Journal: The Journal of Neuroscience

Article Title: Urokinase-Type Plasminogen Activator Promotes Dendritic Spine Recovery and Improves Neurological Outcome Following Ischemic Stroke

doi: 10.1523/JNEUROSCI.5309-13.2014

Figure Lengend Snippet: uPA induces cytoskeletal reorganization in dendritic protrusions. A, Representative micrograph of MAP-2 (green) and phalloidin (white and red) staining in a dendrite of a Wt cerebral cortical neuron incubated 60 min with either 10 nm of uPA (+ uPA) or vehicle (control). Arrows in e denote phalloidin-positive dendritic filopodia. Magnification: 60×. B, Representative micrograph of MAP-2 (green) and profilin (white and red) staining in a Wt cerebral cortical neuron incubated 60 min with either 10 nm of uPA (+ uPA) or vehicle (control). Note that uPA treatment induces the expression of profilin not only in the dendrite but also in the axon. Magnification: 60×. C–G, Representative Western bot analysis and quantification of the mean density of the band of profilin (C) and cofilin (D, E) phosphorylated at serine 3 (p-cofilin) in Wt cerebral cortical neurons incubated 0–60 min with 10 nm uPA (C, D), or 10 nm of uPA's N-terminal fragment (ATF; E), or a combination of uPA and either 5 μm of the Rac inhibitor EHT-1864 (Rac-I; F), or 10 μm of the ROCK inhibitor Y-27632 (ROCK-I; G). C, D, *p < 0.0001 and **p < 0.0001 compared with cells treated 0–5 min with uPA. E, *p < 0.0001 and **p < 0.0001 compared with cells either left untreated or incubated 30 min with uPA or ATF. F, *p < 0.0001 and **p < 0.0001 compared with cells treated with uPA in the presence of the Rac inhibitor EHT-1864. Statistical analysis was performed with one-way ANOVA.

Article Snippet: Experiments were approved by the Institutional Animal Care and Use Committee of Emory University, Atlanta, GA. Recombinant murine uPA and uPA's N-terminal fragment (ATF) were purchased from Molecular Innovations.

Techniques: Staining, Incubation, Expressing, Western Blot

Journal: Journal of medicinal chemistry

Article Title: 6-Substituted Hexamethylene Amiloride (HMA) Derivatives as Potent and Selective Inhibitors of the Human Urokinase Plasminogen Activator for Use in Cancer

doi: 10.1021/acs.jmedchem.8b00838

Figure Lengend Snippet: Inhibition of low molecular weight human uPA by 18 (black) and 27 (red). Data points represent the mean ± SEM (n = 3) from a single representative experiment.

Article Snippet: Experiments with Mouse uPA (active mouse urokinase, HMW, Molecular Innovations Inc., MI, USA) were performed as above with a final enzyme concentration of 20 nM (K m = 580 μ M).

Techniques: Inhibition, Molecular Weight

Journal: Journal of medicinal chemistry

Article Title: 6-Substituted Hexamethylene Amiloride (HMA) Derivatives as Potent and Selective Inhibitors of the Human Urokinase Plasminogen Activator for Use in Cancer

doi: 10.1021/acs.jmedchem.8b00838

Figure Lengend Snippet: Activities of Compounds 4, 5, 18, and 26 against Human and Mouse uPA

Article Snippet: Experiments with Mouse uPA (active mouse urokinase, HMW, Molecular Innovations Inc., MI, USA) were performed as above with a final enzyme concentration of 20 nM (K m = 580 μ M).

Techniques:

Journal: Insect biochemistry and molecular biology

Article Title: Heparan sulfate/heparin glycosaminoglycan binding alters inhibitory profile and enhances anticoagulant function of conserved Amblyomma americanum tick saliva serpin 19

doi: 10.1016/j.ibmb.2016.11.002

Figure Lengend Snippet: List of proteases and their substrates used in substrate hydrolysis assays. Lowest effective protease concentrations (LEPC) and rAAS19 concentrations that inhibit less than 80% of protease activity in specified concentration (IC <80%) are specified.

Article Snippet: The statistical significance of HSGAG binding on rAAS19 inhibitory function was validated using unpaired t-test with Welch's correction in Prism 6 software (GraphPad Software). table ft1 table-wrap mode="anchored" t5 caption a7 Protease (Company) LEPC IC <80% Substrate (Company) Tryptase from Human Lung (Sigma-Aldrich) 4.94 nM NI a N-α-Benzoyl-DL-Arg-pNA (Sigma-Aldrich) Kallikrein from Porcine Pancreas (Sigma-Aldrich) 19.05 nM 1 μM H-D-Pro-Phe-Arg-pNA×2HCl (Chromogenix) Proteinase 3, Human Neutrophil (Athens Research & Technology) 779.31 nM NI a N-Metoxysuccinyl-Ala-Ala-Pro-Val-pNA (Santa Cruz Biotechnology) Elastase from Porcine Pancreas (Sigma-Aldrich) 6.18 nM NI a N-Succinyl-Ala-Ala-Ala-pNA (Sigma-Aldrich) Recombinant Human HMW Urokinase (Molecular Innovations) 9.26 nM NI a CH3SO2-D-CHG-Gly-Arg-pNA×AcOH (Pentapharm) Human tPA, >85% Single Chain (Molecular Innovations) 4.73 nM NI a CH3SO2-D-CHG-Gly-Arg-pNA×AcOH (Pentapharm) Papain (Sigma-Aldrich) 2.14 μM 1 μM N-Benzoyl-Phe-Val-Arg-pNA×HCl (Sigma-Aldrich) Bovine Factor IXa beta (Enzyme Research Laboratories) 31.44 nM 1 μM CH3SO2-D-CHG-Gly-Arg-pNA×AcOH (Pentapharm) Factor Xa Protease (New England Biolabs) 4.65 nM 40 nM Benzoyl-Ile-Glu(γ-OR)-Gly-Arg-pNA×HCl (Chromogenix) Human Factor XIa (Enzyme Research Laboratories) 3.69 nM 200 nM H-D-Pro-Phe-Arg-pNA×2HCl (Chromogenix) Human Factor alpha-XIIa (Enzyme Research Laboratories) 2.03 nM 1 μM H-D-Pro-Phe-Arg-pNA×2HCl (Chromogenix) Plasmin from Human Plasma (Sigma-Aldrich) 11.63 nM 20 nM H-D-Val-Leu-Lys-pNA×2HCl (Chromogenix) Thrombin from Bovine Plasma (Sigma-Aldrich) 27.1 nM 100 nM H-D-Phe-Pip-Arg-pNA×2HCl (Chromogenix) Trypsin from Bovine Pancreas (Sigma-Aldrich) 21.01 nM 4 nM Benzoyl-Ile-Glu(γ-OR)-Gly-Arg-pNA×HCl (Chromogenix) Open in a separate window a No inhibition List of proteases and their substrates used in substrate hydrolysis assays.

Techniques: Activity Assay, Concentration Assay, Recombinant