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V Ellis

Publications and source records attributed to V Ellis.

At least 19 recordsLinked to original sources
PubMed1995

Chemical modification of the urokinase-type plasminogen activator and its receptor using tetranitromethane. Evidence for the involvement of specific tyrosine residues in both molecules during receptor-ligand interaction.

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid anchored receptor (uPAR) is essential for the confinement of plasminogen activation to cell surfaces where it is thought to play an important role in cancer cell invasion and metastasis. The receptor binding site of uPA is retained within its isolated growth factor-like module (GFD; residues 4-43). The NH2-terminal domain of uPAR has a primary role in uPA binding, although maintenance of its multidomain structure has been shown to be necessary for the high affinity of this interaction [Ploug, M., Ellis, V., & Danø, K. (1994) Biochemistry 33, 8991-8997]. To identify residues engaged in the uPAR-uPA interaction, we have performed a "protein-protein footprinting" study on preformed uPAR-GFD complexes by chemical modification with tetranitromethane. All six tyrosine residues in uPAR and the single tyrosine residue in GFD (Tyr24) were susceptible to nitration in the native uncomplexed proteins, whereas in the receptor-ligand complexes both Tyr57 of uPAR and Tyr24 of GFD were protected from modification. Modification of uPAR alone led to a parallel reduction in the potential to bind pro-uPA and 8-anilino-1-naphthalenesulfonate, an extrinsic fluorophore reporting on the accessibility of a hydrophobic site involved in uPA binding. These data clearly demonstrate that Tyr57 in the NH2-terminal domain of uPAR and Tyr24 in uPA are intimately engaged in the receptor-ligand interaction, whereas Tyr87 positioned in the linker region between the first two domains of uPAR does not appear to be shielded by the resulting intermolecular interface.

Amino Acid Sequence↗
PubMed1995

Transcriptional and post-transcriptional regulation of the receptor for urokinase-type plasminogen activator by cytokines and tumour promoters in the human lung carcinoma cell line A549.

The receptor for urokinase-type plasminogen activator (uPAR) is an integral membrane protein that specifically binds urokinase-type plasminogen activator (uPA) and plays a crucial role in cell surface plasmin generation. We have previously found that transforming growth factor-beta, type 1 (TGF-beta 1), increases uPAR gene transcription in the human lung carcinoma cell line A549 and now report that also epidermal growth factor (EGF) and the tumour promoter phorbol 12-myristate 13-acetate (PMA) cause increased uPAR transcription and that PMA and TGF-beta 1 in addition increase the stability of uPAR mRNA, while EGF has no effect on this parameter. All three compounds also increase the uPAR protein level, as measured by cell-binding experiments with radiolabelled ligand. The increase in uPAR protein level was however considerably lower with all three compounds than the increase in mRNA level, suggesting that they also exert a translational or post-translational control. Accompanying the increase in the number of uPAR molecules there was a proportional decrease in their ligand-binding affinity, the mechanism of which is unknown. Platelet-derived growth factor, basic fibroblast growth factor and cyclic AMP analogues did not induce any change in the uPAR mRNA level in A549 cells. Previous studies have shown that expression of uPA and its type-1 inhibitor is regulated by a variety of cytokines in a cell-specific manner. The present study indicates that cytokines in addition influence cell surface plasminogen activation by regulating uPAR expression.

Carcinogens↗
PubMed1995

The receptor for urokinase-type plasminogen activator is not essential for mouse development or fertility.

The urokinase-type plasminogen activator receptor (uPAR) gene was disrupted in mice in order to explore the role of cell surface-associated plasminogen activation in development and hemostasis. Homozygous, uPAR-/- mice were born and survived to adulthood with no overt phenotypic abnormalities. There was no indication of loss of fetal animals based on the Mendelian pattern of transmission of the mutant uPAR gene. uPAR-/- mice carried no detectable uPAR in lung, spleen, and other tissues when measured both immunologically by Western blot analysis and functionally by ligand cross-linking analyses. In addition, activated peritoneal macrophages collected from uPAR-/- mice failed to promote plasminogen activation in vitro. The loss of the receptor also resulted in a redistribution of uPA in some tissues but had no impact on pro-uPA activation in the urogenital tract. Thus, in the absence of other challenging factors such as infection, injury, or other functional deficits, uPAR deficiency does not compromise fertility, development, or hemostasis. These mice provide a means to test the proposed function of uPA/uPAR in wound repair, atherogenesis, and tumor cell invasion in vivo.

Animals↗
PubMed1992

Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain.

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored three-domain membrane protein playing a central role in pericellular plasminogen activation. We have found that urokinase (uPA) can cleave its receptor between domains 1 and 2 generating a cell-associated uPAR variant without ligand-binding properties. In extracts of U937 cells there are two uPAR variants which after complete deglycosylation have apparent molecular masses of 35,000 and 27,000. Analysis with monoclonal antibodies showed that these variants represented the intact uPAR and a two-domain form, uPAR(2+3), lacking ligand-binding domain 1. Trypsin treatment showed that both variants are present on the outside of the cells. Addition to the culture medium of an anticatalytic monoclonal antibody to uPA inhibited the formation of the uPAR(2+3), indicating that uPA is involved in its generation. Purified uPAR can be cleaved directly by uPA as well as by plasmin. The uPA-catalyzed cleavage does not require binding of the protease to the receptor through its epidermal growth factor-like receptor-binding domain, since low molecular weight uPA that lacks this domain also cleaves uPAR. This unusual reaction in which a specific binding protein is proteolytically inactivated by its own ligand may represent a regulatory step in the plasminogen activation cascade.

Antibodies, Monoclonal↗
PubMed1992

The receptor for urokinase-type plasminogen activator is deficient on peripheral blood leukocytes in patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect in bone marrow-derived cells and is clinically associated with intravascular hemolysis, hemoglobinuria, and an increased frequency of venous thrombosis. The common denominator of PNH-affected blood cells appears to be a defect in the membrane attachment of proteins normally anchored by glycosyl-phosphatidylinositol (GPI). We report here that the cellular receptor for urokinase-type plasminogen activator (u-PAR) is deficient on affected peripheral blood monocytes and granulocytes from four individuals with PNH as evidenced by chemical cross-linking analysis as well as by immunofluorescence flow cytometry using a monoclonal anti-u-PAR antibody. In contrast, on normal blood monocytes and granulocytes we find significant amounts of u-PAR, which is attached to the plasma membrane by a GPI-anchor as defined by its sensitivity towards a specific phospholipase treatment. By two-color flow cytometry it was shown that deficiency of u-PAR expression paralleled that of another GPI-anchored protein. As u-PAR is involved in the initiation of pericellular proteolysis, the reduced expression of u-PAR on PNH-affected leukocytes led to an overall reduction in the capacity for plasminogen activation by cell-surface-bound urokinase. Whereas the abnormal susceptibility of PNH-affected erythrocytes to lysis by autologous complement has been related to the low expression of three GPI-anchored complement regulatory proteins on the cell surface, we now propose that lack of u-PAR expression on the surface of peripheral blood leukocytes may be causally related to the high incidence of venous thrombosis observed in PNH patients.

Animals↗
PubMed1991

Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor.

We have raised four monoclonal antibodies recognizing different epitopes within the human cell-surface receptor for urokinase-type plasminogen activator (u-PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro-u-PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP-inactivated [125I]-u-PA to U937 cells, is directed against the u-PA binding NH2-terminal domain of u-PAR, a well-defined fragment formed by limited chymotrypsin digestion of purified u-PAR, demonstrating the functional independence of the u-PA binding domain as well as the critical role of u-PAR in the assembly of the cell-surface plasminogen activation system.

Antibodies, Monoclonal↗
PubMed1991

Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor.

The specific cellular receptor for urokinase-type plasminogen activator (uPA) is found on a variety of cell types and has been postulated to play a central role in the mediation of pericellular proteolytic activity. We have studied the kinetics of plasminogen (Plg) activation catalyzed by uPA specifically bound to its receptor on the human monocytoid cell-line U937 and demonstrate this process to have properties differing widely from those observed for uPA in solution. The solution-phase reaction was characterized by a Km of 25 microM and for the cell-associated reaction this fell 40-fold to 0.67 microM, below the physiological Plg concentration of 2 microM. A concomitant 6-fold reduction in kcat resulted in an increase in the overall catalytic efficiency, kcat/Km, of 5.7-fold. This high affinity Plg activation was abolished in the presence of a Plg-binding antagonist. In contrast to intact cells, purified uPA receptor (isolated from phorbol 12-myristate 13-acetate-stimulated U937 cells) was observed to partially inhibit uPA-catalyzed Plg activation, although activity against low molecular weight substrates was retained. Therefore, the cellular binding of Plg appears to be of critical importance for the efficient activation of Plg by receptor-bound uPA. Plasmin generated in the cell-surface Plg activation system described here was also observed to be protected from its principal physiological inhibitor alpha-2-antiplasmin. Together, these data demonstrate that the cell surface constitutes the preferential site for Plg activation when uPA is bound to its specific cellular receptor, which therefore has the necessary characteristics to play an efficient role in the generation of pericellular proteolytic activity.

Antifibrinolytic Agents↗
PubMed1991

Influence of tryptophan modification upon digestion of antithrombin III by elastase.

Chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNBSB) generates products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl [HNB] incorporated/mole of antithrombin III) but with high or low affinity for heparin-Sepharose. Upon digestion with pancreatic or neutrophil elastase the low affinity forms generate a product of molecular weight form (55 kDa) not seen in digests of native antithrombin III or modified forms with high affinity for heparin. When measured as loss of activity the observed rate of digestion of the latter in the absence of heparin was more rapid than that of native antithrombin III. The differences in digestion are considered to be related to conformation at differences between the various forms.

Antithrombin III↗
PubMed1991

Urokinase-receptor biosynthesis, mRNA level and gene transcription are increased by transforming growth factor beta 1 in human A549 lung carcinoma cells.

We have compared the cell-specific expression and regulation of the receptor for urokinase-type plasminogen activator (u-PAR) by transforming growth factor beta type 1 (TGF-beta 1) in 10 human cell lines derived from both normal and neoplastic tissues. The basal expression of u-PAR mRNA as well as its response to TGF-beta 1 varied strongly between different cell lines; however, five out of the 10 cell lines responded to TGF-beta 1 by an increase in the u-PAR mRNA level. Among these, A549 cells were selected for a detailed elucidation of the molecular mechanism involved in TGF-beta 1 regulation of u-PAR mRNA expression. TGF-beta 1 caused an early increase in u-PAR mRNA level, with a maximal 15-fold enhancement after 24 h of treatment. This was paralleled by an increase in u-PAR protein as detected by crosslinking studies with radiolabeled ligand, and also resulted in an increase in cell surface plasmin generation. The protein synthesis inhibitor cycloheximide also increased the level of u-PAR mRNA in a time-dependent fashion and when both cycloheximide and TGF-beta 1 were used, an additive effect was seen. Nuclear run-on experiments demonstrated only a moderate (3-fold) increase in the u-PAR gene transcription rate after exposure of the cells to TGF-beta 1 for 3 h compared with a 12-fold increase in the mRNA level. TGF-beta 1 also caused an increase of both u-PA and PAI-1 antigens, while there was no detectable effect on t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗
PubMed1990

Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors.

Urokinase-type plasminogen activator (uPA) binds to a specific receptor on various cell types, the bound molecule retaining its enzymatic activity against plasminogen. We have now investigated whether receptor-bound uPA also retains the ability to react with and be inhibited by plasminogen activator inhibitors (PAI-1 and PAI-2). uPA bound to its receptor on human U937 monocyte-like cells was inhibited by PAI-1 (in its active form in the presence of vitronectin fragments) with an association rate constant of 4.5 x 10(6) M-1 s-1, which was 40% lower than that obtained for uPA in solution (7.9 x 10(6) M-1 s-1). The inhibition of uPA by PAI-2 was decreased to a similar extent by receptor binding, falling from 5.3 x 10(5) to 3.3 x 10(5) M-1 s-1. Stimulation of U937 cells with phorbol 12-myristate 13-acetate was accompanied by a further reduction in receptor-bound uPA inhibition by PAI-1 and PAI-2 to 1.7 x 10(6) and 1.1 x 10(5) M-1 s-1, respectively. These constants although lower than those for uPA in solution still represent rather rapid inhibition of the enzyme, and demonstrate that uPA bound to its specific cellular receptor remains available for efficient inhibition by PAI's, which may therefore play a major role in controlling cell-surface plasminogen activation and extracellular proteolytic activity.

Antibodies↗
PubMed1990

Influence of chemical modification of tryptophan residues on the properties of human antithrombin III.

According to the reaction conditions selected, chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5 nitrobenzyl) sulfonium bromide (HNBSB) generated products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl (HNB) incorporated/mole of antithrombin III) but with high or low affinity for heparin. These products were subjected to digestion by cyanogen bromide and shown to be modified equivalently in fragment II containing Trp 189 and Trp 225 and fragment III containing Trp 49. The molar level of incorporation of HNB into these fragments was similar in the high and low affinity forms. Both high and low affinity forms showed loss of heparin cofactor activity. A recovery of heparin cofactor activity towards coagulation factor Xa was observed upon prolonged storage of low affinity forms at -70 degrees C. It is considered that the loss of high affinity for heparin upon modification of antithrombin III arises from change or stabilization of conformation associated with tryptophan modification and is not a singular property of modification of Trp 49.

Antithrombin III↗
PubMed1990

The effect of Orem's self-care model on nursing care in a nursing home setting.

The need of the functionally disabled for nursing care is a primary rationale for institutionalization in long-term care settings. However, maintenance of the self-care abilities that nursing home residents retain is an important component of their quality of life. Nurses are in an important position to encourage and sustain residents' abilities to participate in their care. Nevertheless, without specifying the role of the resident in care, nursing interventions may inadvertently reinforce dependency and apathy at the expense of autonomy, control and well-being. Data from nursing home care records and interviews suggest that nursing staff using Orem's Self-Care Deficit Nursing Theory differ in their nursing assessments and goals of care from staff on a control unit. Further study is recommended of the influence of the model on staff perspectives of care and on interventions which support nursing home residents' self-care ability and well-being.

Aged↗
PubMed1990

The role of adenosine in HgCl2-induced acute renal failure in rats.

It has been proposed that adenosine mediates the renal hemodynamic changes in acute renal failure (ARF) and that these changes are pathogenic in reducing glomerular filtration rate. Consistently, adenosine-receptor antagonists such as theophylline have been shown to have protective effects in several experimental models of ARF. The present experiments were designed to explore the potential role of adenosine in HgCl2-induced ARF in rats. In isolated perfused rat kidneys, HgCl2 increased adenosine production and induced a concentration-dependent vasoconstriction. However, the vasoconstriction was unrelated to adenosine production and was not antagonized by theophylline. During the initiation phase of HgCl2-induced ARF in intact rats (first 4 h after injection), theophylline failed to reverse the reduction in inulin clearance, and this failure could not be attributed to a loss of vascular responsiveness to adenosine, since N6-cyclohexyladenosine, a receptor agonist, produced a further reduction in inulin clearance. Furthermore, theophylline actually had deleterious effects during the maintenance phase of HgCl2-induced ARF in intact unanesthetized rats, as evidenced by higher mean serum creatinine values in theophylline-injected rather than in saline-injected rats, on both the second and third days after HgCl2 injection. Therefore HgCl2 acutely increases renal adenosine production, but increased adenosine does not mediate acute HgCl2-induced renal vasoconstriction, and adenosine-receptor antagonism does not have protective effects during the initiation or the maintenance phases of HgCl2-induced ARF in rats. These results provide no support for the hypothesis that increased adenosine mediates the hemodynamic changes in HgCl2-induced ARF.

Acute Kidney Injury↗
PubMed1989

Effect of a heparan sulphate with high affinity for antithrombin III upon inactivation of thrombin and coagulation factor Xa.

The kinetics of inhibition of human alpha-thrombin and coagulation Factor Xa by antithrombin III were examined under pseudo-first-order reaction conditions as a function of the concentration of heparan sulphate with high affinity for antithrombin III. The maximum observed second-order rate constant was, for the antithrombin III-thrombin reaction, 1.2 x 10(9) M-1.min-1 compared with 2.4 x 10(9) M-1.min-1 in the presence of high-affinity heparin. However, the maximum rate was catalysed by much higher concentrations of heparan sulphate (1.3 microM) than of heparin (0.025 microM). Differences were also observed in the maximal acceleration of the antithrombin III-Factor Xa interaction: 1.2 x 10(9) M-1.min-1 at 0.2 microM-heparin sulphate compared with 2.2 x 10(9) M-1.min-1 at 0.04 microM-heparin. The differences in properties of heparan sulphate and heparin were analysed by using the random bi-reactant model of heparin action [Griffith (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5460-5464]. It was observed that the apparent binding affinity for thrombin was higher for heparan sulphate (180 nM) than for heparin (14 nM). The rate constant for transformation of the antithrombin III-Factor Xa complex into irreversible product differed between heparan sulphate (96 min-1) and heparin (429 min-1). These properties of the high-affinity heparan sulphate may be of importance in consideration of a putative role in the control of intravascular haemostasis.

Antithrombin III↗