Journal: RNA

Article Title: Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding

doi: 10.1261/rna.2338210

Figure Lengend Snippet: The binding of upanap-12 to uPA requires the GFD. The relative association of upanap-12 to different variants of uPA is indicated: The inactive zymogen form of uPA (pro-uPA) active uPA (uPA), uPA without the growth factor domain (ΔGFD), and uPA without the entire ATF (also called low molecular weight uPA; LMW-uPA). The domain structure of the different variants is shown at each bar, while the inset illustrates pro-uPA with domain designations: (GFD) growth factor domain, (KD) kringle domain, and (SPD) serine protease domain. uPA variants were captured on an SPR sensor surface containing immobilized polyclonal anti-uPA antibody to the following levels: pro-uPA (215–280 RU), uPA (210–260 RU), ΔGFD-uPA (210–230 RU), and LMW-uPA (140–160 RU). The binding level of 50 nM upanap-12 to each captured variant was subsequently recorded. Using the molecular weight of the different variants, the binding level of aptamers per mole of captured variant was calculated and presented relative to the pro-uPA result. Open bars represent mean values and standard deviations derived from three independent experiments.

Article Snippet: Human two-chain uPA was from Wakamoto Pharmaceutical Company, while human pro-uPA and mouse uPA were from Molecular Innovations.

Techniques: Binding Assay, Molecular Weight, Variant Assay, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Anti-angiogenic Activity of the Recombinant Kringle Domain of Urokinase and Its Specific Entry into Endothelial Cells

doi: 10.1074/jbc.m212358200

Figure Lengend Snippet: FIG. 2. Inhibition of endothelial cell proliferation by UK1. A, inhibition of BCE cell proliferation induced by bFGF. Anti-proliferative activity was assayed with BCE cells in the presence of 1 ng/ml bFGF for 72 h as described under “Experimental Procedures.” The effects of UK1 (closed circles) were compared with those of angiostatin (open circles) and sc-uPA (closed triangles). Values, given as percentages of inhibi- tion, represent the mean of three determinations (S.E.). B, inhibition of HUVE cell proliferation by UK1. The cells were cultured with 3 ng/ml bFGF, 10 ng/ml VEGF, or 10 ng/ml EGF with or without various amounts of UK1 for 18 h, followed by incubation with [3H]thymidine as described under “Experimental Procedures.” C represents a condition of no growth factor and no inhibitor. Each value represents the mean S.E. C, effects of anti-UK1 and anti-uPAR antibodies on UK1 inhibitory activity. The cells were cultured in the absence or presence of 3 ng/ml bFGF with or without 320 nM UK1 preincubated with control antibody (100 g/ml) or anti-UK1 antibody (100 g/ml). In the case of anti-uPAR antibody, it was added directly to the cells 45 min before addition of UK1. A [3H]thymidine incorporation assay was followed. Results are expressed as the percentages of DNA synthesis in the absence of inhib- itor UK1 (as 100%).

Article Snippet: Recombinant human uPA receptor (uPAR, 870-UK/CF), anti-human uPAR polyclonal antibody (AF807), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) were purchased from R&D system (Minneapolis, MN).

Techniques: Inhibition, Activity Assay, Cell Culture, Incubation, Control, Thymidine Incorporation Assay, DNA Synthesis

Journal: Journal of Biological Chemistry

Article Title: Anti-angiogenic Activity of the Recombinant Kringle Domain of Urokinase and Its Specific Entry into Endothelial Cells

doi: 10.1074/jbc.m212358200

Figure Lengend Snippet: FIG. 6. Real-time interaction analysis of UK1 binding to immo- bilized recombinant uPA receptor. Ligand binding curves were obtained by automated measurements of surface plasmon resonance (BIAcore Technology). Recombinant uPAR was covalently coupled to a carboxymethyl-type sensor chip as described under “Experimental Pro- cedures.” Immobilization of uPAR was performed in flowcell 2, while flowcell 1 was immobilized with bovine serum albumin. Samples of uPA (A and C) and UK1 (B and C) were subjected to real-time interaction analysis with the coupled sensor chips, using a BIAcore 2000 instru- ment. For the competition analysis, the concentration of tc-uPA was fixed at 150 nM, and the concentration of UK1 was increased from 150 to 600 nM (C). A different level of immobilization was applied for the kinetics (Rmax 780, A and B) and for the competition analysis (C).

Article Snippet: Recombinant human uPA receptor (uPAR, 870-UK/CF), anti-human uPAR polyclonal antibody (AF807), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) were purchased from R&D system (Minneapolis, MN).

Techniques: Binding Assay, Recombinant, Ligand Binding Assay, SPR Assay, Concentration Assay