The effect of small ubiquitin-like modifier-1 modification on the formation of Lewy body-like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of alpha-synuclein / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the effect of small ubiquitin-like modifier (SUMO-1) modification on the formation of Lewy body like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of alpha-synuclein.</p><p><b>METHODS</b>cDNA encoding the human alpha-synuclein without the stop codon was cloned into a pGEM T-easy vector. Restriction enzyme mapping and DNA sequencing were performed to analyze the plasmid, which was then subcloned into a pEGFP-N1 vector. The recombinant plasmid alpha-synuclein-pEGFP was transfected into HEK293 cells by lipofectamin method. Inclusions in the cultured cells were identified with HE staining. Apoptosis of the HEK293 cell was measured by Hoechst 33258 staining, MTT and Annexin V-PE flow cytometry.</p><p><b>RESULTS</b>The Lewy-body like inclusions were found in cytoplasm of cultured cells. Hoechst staining showed that the nuclei of cells were enlarged in the wild-type and A53T mutation groups 48 h after transfection, chromatin were accumulated and appeared spot-like. The nucleus stain was equitable in the K96R and K96R-A53T groups. MTT assay showed that the viability of cells transfected with empty plasmid was 96.2%, but it dropped to 53.4% and 56.1% in cells transfected with wild-type alpha-synuclein-pEGFP and A53T mutant group, respectively. The viability was 72.3% and 69.8% in cells transfected with K96R and K96R-A53T, respectively (P<0.05). Forty eight hours after transfection, the apoptosis rate was 3.9% in empty plasmid group, 32.2% and 34.1% in cells transfected with wild-type and mutant alpha-synuclein-pEGFP, 19.4% and 20.3% in the K96R and K96R-A53T transfected cells. There was significant difference between the two groups (P<0.05).</p><p><b>CONCLUSION</b>SUMO-1 modification did not have influence on the Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro, but had a toxic effect which could increase the apoptosis induced by wild type overexpression and mutation of alpha-synuclein.</p>

A related analysis for alpha, beta fibrinogen gene haplotypes and nucleotide polymorphisms associated with the ischemic stroke in Hainan Han population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the association of that the polymorphisms and haplotypes of Taq I site in beta fibrinogen gene and the single nucleotide sites -455 G/A, -249 C/T, -148 C/T, +1689T/G, Bsm A I G/C, 448 G/A, Bcl I G/A, Hinf I A/C in beta-fibrinogen gene are linked up with the ischemic stroke(IS).</p><p><b>METHODS</b>Turbidmetric assay was used to measure the plasma fibrinogen level of one hundred and sixty cases with ischemic stroke and one hundred and thirty healthy individuals from Hainanese Han population. The polymorphisms and genotypes were characterized by PCR-RFLP. Hardy-Weinberg equilibrium and statistical differences of allelic, genotype and haplotype frequencies were obtained by Chi-square test. Pairwise linkage disequilibrium was calculated and haplotypes of nine or four polymorphisms were estimated by the EH + program.</p><p><b>RESULTS</b>There were highly significant differences in genotype frequencies and allelic frequencies of the polymorphisms -455 G/A, -148 C/T, 448 G/A, which happened between the IS group and control subjects (P< 0.01). However, the significant differences of the allelic frequencies in the other six polymorphisms were not found between the IS group and the control (P> 0.05). The odds ratio(OR) with the rare alleles of A -455, T -148 and A 448 is 2.46, 2.30 and 2.08 (95% confidence interval 1.153%-3.924%, 1.429%-3.694% and 1.298%-3.329%) respectively. No definite haplotype block was found by linkage disequilibrium analysis in the control group and the IS group. Association of haplotypes constructed from the nine polymorphisms with IS was not found. Among the haplotypes constructed from four polymorphisms including -455 G/A, -148 C/T, 448 G/A alleles, haplotype differences were found between the control group and the IS group. Haplotypes with G -455, C -148, G448 alleles appeared more frequently in control group(P< or = 0.01), whereas haplotypes with A -455, T -148, A 448 occurred more frequently in the IS group(P< 0.01).</p><p><b>CONCLUSION</b>The results of multi-allele and haplotype analysis indicated that the polymorphisms -455 G/A, -148 C/T, 448 G/A in beta fibrinogen gene were the possible risk factors associated with the occurrence of ischemic stroke in Hainan Han population.</p>