986

Effects of phenothiazines on binding and processing of epidermal growth factor in 3T3 cells.

Chlorpromazine (CPZ) or the functionally related N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide caused a rapid decrease in binding of 125I-epidermal growth factor (EGF) that was due to a specific decrease in receptor affinity. The decrease in ligand binding was observed when cells were exposed to CPZ at either 4 degrees C or 37 degrees C but a rapid reversal of CPZs effects was observed only during a 37 degrees C incubation. In contrast to the decrease in 125I-EGF binding seen after short (30 min) accumulations at 37 degrees C, the presence of CPZ caused a large increase in the amount of cell-associated radioactivity after longer periods (over 1 h) of accumulation. Although the CPZ-induced effect was similar in extent to that observed after the addition of methylamine, the increased accumulation after CPZ was probably not due to a nonspecific ionic neutralization of the lysosomes. CPZ did not lower EGF binding in cultures chronically treated with a phorbol ester to reduce protein kinase C levels, although the CPZ-induced increases in accumulation were still observed in cells with reduced protein kinase C activity.

Animals↗

PubMed1985

Late G1 amino acid restriction point in human dermal fibroblasts.

Human dermal fibroblasts arrested in G0 by maintenance in medium supplemented with 0.1% serum were not restimulated to divide when fresh medium containing 10% dialyzed serum but lacking group B amino acids (cystine, isoleucine, lysine, phenylalanine and tyrosine) was added. Unlike rodent cells, the addition of fresh serum-supplemented medium lacking only isoleucine did not cause a growth arrest. The amino acid sensitive growth arrest in human fibroblasts was dependent both on presynchronization in G0 as well as a prestarvation for amino acids prior to stimulation with high serum. When cells were restimulated in the absence of amino acids, they arrested predominantly in G1, although a small percentage of cells entered early S phase. When medium containing a complete complement of amino acids was then added, cells initiated DNA synthesis following a minimum lag of 2-3 hr. Growth arrested cells initiated DNA synthesis even when complete unsupplemented medium was added, although the addition of high concentrations of insulin or 10% serum increased the rate of entry.

Amino Acids↗

PubMed1985

Chloroquine allows the secretion of internalized 125I-epidermal growth factor from fibroblasts.

Incubation of cells with labelled hormone in the presence of the lysosomotropic agent chloroquine produces an enhanced intracellular accumulation of hormone and receptor. Using a pulse-chase paradigm in which cell surface receptors were labelled with 125I-EGF at 4 degrees C, it was found that when 100 microM chloroquine was present in the 37 degrees C chase medium intact hormone was accumulated in the medium. Without chloroquine, low molecular weight (mw) degradation products were found in the medium. The processes of receptor-mediated endocytosis and subcellular distribution of 125I-EGF-receptor complexes were unchanged by chloroquine. The source of the intact hormone accumulating in the medium was therefore an intracellular compartment(s). The 125I-EGF released from the cells could rebind to surface receptors and be re-internalized; rebinding was inhibited by unlabelled EGF or Concanavalin A in the incubation medium. The concentration of unlabelled EGF required to inhibit rebinding was more than three orders of magnitude greater than the amount of 125I-EGF whose rebinding was inhibited. Thus, the 125I-EGF released from intracellular sites was rebound preferentially over exogenous EGF. The possible pathways for secretion of intact 125I-EGF and mechanisms of its preferential rebinding are discussed.

Animals↗

PubMed1985

Insulin-like growth factor II binding to cultured human chondrosarcoma cells.

Cultured cells originally derived from a human chondrosarcoma (A1684) were used to investigate somatomedin binding in terms of kinetics and specificity. In this study, the rat somatomedin, multiplication-stimulation activity (MSA) was utilized. While the human chondrosarcoma cells did not exhibit a mitogenic response to MSA, the rate of transport of glucose and amino acids was significantly increased. In competitive binding experiments a specific insulin-insensitive MSA receptor was identified which showed half maximal displacement of tracer at a concentration of 250 ng/ml of MSA using whole cells. This receptor had an affinity constant of 4.8 X 10(7) M-1. Kinetic analysis of MSA binding to membrane preparations and to Triton X-100 solubilized membranes revealed an increase in the binding affinity to 1.28 X 10(8) M-1 and 2.8 X 10(8) M-1, respectively. Of particular significance is the observation that these cells have especially high levels of MSA receptors. Determination of binding capacity revealed that these cells contain approximately 1.9 X 10(6) MSA receptors per cell and therefore are an excellent model system for the characterization and purification of somatomedin receptors. Affinity labeling of the MSA receptor using the chemical crosslinking reagent, disuccinimidyl suberate, confirmed that this receptor was of the type II class of somatomedin receptors and exhibited a molecular weight of 218,000 under nonreducing conditions.

Affinity Labels↗

PubMed1977