Identification and functional significance of troponin I isoforms in neonatal rat heart myofibrils

We investigated the mechanism(s) responsible for differences in the effects of acidic pH on Ca2+ activation of the activity of adult and neonatal rat heart myofilaments. Studies on preparations of myofilaments reconstituted with adult troponin-tropomyosin (Tn-Tm) and either adult or neonatal thick filaments indicated that the difference in effect of acidic pH is related to differences in Tn-Tm and not other myofilament proteins. Immunoblotting analysis showed that development of the rat heart myofibrils is associated with isoform switching from slow skeletal TnI to cardiac TnI and from a slow mobility isoform of TnT (TnT1) to a faster Mr isoform (TnT2. Expression of slow skeletal TnI was associated with a relative insensitivity of myofilament Ca2+ activation to deactivation by acidic pH. Moreover, the effect of acidic pH on Ca2+ activation of ATPase activity of soleus myofibrils, which contain cardiac TnC and slow skeletal TnI, was essentially the same as the effect of acidic pH on rat cardiac myofibrils in the early neonatal period. Neonatal myofilaments also contained a relative abundance of a set of polypeptides copurifying with the thin filaments. We have identified these proteins as histones. The relative amount of histones among a variety of preparations from different species was not correlated with the pH sensitivity of myofibrillar Ca2+ activation. Shifts in TnT isoforms among these species were also not correlated with an altered response to acidic pH. Our data provide evidence in support of the hypothesis that the relative insensitivity of neonatal myofilament activity to acidic pH is due to the presence of slow skeletal TnI in the thin-filament regulatory complex.     

荧光原位杂交技术分析人结肠菌群方法研究

建立荧光原位杂交技术分析人体内结肠菌群的方法。取受试者新鲜粪便 ,选用 5种特异性的 16SrRNA寡核苷酸探针 ,检测粪便样本收集后的保存时间、温度 ,离心条件及样本固定液存放时间对杂交计数结果的影响。结果建立最佳实验条件为 :粪便样本收集后应尽快在 4℃下保存 ,放置时间不要超过 12小时即作处理 ;样本的适宜离心条件为 70 0g 2分钟 ;样本用多聚甲醛固定后在 - 80℃下存放时间不要超过 5个月。该方法具有较好的稳定性 ,可以有效地检出个体之间结肠菌群的差异。