| In vitro |
YM-90709 potently inhibits the binding of 100 pM [125I]-IL-5 to IL-5R on human peripheral eosinophils and eosinophilic HL-60 clone 15 cells with IC50 values of 1.0±0.40 and 0.57±0.21 μM, respectively. YM-90709 inhibits the 4 pM IL-5-induced effect in a concentration-dependent manner with an IC50 value of 0.45±0.024 μM. YM-90709 also inhibits the higher concentrations (12 and 40 pM) of IL-5-induced effects with IC50 values of 0.89±029 and 1.0±0.22 μM, respectively. [1] YM-90709 is a novel interleukin-5 receptor antagonist, YM-90709 inhibits antigen-induced eosinophil recruitment into the airway, the same as anti-IL-5 mAb does. YM-90709 inhibits the binding of IL-5 to IL-5R on human eosinophils, but did not inhibit the binding of GM-CSF to GM-CSFR. In addition, YM-90709 inhibits IL-5-induced, but not GM-CSF-induced, eosinophil survival as well as the tyrosine phosphorylation of Janus kinase 2. [2]
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| キナーゼ試験 |
Nuclease assay: Reactions with oligonucleotide nonhairpin substrates contains 25 mM MOPS (pH 7.0), 60 mM KCl, 0.2% Tween 20, 2 mM DTT, 1 mM or 5 MnCl2 (or 5 mM MgCl2, or 5 mM CaCl2), 0.1 pmol of DNA substrate, and 0.3 pmol of Mre11 (or an equivalent amount of Mre11 complexed with Rad50) in a volume of 10 μl, and are incubated at 37°C for 30 min. SDS, EDTA, and proteinase K are then added to final concentrations of 0.2%, 5 mM, and 0.1 mg/ml, respectively, and incubated for another 15 min. 4 μl of each reaction is mixed with 4 μl of formamide loading buffer, and then loaded onto a sequencing gel containing 10% acrylamide and 7 M urea. After the run, each gel is analyzed using a phosphorimaging system. Reactions containing hairpin substrates are identical to those with nonhairpin substrates except that 3 pmol of Mre11 is added to reactions as indicated, and the reactions are incubated at room temperature overnight. Nonhomologous end-joining reactions contains 25 mM MOPS (pH 7.0), 60 mM KCl, 0.2% Tween 20, 2 mM DTT, 4 mM MgCl2, 2 mM MnCl2, 0.5 mM ATP, 4 ng of plasmid DNA, 10% polyethylene glycol, 0.01 pmol of human DNA ligase I, and 0.06 pmol of Mre11 or 0.1 units of E. coli exonuclease III (GIBCO-BRL), in a volume of 10 μl. After incubation at 37°C for 25 min, Tween 20 is added to a final concentration of 0.5%, and a 2.5 μl aliquot is amplified by PCR using primers DAR5 and DAR147. PCR products are cloned using the TA cloning kit and sequenced using an automated ABI Capillary Genetic Analyzer. |
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