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D D Cunningham

Publications and source records attributed to D D Cunningham.

At least 73 records · Page 4Linked to original sources
PubMed1986

A simple two-step purification of protease nexin.

This paper describes a simple purification procedure for protease nexin, a serine proteinase inhibitor secreted by cultured human fibroblasts that regulates proteinase activity at and near the cell surface. The first step in the procedure takes advantage of the high-affinity binding of protease nexin to dextran sulphate-Sepharose. This step eliminates the need for prior concentration of the serum-free fibroblast-conditioned medium, since protease nexin binds to the resin in the presence of physiological saline. The use of dextran sulphate also provides an affinity resin with considerably less variability than the heparin-based resins previously used. Final purification to homogeneity involves a combination of DEAE-Sepharose in-line with dextran sulphate-Sepharose to simultaneously purify and concentrate the protein. Purified protease nexin is shown by Ouchterlony analysis and peptide mapping to be immunologically and structurally distinct from antithrombin III and heparin cofactor II, two plasma proteinase inhibitors with similar properties.

Amyloid beta-Protein Precursor↗
PubMed1986

Interactions of serine proteases with cultured fibroblasts.

This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN-1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes.

Amyloid beta-Protein Precursor↗
PubMed1986

Human fibroblasts accelerate the inhibition of thrombin by protease nexin.

Protease nexin (PN) is a protein protease inhibitor secreted by human fibroblasts in culture that complexes and inhibits certain regulatory serine proteases. The PN-protease complexes then bind to these cells and are rapidly internalized and degraded. This report shows that the fibroblast surface accelerates the formation of PN-thrombin complexes. In contrast, it did not accelerate the formation of complexes between thrombin and antithrombin III, a closely related protease inhibitor found in plasma. These results support a role for PN in the regulation of certain proteases in the extravascular compartment at and near the surface of tissue cells. The activity that accelerated PN-thrombin complex formation was membrane-associated, since fixed cells, purified membranes, and extracellular matrix preparations all contained this activity. The ability of cells to accelerate the reaction between PN and thrombin was inhibited by protamine, suggesting that the activity was similar to that of heparin. Heparitinase digestion of plasma membranes prior to assay reduced the activity by about 80%, suggesting that heparan sulfate may account for most of the accelerative activity.

Amyloid beta-Protein Precursor↗
PubMed1985

Intracellular processing of epidermal growth factor and its effect on ligand-receptor interactions.

When normal human fibroblasts are brought to a steady state with 125I-labeled epidermal growth factor (125I-EGF), greater than 90% of the radioactivity is intracellular. We investigated this material to determine whether the 125I-EGF is intact or degraded. Our results show that 125I-EGF is rapidly processed after internalization and can be resolved into four peaks by native gel electrophoresis. These different forms were isolated and tested for their ability to bind to cell-surface EGF receptors. The first processed form was fully capable of binding to EGF receptors, but the second processed form could not. The third form was a collection of small degradation products. We calculated that at steady state about 60% of internalized "125I-EGF" was in a form still able to bind to EGF receptors. We then investigated the ability of different reported inhibitors of EGF "degradation" to block the processing of EGF. Although inhibitors of cathepsin B (leupeptin, antipain, N alpha-p-tosyl-L-lysine chloromethyl ketone, and chymostatin) were able to inhibit the release of monoiodotyrosine from treated cells in a time- and concentration-dependent manner, they had little effect on the processing step that apparently inactivates 125I-EGF. In contrast, agents that raised intravesicular pH, such as methylamine and monensin, inhibited the initial steps in EGF processing as well as the later steps. Low temperatures inhibited the transfer of 125I-EGF to the lysosomes and inhibited the conversion of EGF to a nonbindable form, but had little effect on the initial processing. We conclude that the intracellular processing of EGF is a multistep process that is initiated prior to lysosomal fusion, involves cathepsin B activity, and requires an acidic pH. In addition, many of the protease inhibitors that have been utilized to investigate the role of EGF degradation in mitogenesis do not block the conversion of EGF to a form that is apparently unable to interact with its receptor.

Animals↗
PubMed1985

Effects of EGF and thrombin on inositol-containing phospholipids of cultured fibroblasts: stimulation of phosphatidylinositol synthesis by thrombin but not EGF.

The effects of growth factors on inositol-containing phospholipids were investigated to test the hypothesis that alterations in their metabolism are involved in mitogenic stimulation. Thrombin and EGF stimulated comparable increases in the synthesis (30-50%) and degradation (20-40%) of phosphatidylinositol 4-monophosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) in a cell line which is mitogenically responsive to both growth factors. The increases in synthesis were time and dose dependent in a manner which was consistent with their involvement in mitogenesis; the increases were observed only under conditions where a mitogenic response occurred. While it has been suggested that an increased synthesis of phosphatidylinositol (PI) is coupled to the stimulation of DPI and TPI synthesis, we found that thrombin stimulated an early synthesis PI but EGF did not. To further evaluate the involvement of PI in thrombin-stimulated cell division we determined the time and dose dependence of the stimulated PI synthesis and found that it also occurred in a manner which was consistent with its involvement in thrombin-stimulated cell division. Furthermore, the stimulated PI synthesis was not observed with nonmitogenic proteases or in cell lines which were not responsive to thrombin. These results demonstrate that the metabolism of DPI and TPI appears closely related to the mitogenic response generated by EGF and thrombin. However, an early stimulation of PI synthesis is not coupled to this metabolism and is not necessary for mitogenic stimulation by EGF. Thus, a stimulation of PI synthesis is not a valid measure of alterations in inositol-containing phospholipids and what has been termed the "PI response."

Animals↗
PubMed1984

Epidermal growth factor immunoreactive material in the central nervous system: location and development.

Epidermal growth factor (EGF) is a potent mitogen with hormonal activity in the gastrointestinal tract. Material cross-reacting with EGF was detected in the central nervous system of the developing and adult albino rat by the indirect immunofluorescence technique. High concentrations of EGF-cross-reacting material were identified in forebrain and midbrain structures of pallidal areas of the brain. These include the globus pallidus, ventral pallidum, entopeduncular nucleus, substantia nigra pars reticulata, and the islands of Calleja . Thus, EGF may represent another gut-brain peptide with potential neurotransmitter-neuromodulator functions in pallidal structures of the extrapyramidal motor systems of the brain.

Animals↗
PubMed1984

Relationship between epidermal growth factor receptor occupancy and mitogenic response. Quantitative analysis using a steady state model system.

Because mitogenic stimulation by epidermal growth factor (EGF) requires at least an 8--12-h exposure to commit the majority of the cells to divide, a number of complicating processes must be evaluated in an analysis of the relationship between receptor occupancy and the biological response. During this period, the number of cellular EGF receptors changes and the cells continuously bind, internalize, and degrade the hormone. To deal with these changes, we utilized our previously described steady state model that permits an analysis of EGF interactions with cells under steady state conditions. For these studies we derived and measured a number of steady state rate constants to describe the interaction of 125I-EGF with cultured human and mouse fibroblast-like cells. The validity of these constants was established by demonstrating the close agreement between experimentally determined results and predicted results from computer simulations using these constants. These approaches in conjunction with experiments on the mitogenic stimulation of the cells with EGF led to the conclusion that there is a linear relationship between mitogenic stimulation and EGF receptor occupancy at steady state, with no apparent "spare" EGF receptors.

Animals↗
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

Protease nexins: cell-secreted proteins that mediate the binding, internalization, and degradation of regulatory serine proteases.

The protease nexins (PN-I, Mr approximately 38,000; PN-II, Mr approximately 95,000; and PN-III, Mr approximately 31,000) are recently described cell-secreted proteins that selectively link to regulatory serine proteases in the extracellular environment and mediate their cellular binding, internalization, and degradation. In the present studies we compared the protease nexins with respect to protease specificity, heparin sensitivity, and general mode of action. By competitive binding assays using [125I]-thrombin, [125I]-nerve growth factor-gamma (125I-NGF-gamma), and [125I]-epidermal growth factor binding protein (125I-EGF-binding protein), we characterized the nexins in terms of protease specificity and determined that PN-I links to and mediates the cellular binding of thrombin or urokinase, whereas PN-II and PN-III preferentially link to and mediate the cellular binding of the EGF binding protein and NGF-gamma, respectively. In addition, whereas the ability of PN-I to link to thrombin is strongly modulated by heparin, PN-II and PN-III are essentially unaffected by heparin. The linkage of each of the nexins to their respective proteases requires the catalytic site serine of the protease, judged by the inability of diisopropylphospho (DIP) derivatives of the proteases tested to link to their respective nexins. Subsequent to linkage, the nexin:protease complexes are bound to cells, rapidly internalized, and ultimately degraded via a monensin-sensitive apparently lysosomal pathway, although each nexin:protease complex is degraded at its own characteristic rate. Importantly, the protease nexins provide the major pathway through which human fibroblasts interact with each of the serine proteases studied. Taken together, these data suggest that the nexins are a unique class of cell-secreted proteins that enable cells to monitor and selectively regulate specific serine proteases in their environment.

Amyloid beta-Protein Precursor↗
PubMed1983

A reevaluation of the response of human umbilical vein endothelial cells to certain growth factors.

Human umbilical vein endothelial cells (HUV-EC) were isolated and maintained in pure culture on a fibronectin matrix with hypothalamic derived endothelial cell growth factor included in the culture medium. HUV-EC maintained under these conditions displayed only slight alterations in morphological appearance and continued to produce Factor VIII antigen. The cells showed an unaltered growth response to serum and added growth factors up to passage 16 (greater than 30 population doublings). We found that epidermal growth factor (EGF) was potently mitogenic for HUV-EC, but only in the presence of endothelial cell growth factor. Also in contrast to a previous report, we were unable to demonstrate a potentiation of this response by human alpha-thrombin. Because of these discrepancies, we performed studies to determine if they might be explained by a difference in the interaction of our HUV-EC with EGF. In studies utilizing 125I-EGF as tracer probe, we determined that our HUV-EC have an EGF receptor number of 13,000 sites/cell with an apparent Kd-4.0 X 10(-9) M. In addition, receptor-bound 125I-EGF was rapidly internalized and degraded presumably by a lysosomally mediated pathway since degradation was complete to the amino acid level. These results are in agreement with those previously published and thus do not provide a basis on which to resolve discrepancies regarding the growth response of HUV-EC to various growth factors.

Cell Division↗
PubMed1983

The relationship of disc position to zone-of-inhibition size in the disc-agar diffusion test.

One hundred sixteen clinical isolates of Enterobacter sp., Escherichia coli, Klebsiella sp., Proteus mirabilis, Staphylococcus aureus and S. epidermidis were tested against cefoperazone, cefoxitin, cefamandole and piperacillin by disc-agar diffusion. Zones-of-inhibition of discs dropped near the center of the plate were compared with those dropped near the periphery. In 28% of the comparisons the inner and outer zones differed by 2 mm or more. Of those, the inner zone was larger 2.5 times more often than the outer zone. Results suggest that the chances of obtaining a susceptible result are greater when discs are placed near the center as opposed to the periphery of the plate.

Agar↗
PubMed1982

Cells regulate their mitogenic response to thrombin through release of protease nexin.

We previously reported that human and mouse fibroblast-like cells release into their growth medium a protein that we termed protease nexin. Protease nexin forms a covalent acyl linkage with thrombin and certain other serine proteases via the protease active site and mediates their binding, internalization and degradation by cells. Binding of thrombin-protease nexin to cells is mediated by the protease nexin portion of the complex to a high-affinity cellular binding site. As thrombin is a potent mitogen for a variety of fibroblast-like cells in culture, we examined whether protease nexin itself regulates thrombin-stimulated cell division. Recently, we showed that heparin virtually blocked the binding of thrombin-protease nexin complexes to both mouse and human cells without affecting the ability of these cells to respond to thrombin. Thus, protease nexin does not appear to be a positive modulator in thrombin-induced cell division. Here, we show that protease nexin negatively regulates the mitogenic response of cells in culture to thrombin.

Amyloid beta-Protein Precursor↗