Calliari A, Sotelo-Silveira J, Costa M C, Nogueira J, Cameron L C, Kun A, Benech J, Sotelo J R
Departament of Molecular and Cell Biology, Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay.
Cell Motil Cytoskeleton. 2002 Apr;51(4):169-76. doi: 10.1002/cm.10017.
The presence of Myosin Va (an actin-based molecular motor) in the peripheral nervous system was examined and its subcellular distribution within the axons of the sciatic nerve was demonstrated via immunocytochemistry. Myosin Va (M-Va) in the nerve was detected by using SDS-PAGE and Western blot techniques with a polyclonal antibody specifically raised against the M-Va globular tail domain. In addition, purification of M-Va from the rat sciatic nerve prior to immunoblotting yielded a M-Va standard band. Likewise, optical immunocytochemical procedures revealed the presence of M-Va, particularly in the cortical axoplasmic territory, but also in the Schwann cell soma. The above experiments were carried out both on intact as well as on severed sciatic nerves with similar results. The proximal stumps of severed sciatic nerves (from 0 to 72 h after injury) were labelled in vivo with (35)S-methionine. SDS-PAGE autoradiography of the immunoabsorbed M-Va from the radiolabelled homogenized nerve tissue showed a significant increment of the radioactive intensity of M-Va heavy chain band through time. Moreover, a significant increment of transcripts coding for M-Va heavy chain was detected through time using RT-PCR after nerve injury and compared to intact nerves. This data suggest that M-Va is up-regulated in a time-dependent manner. The latter suggests a possible involvement of M-Va in nerve regeneration processes.
研究了肌球蛋白Va（一种基于肌动蛋白的分子马达）在外周神经系统中的存在情况，并通过免疫细胞化学方法证明了其在坐骨神经轴突内的亚细胞分布。通过使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹技术，用针对肌球蛋白Va球状尾部结构域特异性制备的多克隆抗体检测神经中的肌球蛋白Va（M-Va）。此外，在免疫印迹之前从大鼠坐骨神经中纯化M-Va产生了一条M-Va标准带。同样，光学免疫细胞化学方法揭示了M-Va的存在，特别是在皮质轴浆区域，但在雪旺细胞胞体中也有。上述实验在完整的以及切断的坐骨神经上均进行，结果相似。切断的坐骨神经近端残端（损伤后0至72小时）在体内用（35）S-蛋氨酸标记。对来自放射性标记的匀浆神经组织的免疫吸附M-Va进行SDS-PAGE放射自显影显示，随着时间的推移，M-Va重链带的放射性强度显著增加。此外，在神经损伤后通过逆转录-聚合酶链反应（RT-PCR）并与完整神经相比，检测到编码M-Va重链的转录本随着时间的推移显著增加。这些数据表明M-Va以时间依赖性方式上调。后者表明M-Va可能参与神经再生过程。
