ABSTRACT
Objective To investigate the effects of 14-3-3 protein overexpression on the 1-methyl-4-phenylpyridinium (MPP(+)) induced pheochromocytoma (PC12) cell death and the potential mechanisms. Methods pcDNA3.1(+)-14-3-3 plasmids, which could be expressed in mammalian cell, were constructed and transfected into PC12 cells with Lipofectamine 2000. The expression of 14-3-3 protein, Bcl-2 protein, and BAD protein were determined by western blot. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, microplate reader, and flow cytometric analysis were used to measure cell viability, the caspase activity, and apoptotic ratio respectively. Results (1) The expression of 14-3-3 protein increased significantly three weeks after pcDNA3.1 (+)-14-3-3 plasmids transfected into PC12 cells. (2) MPP(+) caused a decrease of cell viability in a dose-dependent manner. At 100 mu mol/L MPP(+), cell viability reduced approximately 50%. (3) The caspase activity increased along with the MPP(+) concentrations rising and reached its maximum value (0.34 mu mol/mg protein) at 100 mu mol/L MPP(+). However caspase activity decreased significantly when the MPP(+) concentration exceeded 100 mu mol/L. (4) Overexpression of 14-3-3 protein decreased the apoptosis ratio of PC12 cells treated with 100 mu mol/L MPP(+) from 26.5% to 8.6%. (5) Bcl-2 protein tended to decrease but BAD protein tended to increase after treatment of PC12 cells with 100 mu mol/L MPP(+). Overexpression of 14-3-3 protein significantly increased the cellular level of Bcl-2 protein and decreased that of BAD protein. Conclusion Overexpression of 14-3-3 protein may reduce MPP(+)-induced apoptotic cell death in PC12 cells by up-regulating the Bcl-2 expression and down-regulating the BAD expression. These results may provide a promising target for treatment of Parkinson' s disease.
ABSTRACT
<p><b>OBJECTIVE</b>To analyze the susceptibility genes of a Parkinson's Disease (PD) family.</p><p><b>METHODS</b>The blood samples of a four-generation classic idiopathic PD family were collected. Two-point LOD score method was applied to analyze the linkage disequilibrium between the disease locus and microsatellite markers.</p><p><b>RESULTS</b>We studied 13 markers near the 9 genes that had been reported to be associated with PD. No obvious evidence showed that the selected markers had anything correlation with PD locus.</p><p><b>CONCLUSION</b>These 9 genes are not the susceptibility genes of PD in this family.</p>
Subject(s)
Adult , Aged , Female , Humans , Middle Aged , China , Genetic Linkage , Genetic Predisposition to Disease , Genotype , Microsatellite Repeats , Parkinson Disease , Genetics , PedigreeABSTRACT
Objective To investigate the protective effects of the 14-3-3 protein overexpression on the injury of PC12 cell induced by MPP~+ and its mechanisms.Methods For expression in mammalial cells, pcDNA3.1(+)-14-3-3 plasmid was constructed and transfeeted into PC12 cell with Lipofectamine~(TM)2000. The overexpression of transfected 14-3-3 gene in PC12 cell was determined by immunofluorescence and Western blotting.The effects of 14-3-3 overexpressing on the cells viability,apoptotie ratio and the activity of superoxide dismutase(SOD)as well as glutathione peroxidase(GSH-Px)of PC 12 cell treated with MPP~+ were measured by MTT assay,flow cytometry analysis and microplate reader respectively.Results The expression of 14-3-3 protein in transfection group(1.19?0.06)increased evidently compared with control group(0.75?0.05).And the antioxidant enzyme activity assession,MTT assay and flow cytometry analysis shows that the overexpression of 14-3-3 protein elevates the activity of SOD(transfection group:(9.13? 0.41)U/mg protein,MPP~+ group:(6.45?0.52)U/mg protein)and GSH-Px(transfection group: (89.66?3.42)?mol/mg,protein MPP~+ group:(82.73?4.15)?mol/mg protein),increases the cell viability(transfection group:0.78?0.06,MPP~+ group:0.54?0.07),and inhibits cell apoptosis (transfeetion group:11.87%?3.26%,MPP~+ group:36.30%?2.39%)of PC12 induced by MPP~. Conclusion The overexpression of 14-3-3 protein could elevate the activity of antioxidant enzymes SOD and GSH-Px,reduce oxidant stress,alleviate MPP~+ toxicity,and thus inhibit the apoptosis of PC12 cell induced by MPP~+.