ABSTRACT
The modified asparaginase Was79 was derived from the recombinant wild-type L-asparaginase of Wolinella succinogenes. The Was79 contains the amino acid substitutions V23Q and K24T responsible for the resistance to trypsinolysis and the N-terminal heparin-binding peptide KRKKKGKGLGKKR responsible for the binding to heparin and tumor K562 cells in vitro. When tested on a mouse model of Fischer lymphadenosis L5178Y, therapeutic efficacy of Was79 was significantly higher than that of reference enzymes at all single therapeutic doses used (125-8000 IU/kg). At Was79 single doses of 500-8000 IU/kg, the complete remission rate of 100 % was observed. The Was79 variant can be expressed intracellularly in E. coli as a less immunogenic formyl-methionine-free form at high per cell production levels.
Subject(s)
Antineoplastic Agents/administration & dosage , Asparaginase/genetics , Asparaginase/metabolism , Heparin/metabolism , Leukemia L5178/drug therapy , Wolinella/enzymology , Amino Acid Substitution , Animals , Antineoplastic Agents/pharmacology , Asparaginase/administration & dosage , Asparaginase/pharmacology , Bacterial Proteins/administration & dosage , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Humans , K562 Cells , Mice , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Wolinella/genetics , Xenograft Model Antitumor AssaysABSTRACT
The fluorescent probe chlortetracycline (CTC) was used to investigate redistribution of intracellular Ca2+ in concanavalin A (Con A)-stimulated human peripheral blood lymphocytes. The addition of the mitogen to CTC-equilibrated lymphocytes induced (within 10 to 15 minutes) a Con A-concentration dependent decrease in CTC fluorescence indicating the release of membrane-bound Ca2+. The effect was independent of the level of extracellular Ca2+ and could be observed in the presence of EGTA; it was suppressed by the metabolic inhibitors FCCP, antimycin and sodium cyanide. Analysis of the excitation spectra of CTC fluorescence indicated that the observed effect is caused by redistribution of intracellular Ca2+ rather than Mg2+. Thus the lectin interaction with the lymphocyte plasma membrane results in Ca2+ release into the cytosol from the intracellular stores.
