Interaction of a tyrosine kinase type receptor and its ligand induces receptor-dimerization or -oligomerization followed by transphosphorylation and activation of its intrinsic kinase, which leads to a series of intracellular signals. We have previously reported that the membrane-bound form of Steel factor (SLF) induces more persistent tyrosine kinase activation and longer life span of c-kit encoded protein (KIT) than its soluble form (Miyazawa et al, Blood 85:641, 1995). In this study, we used YB5.B8 monoclonal antibody (MoAb) that recognizes the extracellular domain of KIT to investigate whether immobilized anti-KIT MoAb can substitute for SLF as a potent activator of KIT by cross-linking receptors and further compared its effect with each SLF isoform in a factor-dependent cell line M07e. YB5.B8 MoAb in a soluble state suppressed SLF-induced M07e cell proliferation in a dose- dependent manner. By contrast, once this antibody was immobilized on the goat-antimouse MoAb (GAM)-coated culture plates, it supported the growth of M07e cells in the absence of any growth factors, whereas culture the cells in GAM alone or YB5.B8 without GAM-coated plates resulted in rapid cell-death within 24 hours. As with the natural ligand SLF, immobilized YB5.B8 MoAb synergized with granulocyte- macrophage colony-stimulating factor (GM-CSF) in inducing cell proliferation compared with either YB5.B8 MoAb or GM-CSF alone. Immunoblotting with antiphosphotyrosine MoAb showed that interaction of M07e cells with immobilized YB5.B8 induced tyrosine phosphorylation of a series of intracellular proteins including KIT (145 kD). In addition, cross-linking studies using a water-soluble cross linking reagent bis- sulfosuccinimidyl-suberate showed that immobilized YB5.B8 MoAb induced dimerization and activation of KIT. However, as with stimulation by the membrane-bound form of SLF, the kinetics of KIT activation with YB5.B8 MoAb was more prolonged compared with the cells treated with recombinant soluble SLF. Flow cytometry showed that, unlike the cells treated with soluble SLF, no downmodulation of cell-surface KIT expression was observed in M07e cells cultured with immobilzed YB5.B8 MoAb. These data suggest that immobilized antibodies against hematopoietic receptors may replace their ligand-stimulators; however, their activities may resemble the membrane-bound form rather than the soluble form of natural ligands.

No abstract

Ion exchange and chelate properties of poly(ethyleneimine) resin (PEI) were studied. Adsorption of neucoccin anions (NC−) (maximum 1.7×10−4 mol/g) was greater than that of Crystal Violet cations (CV+) (maximum 0.17×10−4 mol/g), indicating that PEI is an anion exchanger. CV+ adsorption increased with the addition of sodium polyacrylate due to its bridging action between CV+ ions and PEI. Adsorption of CrO42− was confirmed to be much greater than that of NC− ions amounting to 4×10−3 mol/g at a sufficiently concentrated solution of CrO42−. The acid exchange capacity of PEI was found to be about the same as the maximum amount of adsorption of CrO42−. Kinetics of adsorption of CrO42− by PEI was studied at various temperatures. Adsorption followed a bimolecular reaction. Activation energy of 28.03×103 J/mol obtained is a right order of magnitude as an ion exchange adsorption. Chelating adsorption of Fe3+ and Cu2+ ions by PEI studied, the maximum amounts of adsorption being 3.1×10−3 mol/g and 3.3×10−3 mol/g, respectively. The values are greater than those for other synthetic polyethylene polyamine. PEI can be used as an anion and metallic cation adsorbent.

No abstract