Regulation of muscle creatine kinase by phosphorylation in normal and diabetic hearts

Protein kinase C (PKC) is an important signaling molecule in the heart, but its targets remain unclear. Using a PKC substrate antibody, we detected a 40-kDa phosphorylated cardiac protein that was subsequently identified by tandem mass spectroscopy as muscle creatine kinase (M-CK) with phosphorylation at serine 128. The forward reaction using ATP to generate phosphocreatine was reduced, while the reverse reaction using phosphocreatine to generate ATP was increased following dephosphorylation of immunoprecipitated M-CK with protein phosphatase 2A (PP2A) or PP2C. Despite higher PKC levels in diabetic hearts, decreased phosphorylation of M-CK was more prominent than the reduction in its expression. Changes in CK activity in diabetic hearts were similar to those found following dephosphorylation of M-CK from control hearts. The decrease in phosphorylation may act as a compensatory mechanism to maintain CK activity at an appropriate level for cytosolic ATP regeneration in the diabetic heart. Received 15 September 2008; received after revision 30 September 2008; accepted 13 October 2008     

家兔在发育过程中外侧腓肠肌内MyHCs变化与诱发电位的关系

 为研究在生后不同年龄组家兔外侧腓肠肌各亚体肌纤维MyHCs变化与诱发电位的关系,探讨生后发育期间肌纤维MyHCs组成和作用差异与表型之间的相关联系,采用电生理记录仪结合十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS PAGE）检测外侧腓肠肌各亚体。结果表明中间亚体在刺激时各年龄组的峰值电压基本相同,而外侧亚体和内侧亚体分别有些变化。同时发现成年家兔的持续时间都较幼年的长,这可能与各亚体的肌纤维型构成比例有很大关系。外侧腓肠肌各亚体的肌球蛋白重链异构体（MyHCs）电泳条带分别显示MyHCsⅡa、Ⅱd（或Ⅱx）、Ⅱb、Ⅰ共4种,对应于骨骼肌纤维表型ⅡA型（FOG型）、ⅡX（FO型）、ⅡB型（FG型）及Ⅰ型（SO型）4类。     

金属硫蛋白清除羟自由基功能的研究

引言金属硫蛋白（Ｍｅｔａｌｏｔｈｉｏｎｅｉｎ,简称ＭＴ）是一类低分子量、富含半胱氨酸的金属结合蛋白。１９５７年Ｍａｒｇｏｓｈｅｓｅｔａｌ首次从马肾中分离出该蛋白,现发现ＭＴ几乎存在于所有生物体中。哺乳类动物的ＭＴ通常是一条６１个氨基酸的肽链,其中含有...     

Changes of levels of glutamine synthetase isoforms in roots and leaves in response to nitrogen fertilizer application at different growth stages in irrigated rice

Nitrogen is a key element to control the growth and yield of crops. Fertilizer urea nitrogen (N) 60,45, and 30 kg/hm² was applied at three different stages, midtillering, panicle initiation, and flowering, of the growth and development of rice plants, respectively. At both midtillering and panicle initiation, the total activity of glutamine synthetase (GS) in rice roots and leaves was incrased remarkably as a result of a large amount of ammonia absorbed by roots. Native-PAGE and activity staining showed that the increase of total activity in rice roots and leaves was due to the synthesis of GSrb in roots and GS2 in leaves and that the activity of GSra in roots and GS1 in leaves remained constant. The results showed that the assimilation of external nitrogen was carried out by GSrb but not GSra in rice roots and that the activitry of GS2 was induced also by the external nitrogen, and that GSrb played main role in meeting the needs of the rapid tillering for nitrogen. At flowering, the activity of GS in rice roots and leaves did not change almost after topdressing. These rssults suggest that the change of GS activity in rice roots may use as a measure of the utilization efficiency of the fertilizer. Supported by the National Natural Science Foundation of China, Natural Science Foundation of Hubei Province, and International Rice Institute, P. O. Box 933 1099 Manila, Philippines Zhang Chufu: born in 1946, Professor, to whom Correspondence should be addressed     

Prokaryotic expression and characterization of a pea actin isoform （PEAcl） fused to GFP

Actins widely exist in eukaryotic cells and play important roles in many living activities. As there are many kinds of actin isoforms in plant cells,it is difficult to purifyeach actin isoform in sufficient quantities for analysing itsphysicochemical properties. In the present study, apea(pisum Sativum L.)actin isoform (PEAc1)fused to His-tag at its amino terminus and GFP(green fluorescent protein)atits Carboxyl terminus were expressed in E. coli in inclusionbodies. The fusion protein (PEAc1-GFP)was highly purifiedwith the yield of above 2 mg/L culture by dissolving inclu-sions in 8 mol/L urea,renaturing by dialysis in a gradient of urea,and affinity binding to Ni-resin. The purified mono-meric PEAc1-GFP could efficiently bind on DNase I andinhibit the latter抯 enzyme activity. PEAc1-GFP could po-lymerise into green fluorescent filamentous structures(F-PEAc1-GFP),which could be labelled byTRITC-phalloidin,a specific agent for observing microfila-ments. The PEAc1-GFP polymerlzation curve was identicalwith that of chicken skeletal muscle actin. The critical con-centration for PEAc1-Gfp to polymerise into filaments is 0.24 μmol/L.The F-PEAc1-GFP could stimulate myosinMg-ATPase activity in a protein concentration dependantmanner (about 4 folds at 1 mg/mL F-PEAc1-GFP). The re-sults above show that the PEAc1 fused to GFP retained theassembly characteristic of actin, indicating that gene fusion,prokaryotic expression, denaturation and renaturation,andaffinity chromatography is a useful strategy for obtainingplant actin isoform proteins in a large amount.     

拟南芥PP2A B′调节亚基β亚型抗体的制备及鉴定

拟南芥中,蛋白磷酸酶2A(PP2A)B′调节亚基有9个亚型,其中α亚型和β亚型是油菜素内酯(BR)信号通路的正调节子,它们能使转录因子BZR1脱磷酸化.选择β亚型特异的一段多肽P109,利用大肠杆菌表达系统表达并纯化了连有HIS标签和GST标签的融合蛋白HIS-P109和GST-P109.以HIS-P109作为抗原,免疫新西兰兔,获得抗体,然后用GST-P109对抗体进行了亲和纯化.利用此纯化的抗体在不同的拟南芥材料中免疫印迹检测β亚型蛋白的表达,证实制备的抗体能与拟南芥PP2AB′调节亚基β亚型特异性反应,为深入研究PP2A在BR信号转导途径中的功能提供了有力工具.     

Prokaryotic expression and characterization of a pea actin isoform (PEAc1) fused to GFP

~~Prokaryotic expression and characterization of a pea actin isoform (PEAc1) fused to GFP~~     

植物谷氨酰胺合成酶研究进展

谷氨酰胺合成酶(GS)是参与高等植物氨同化过程的关键酶．介绍了高等植物谷氨酰胺合成酶及其同工酶的种类、性质、生理作用和分子生物学等方面的研究进展及在植物抗盐性方面的作用。