Fisher B R, Heredia D J, Brown K M
Division of Life Sciences, Center for Devices and Radiological Health, Food and Drug Administration, Rockville, Maryland 20857, USA.
Teratog Carcinog Mutagen. 1996;16(1):49-64. doi: 10.1002/(SICI)1520-6866(1996)16:1<49::AID-TCM6>3.0.CO;2-G.
Previous work from this laboratory has demonstrated that heat exposure on gestation day 10 (GD10) resulted in disrupted somite development 24 hr after exposure and subsequent thoracic skeletal malformations in neonates. The purpose of the present study was to examine the effects of in vitro heat shock on de novo protein synthesis and on cytoskeletal protein levels in developing rat embryos. Explanted GD10 embryos were exposed to temperatures of 42-42.5 degrees C for 15 min. At various times postexposure (0-27 hr). embryos were labeled with 35S-methionine and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Transient enhanced de novo synthesis of 70- and 90-kD proteins was observed 1-8 hr after exposure. The 70-kD protein was identified as a eukaryotic stress protein and the presence of this protein was detected between 2 and 27 hr posttreatment. Western blot analysis was used to detect quantitative changes in total actin (microfilaments), tubulin (microtubules), and vimentin (intermediate filaments). Immediately following exposure, a reduction of total vimentin to minimal detectable levels was observed in heat-treated embryos. Levels of total vimentin remained depressed for more than 2 hr and gradually returned to control levels 4-8 hr postexposure. No change in total actin or tubulin was detected in treated embryos. The data demonstrate that heat-induced alterations in proteins comprising intermediate filaments occur concomitantly with the induction of stress proteins and precede aberrant somite morphology. These alterations in embryonic proteins may help elucidate the mechanism(s) by which skeletal malformations are produced.
该实验室之前的研究表明，在妊娠第10天（GD10）进行热暴露会导致暴露后24小时体节发育紊乱，并在新生儿中出现随后的胸廓骨骼畸形。本研究的目的是检测体外热休克对发育中的大鼠胚胎从头合成蛋白质以及细胞骨架蛋白水平的影响。将GD10期的胚胎移出后，暴露于42 - 42.5摄氏度的温度下15分钟。在暴露后的不同时间点（0 - 27小时），用35S - 蛋氨酸标记胚胎，并进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）分离处理。暴露后1 - 8小时观察到70kD和90kD蛋白质的瞬时从头合成增强。70kD蛋白质被鉴定为一种真核应激蛋白，并且在处理后2至27小时检测到该蛋白的存在。采用蛋白质免疫印迹分析来检测总肌动蛋白（微丝）、微管蛋白（微管）和波形蛋白（中间丝）的定量变化。暴露后立即观察到，热处理的胚胎中总波形蛋白减少至最低可检测水平。总波形蛋白水平在超过2小时内持续降低，并在暴露后4 - 8小时逐渐恢复到对照水平。在处理的胚胎中未检测到总肌动蛋白或微管蛋白的变化。数据表明，热诱导的组成中间丝的蛋白质变化与应激蛋白的诱导同时发生，并先于异常的体节形态出现。胚胎蛋白的这些变化可能有助于阐明产生骨骼畸形的机制。
