Mermelstein C dos S, Costa M L, Chagas Filho C, Moura Neto V
Departmento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil.
J Muscle Res Cell Motil. 1996 Apr;17(2):199-206. doi: 10.1007/BF00124242.
The cocarcinogenic phorbol ester 13-tetradecanoyl-O-phorbol acetate selectively and reversibly inhibits the ongoing differentiation programme of chick muscle cells in culture. 13-tetradecanoyl-O-phorbol acetate promptly blocks spontaneous contractions in mature myotubes and induces them to retract, forming giant myosacs and concurrently stress fibre-like structures are assembled. Using indirect immunofluorescence to localise desmin, the muscle specific intermediate filament protein, it was shown that its distribution is longitudinally oriented in mature myotubes. In myosacs, desmin has a reticular pattern although not as linearly oriented as in control myotubes. Using gel electrophoresis of control and 13-tetradecanoyl-O-phorbol acetate treated cell extracts, three major protein bands were observed with molecular weight of 43, 50 and 55 kDa. They migrate as actin, desmin and vimentin, respectively. The 50 kDa and 55 kDa proteins were expressed more in 13-tetradecanoyl-O-phorbol acetate-treated cells. The 50 kDa band was confirmed as desmin by immunoblotting using anti-chicken desmin antibody. Two-dimensional gel electrophoresis analysis showed the appearance of more acidic isoforms of the 50 and 55 kDa proteins 13-tetradecanoyl-O- phorbol in acetate-treated cells. The 43 kDa protein was seen as three distinct isoforms in control cells and as only two isoforms in 13-tetradecanoyl-O-phorbol acetatetreated cells.
促癌剂佛波酯13-十四酰佛波醇乙酸酯可选择性且可逆地抑制培养的鸡肌肉细胞正在进行的分化程序。13-十四酰佛波醇乙酸酯能迅速阻断成熟肌管中的自发收缩，并诱导它们回缩，形成巨大的肌囊，同时组装应力纤维样结构。利用间接免疫荧光法对肌肉特异性中间丝蛋白结蛋白进行定位，结果表明其在成熟肌管中呈纵向分布。在肌囊中，结蛋白呈网状分布，尽管不像对照肌管中那样呈线性排列。通过对对照细胞和经13-十四酰佛波醇乙酸酯处理的细胞提取物进行凝胶电泳，观察到三条主要蛋白带，分子量分别为43、50和55 kDa。它们分别迁移为肌动蛋白、结蛋白和波形蛋白。50 kDa和55 kDa的蛋白在经13-十四酰佛波醇乙酸酯处理的细胞中表达更多。通过使用抗鸡结蛋白抗体进行免疫印迹，确认50 kDa的条带为结蛋白。二维凝胶电泳分析显示，在经13-十四酰佛波醇乙酸酯处理的细胞中，50 kDa和55 kDa的蛋白出现了更多酸性异构体。43 kDa的蛋白在对照细胞中表现为三种不同的异构体，而在经13-十四酰佛波醇乙酸酯处理的细胞中仅表现为两种异构体。
