Construction and expression of various human prion proteins mutants with modified N-glycosylation sites in mammalian cells / 生物工程学报

To study the biological function of the N-glycosylation modification of prion proteins (PrP), various eukaryotic expression vectors for the mutants with N-glycosylation modification of human PrP had been constructed and expressed. With site-direct mutation technique, human PRNP gene was mutated and the obtained mutants were subcloned into eukaryotic expressing plasmid pcDNA3.1 and transiently expressed in Hela cervical adenocarcinoma cell. The expression products of the mutated PrP were identified with Western blotting assay and the PNGase digestion assay. Several mutants with specific glycosylation modification were identified from the expressed products by Western blot, including two mutants with one glycosylation site mutated and one without any mutation at glycosylation sites. The expressed products were digested with PNGase F. The wild type proteins and those with one of glycosylation sites mutated were digested, resulting in their molecular weights reduced, while the molecular weights of products with mutations at both glycosylation sites were not changed. The mutant of wild type human PRNP gene at N-glycosylation modification sites and six modified mutants with mono- or non-N-glycosylation had been obtained successfully in the study. Moreover, the modified PrP with mono- and non-N-glycosylation were able to be expressed transitantly in Hela cells, which could be a useful means for studying prions.

A colorimetric method of HEC toxin hemolysis test for diagnosis of paroxysmal nocturnal hemoglobinuria / 中国实验血液学杂志

The purpose of the study is to establish a colorimetric method of HEC toxin hemolysis test for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). RBCs from normal persons and patients with PNH and non-PNH anemia were treated with HEC toxin secreted by Aeromonas hydrophila J-1 strain and the absorbance at 630 nm was measured to quantitate the extent of hemolysis. The results demonstrated that the RBCs from PNH patients showed resistance to the toxin hemolysis, which was in accord with the percentages of CD59(-) cells, while the RBCs from normal persons and non-PNH anemic patients were nearly totally lysed. It is concluded that the method can be considered as a simple, specific and reliable method for the diagnosis of PNH.

GPI-Pr Deficiency and Apoptosis of PNH Granulocytes / 中国实验血液学杂志

To study the relationship of Glycosyl phosphatidylinositol anchored proteins (GIP-Pr) and apoptosis of paroxysmal nocturnal hemoglobinuria (PNH) cells, we isolated peripheral granulocytes from 10 patients with PNH and 10 normal controls and measured apoptosis induced by serum starvation. The FCM analysis of phosphotidylserine (ps) externalization in granulocytes was determined using Annexin-V-FLUOS labeling. After the cells were induced for apoptosis in serum-free medium for 20 hours, the percentage of externalization was 78.6% in normal control cells but 39.5% in PNH cells. The results of FCM analysis of PI stained granulocytes showed that the PI positive rate was 51.5% in control cells and 30.2% in PNH cells. The gel electrophoresis analysis of DNA fragmentation all indicate that PNH granulocytes were relatively resistant to apoptosis as compared with normal controls. This resistance to apoptosis might not be related to the percentage of CD59 deficient granulocytes.