目的探讨Destrin蛋白在氧化型低密度脂蛋白(oxygenized low density lipoprotein,oxLDL)损伤人颈动脉内皮细胞过程中的作用和机制。方法人颈动脉内皮细胞给予剂量梯度的oxLDL(0、5、25、100μg/ml)刺激24h,检测细胞中Destrin蛋白的表达...目的探讨Destrin蛋白在氧化型低密度脂蛋白(oxygenized low density lipoprotein,oxLDL)损伤人颈动脉内皮细胞过程中的作用和机制。方法人颈动脉内皮细胞给予剂量梯度的oxLDL(0、5、25、100μg/ml)刺激24h,检测细胞中Destrin蛋白的表达变化;通过RNA干扰的方式下调颈动脉内皮细胞中Destrin的表达,观察其对oxLDL刺激诱导的颈动脉内皮细胞损伤的影响,同时检测单核细胞趋化蛋白1(MCP-1)和细胞间黏附分子1(ICAM-1)的表达水平。结果与oxLDL刺激对照RNAi比较,oxLDL刺激RNAi MCP-1和ICAM-1蛋白表达明显增加[(734.2±113.4)pg/ml vs(502.1±96.7)pg/ml,(152.5±22.0)pg/ml vs(94.2±16.5)pg/ml,P<0.05];细胞活力明显减低[0.41±0.05 vs 0.62±0.06,P<0.05],细胞凋亡明显增高[(50±10)%vs(32±8)%,P<0.05]。结论oxLDL损伤颈动脉内皮细胞的过程中,Destrin蛋白表达下调是oxLDL的损伤机制之一。展开更多
Destrin, also called actin-depolymerizing factor (ADF), exists in resting parotid tissue as phosphorylated (inactive) and dephosphorylated (active) forms, and β-adrenergic stimulation of this tissue induces dephospho...Destrin, also called actin-depolymerizing factor (ADF), exists in resting parotid tissue as phosphorylated (inactive) and dephosphorylated (active) forms, and β-adrenergic stimulation of this tissue induces dephosphorylation of destrin. It is suggested that destrin dephosphorylation is involved in cortical F-actin disruption observed in parallel with β-agonist-induced amylase secretion. At present, the phosphorylation/dephosphorylation mechanism of destrin in parotid tissue is not known. We previously detected, in a crude rat parotid extract, a constitutively active protein kinase catalyzing phosphorylation of destrin;however, its identification has been hampered by difficulty in its enrichment. The purpose of this study was to explore a simple purification method(s) for this enzyme. To this end, we first developed a high-throughput dot-blot assay for the kinase with an anti-phosphodestrin antibody and then studied its purification by column chromatography on several media. We found that the kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxyapatite columns. In each chromatography, however, the kinase could be eluted, at the cost of resolution, only by sharp increases in the elution power of the eluent;gradual increases in the elution power resulted in unacceptably poor recovery. We confirmed that enzymatic properties of the kinase were not basically altered during the purification. Further purification of the kinase was achieved by native polyacrylamide gel electrophoresis (PAGE), which resolved the kinase activity into two bands and separated the activity from most proteins (the kinase activity after PAGE was detected with destrin-coated polyvinylidene difluoride membranes and the anti-phosphodestrin antibody). The two bands seem to constitute the major destrin-phosphorylating activity in the resting rat parotid gland. We here report its partial purification and characterization together with the detection methods.展开更多
文摘目的探讨Destrin蛋白在氧化型低密度脂蛋白(oxygenized low density lipoprotein,oxLDL)损伤人颈动脉内皮细胞过程中的作用和机制。方法人颈动脉内皮细胞给予剂量梯度的oxLDL(0、5、25、100μg/ml)刺激24h,检测细胞中Destrin蛋白的表达变化;通过RNA干扰的方式下调颈动脉内皮细胞中Destrin的表达,观察其对oxLDL刺激诱导的颈动脉内皮细胞损伤的影响,同时检测单核细胞趋化蛋白1(MCP-1)和细胞间黏附分子1(ICAM-1)的表达水平。结果与oxLDL刺激对照RNAi比较,oxLDL刺激RNAi MCP-1和ICAM-1蛋白表达明显增加[(734.2±113.4)pg/ml vs(502.1±96.7)pg/ml,(152.5±22.0)pg/ml vs(94.2±16.5)pg/ml,P<0.05];细胞活力明显减低[0.41±0.05 vs 0.62±0.06,P<0.05],细胞凋亡明显增高[(50±10)%vs(32±8)%,P<0.05]。结论oxLDL损伤颈动脉内皮细胞的过程中,Destrin蛋白表达下调是oxLDL的损伤机制之一。
文摘Destrin, also called actin-depolymerizing factor (ADF), exists in resting parotid tissue as phosphorylated (inactive) and dephosphorylated (active) forms, and β-adrenergic stimulation of this tissue induces dephosphorylation of destrin. It is suggested that destrin dephosphorylation is involved in cortical F-actin disruption observed in parallel with β-agonist-induced amylase secretion. At present, the phosphorylation/dephosphorylation mechanism of destrin in parotid tissue is not known. We previously detected, in a crude rat parotid extract, a constitutively active protein kinase catalyzing phosphorylation of destrin;however, its identification has been hampered by difficulty in its enrichment. The purpose of this study was to explore a simple purification method(s) for this enzyme. To this end, we first developed a high-throughput dot-blot assay for the kinase with an anti-phosphodestrin antibody and then studied its purification by column chromatography on several media. We found that the kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxyapatite columns. In each chromatography, however, the kinase could be eluted, at the cost of resolution, only by sharp increases in the elution power of the eluent;gradual increases in the elution power resulted in unacceptably poor recovery. We confirmed that enzymatic properties of the kinase were not basically altered during the purification. Further purification of the kinase was achieved by native polyacrylamide gel electrophoresis (PAGE), which resolved the kinase activity into two bands and separated the activity from most proteins (the kinase activity after PAGE was detected with destrin-coated polyvinylidene difluoride membranes and the anti-phosphodestrin antibody). The two bands seem to constitute the major destrin-phosphorylating activity in the resting rat parotid gland. We here report its partial purification and characterization together with the detection methods.
