Objective Study the effect of NMDA receptor antagonist GAPA on the [Ca 2+ ]i of the bilirubin precipitated brain tissue. Methods 10 ug/g of GAPA was administrated peritoneally to Gunn rat with bilirubin induced encephalopathy, brain tissue suspensions was prepared, Fura 2/AM was loaded. the neural cytosolic Ca 2+ was measured by flurescence imaging analysis. Results The concentration of neural cytosolic Ca 2+ in bilirubin precipitated brain tissue was significantly more than that in the control group; NMDA receptor antagonist GAPA could significantly decrease the cytosolic Ca 2+ overload. Conclusion Cytosolic Ca 2+ overload was found in the bilirubin precipitated brain tissue. NMDA receptor antagonist GAPA could prevent the cytosolic Ca 2+ overload in bilirubin induced brain tissue.

Objective To demonstrate that heme oxygenase (HO) isoforms exist in rat spleen treated with hematin and phenylhydrazine and to confirm that the isolated cDNA actually encodes HO-1 by expressing cDNA in monkey kidney cells (COS-1 cells ) in order to prepare HO-1 mutant for inhibiting the natural enzyme.Methods The rat spleen microsomal fractions were first purified by diethylaminoethyl (DEAE)-Sephacel and hydroxylapatite. The activity of two isoforms (HO-1 and HO-2) of enzyme and their apparent molecular weight on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) were measured. Secondly, using the isolated HO-1 cDNA clone, the expression plasmid pcDNA3HO1 was constructed and transfected into cultured COS-1 cells. The transfected cells were collected and disrupted by sonication, and the microsomes were prepared by ultracentrifugation. The activity of HO-1 was measured.Conclusion The activity of expressed HO-1 in COS-1 cells is higher than that of purified enzyme from rat spleen tissue. It is suggested that this clone having an insert of 1030 base-pairs encodes HO-1 and that we can prepare HO-1 mutant by site-directed mutagenesis of HO-1 cDNA to prevent and treat hyperbilirubinemia.