本文探讨了沙蚕活性蛋白酶(Nereis active protease,NAP)诱导SPC-A-1细胞凋亡机制。采用MTT法检测NAP对SPC-A-1细胞的抑制作用,倒置显微镜及AO/EB染色观察SPC-A-1细胞的形态学的变化,采用流式细胞术检测细胞早期凋亡率和细胞膜电位的变...本文探讨了沙蚕活性蛋白酶(Nereis active protease,NAP)诱导SPC-A-1细胞凋亡机制。采用MTT法检测NAP对SPC-A-1细胞的抑制作用,倒置显微镜及AO/EB染色观察SPC-A-1细胞的形态学的变化,采用流式细胞术检测细胞早期凋亡率和细胞膜电位的变化;并通过Western Blotting检测细胞中凋亡相关蛋白的表达变化。NAP对SPC-A-1细胞活性具有明显的抑制作用且呈现剂量和时间的依赖性;NAP作用后细胞出现凋亡的形态学特征;经流式细胞术检测结果显示,随着NAP作用浓度的增加,SPC-A-1细胞的早期凋亡率从13.50%提高到22.98%,且线粒体膜电位下降所占的百分率从12.95%提高到25.28%。Western Blotting结果显示,50μg/mL NAP作用24h后,Bax/Bcl-2的比率相对对照组明显增加了6.05倍;Cyt-C、Cleaved-Caspase 9、Cleaved-Caspase 3、Cleaved-PARP等含量显著上调,相对表达量分别达到对照组的2.32、3.07、3.68、1.36倍。NAP诱导SPC-A-1细胞凋亡的作用机理有可能是通过下调Bcl-2蛋白的表达、上调Bax蛋白的表达,进而诱导线粒体膜电位的下降,促使Cyt-C的转移以及激发Caspase家族发生级联反应最终导致SPC-A-1细胞的凋亡。展开更多
目的从海洋样品中筛选产蛋白酶海洋细菌及可以降解南极磷虾原肌球蛋白(tropomyosin,TM)的蛋白酶。方法采用透明圈法筛选产蛋白酶菌株,并根据聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE)和酶联免疫吸附测定法(enzyme-l...目的从海洋样品中筛选产蛋白酶海洋细菌及可以降解南极磷虾原肌球蛋白(tropomyosin,TM)的蛋白酶。方法采用透明圈法筛选产蛋白酶菌株,并根据聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE)和酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)确定降解效果最佳菌株,对其产的蛋白酶的酶学特性及降解原肌球蛋白条件进行研究。结果最佳为菌株4sp5,经16SrDNA鉴定为Bacillus sp.。蛋白酶的最适作用温度和pH分别为60℃和9。Na^(+)和K^(+)对蛋白酶活有促进作用,其对有机溶剂敏感,表面活性剂对其影响较小。3种抑制剂均可抑制酶的活性。当加酶量为24U/mL时,7.0h能够有效地降解南极磷虾原肌球蛋白,经ELISA试剂盒测定,其致敏性消除率达98.63%。结论菌株4sp5及其产生的蛋白酶在南极磷虾原肌球蛋白脱敏作用中具有应用前景。展开更多
The pharmaceutical and anticoagulant agent heparin,a member of the glycosaminoglycan family of carbohydrates,has previously been identified as a potent inhibitor of a key Alzheimer’s disease drug target,the primary n...The pharmaceutical and anticoagulant agent heparin,a member of the glycosaminoglycan family of carbohydrates,has previously been identified as a potent inhibitor of a key Alzheimer’s disease drug target,the primary neuronalβ-secretase,β-site amyloid precursor protein cleaving enzyme 1(BACE1).The anticoagulant activity of heparin has,however,precluded the repurposing of this widely used pharmaceutical as an Alzheimer’s disease therapeutic.Here,a glycosaminoglycan extract,composed predominantly of 4-sulfated chondroitin sulfate,has been isolated from Sardina pilchardus,which possess the ability to inhibit BACE1(IC50[half maximal inhibitory concentration]=4.8μg/mL),while displaying highly attenuated anticoagulant activities(activated partial thromboplastin time EC50[median effective concentration]=403.8μg/mL,prothrombin time EC50=1.3 mg/mL).The marine-derived,chondroitin sulfate extract destabilizes BACE1,determined via differential scanning fluorimetry(ΔTm–5°C),to a similar extent as heparin,suggesting that BACE1 inhibition by glycosaminoglycans may occur through a common mode of action,which may assist in the screening of glycan-based BACE1 inhibitors for Alzheimer’s disease.展开更多
基金financially supported by grants from the Engineering and Physical Sciences Research Council,UK,the Biotechnology and Biological Sciences Research Council,UK,the Medical Research Council,UK,Intellihep Ltd.,UK,MI Engineering Ltd.,UK and Financiadora de Estudos e Projetos
文摘The pharmaceutical and anticoagulant agent heparin,a member of the glycosaminoglycan family of carbohydrates,has previously been identified as a potent inhibitor of a key Alzheimer’s disease drug target,the primary neuronalβ-secretase,β-site amyloid precursor protein cleaving enzyme 1(BACE1).The anticoagulant activity of heparin has,however,precluded the repurposing of this widely used pharmaceutical as an Alzheimer’s disease therapeutic.Here,a glycosaminoglycan extract,composed predominantly of 4-sulfated chondroitin sulfate,has been isolated from Sardina pilchardus,which possess the ability to inhibit BACE1(IC50[half maximal inhibitory concentration]=4.8μg/mL),while displaying highly attenuated anticoagulant activities(activated partial thromboplastin time EC50[median effective concentration]=403.8μg/mL,prothrombin time EC50=1.3 mg/mL).The marine-derived,chondroitin sulfate extract destabilizes BACE1,determined via differential scanning fluorimetry(ΔTm–5°C),to a similar extent as heparin,suggesting that BACE1 inhibition by glycosaminoglycans may occur through a common mode of action,which may assist in the screening of glycan-based BACE1 inhibitors for Alzheimer’s disease.
