高糖下调大鼠许旺细胞calpainⅡ的表达

目的：观察高糖对体外培养的大鼠许旺细胞calpainⅡ表达的影响。 方法： 体外培养大鼠许旺细胞，用MTT检测许旺细胞的存活力，用原位杂交和免疫细胞化学方法分别测定许旺细胞calpainⅡmRNA和蛋白的表达。 结果： 高糖（50 mmol/L）培养24 h，许旺细胞存活力明显低于对照组（30 mmol/L）（73.95% vs 100%， P＜0.01）。许旺细胞calpainⅡ mRNA阳性颗粒和calpainⅡ蛋白阳性颗粒也明显少于对照组。 结论： 高糖培养下调许旺细胞calpainⅡ表达。     

重组Calpain蛋白的免疫原性及其诊断上的应用研究

目的研究在大肠杆菌中表达的日本血吸虫Calpain蛋白的免疫原性及其在日本血吸虫诊断上的应用. 方法用重组Calpain蛋白免疫BALB/c小鼠,ELISA法测定特异性IgG抗体的动态变化及其IgG亚型(IgG1, IgG2a, IgG2b, IgG3)产生特征;同时用重组Calpain蛋白作为诊断抗原,粪检血吸虫病阳性患者血清作为一抗,肝吸虫阳性患者血清作为考核交叉反应血清. 结果重组Calpain抗原免疫小鼠后,产生了一个极高的抗Calpain特异性抗体,免疫4周后IgG类抗体达到高峰,与对照组鼠相比较,免疫鼠血清中IgG1, IgG2a, IgG2b特异性抗体也有显著性的上升; Calpain重组抗原血清诊断日本血吸虫和粪检的符合率为100%, 粪检阳性EPG低的个体抗Calpain抗体滴度高, EPG高的患者抗Calpain抗体滴度反而低, 与肝吸虫的交叉反应率为37%. 结论日本血吸虫Calpain是一个具有免疫原性的蛋白,能够激发宿主产生高水平的免疫球蛋白,能够敏感地测定日本血吸虫的感染, 说明Calpain可以发展成为日本血吸虫病的诊断抗原和日本血吸虫疫苗候选分子.     

日本血吸虫疫苗候选分子Calpain的原位表达

目的　探讨日本血吸虫疫苗候选分子———钙离子激活的中性蛋白激酶 (Calpain)在日本血吸虫体内的表达部位及其特征。方法　用重组纯化的Calpain抗原免疫大鼠 ,制备抗Calpain血清 ,从感染鼠灌流分离的日本血吸虫成虫被切片 ,用羊抗大鼠IgG抗体作为二抗进行免疫组织化学分析 ,观察Calpain蛋白在日本血吸虫成虫体内的表达部位。 结果　重组Calpain抗原免疫大鼠后 ,产生了一个极高的抗Calpain特异性抗体 ,并在日本血吸虫成虫切片上识别自然的日本血吸虫Calpain ,且大多数表达在成虫真皮层。结论　日本血吸虫钙离子激活的中性蛋白激酶 (Calpain)主要分布在成虫肌肉和真皮层 ,提示Cal pain可能是日本血吸虫的一个疫苗候选分子 ,并提示这个分子的重组抗原有可能应用于血吸虫病诊断。     

钙蛋白酶参与心力衰竭患者心肌重构的信号传导

目的 探讨钙敏感的信号物质钙蛋白酶(calpain)对心力衰竭(心衰)患心肌重构信号传导的调控。方法选择因二尖瓣狭窄伴关闭不全心脏病接受二尖瓣置换术的心衰患39例,正常对照38例(其中8例来自意外伤亡的器官捐献)。彩色多普勒超声心动图检测心功能参数,放免法检测心衰患血浆及心肌组织血管紧张素Ⅱ含量,免疫印迹法检测心肌组织calpain、钙调神经磷酸酶(CaN)的抑制蛋白(cain/cabin 1),cain／cabin 1降解产物(cain／cabin 1△)蛋白表达、CaN磷酸化。结果心衰患血浆及心肌组织血管紧张素Ⅱ浓度、心肌组织u-calpain、m-calpain、cain／cabin 1降解产物cain／cabin 1△蛋白表达及CaN磷酸化明显高于对照组,且随心功能恶化逐渐增加,cain／cabin 1蛋白表达随心功能恶化逐渐降低。结论心衰患通过calpain降解cain／cabin 1进而激活CaN信号通路,提示其在肾素一血管紧张素系统等介导的心肌重构中起一定作用。     

A mutation in the N-terminus of troponin I that is associated with hypertrophic cardiomyopathy affects the Ca(2+)-sensitivity, phosphorylation kinetics and proteolytic susceptibility of troponin

The first human cardiac troponin I (hcTnI) mutation in the N-terminal 32 residue region, R21C (arginine residue number 21 mutated to cysteine), which has been linked to hypertrophic cardiomyopathy (HCM), has recently been reported. The effect of this mutation on the physiological function of hcTnI was investigated. Human cTnI R21C (in the absence or presence of troponin T and troponin C) was phosphorylated by protein kinase A (PKA) at a significantly slower rate than wild-type hcTnI. In skinned fiber studies, the TnI R21C mutant showed a large increase in Ca(2+)-sensitivity of force development when compared to wild-type TnI (DeltapCa(50)=0.33). Phosphorylation of skinned fibers containing TnI R21C by PKA resulted in a significantly smaller decrease in the Ca(2+)-sensitivity of force development when compared to phosphorylation of fibers containing wild-type TnI. The decreased sensitivity of TnI R21C to PKA is most likely due to a decreased ability of PKA to phosphorylate this TnI rather than conformational problems within this TnI. In addition, skinned fibers were found to contain an endogenous kinase that is capable of phosphorylating wild-type TnI. However, the endogenous kinase activity did not affect the Ca(2+)-sensitivity of force development, the Hill coefficient or maximal force of these skinned fibers. Actomyosin ATPase assays showed that the R21C mutation did not affect the inhibitory properties of TnI or the maximal ATPase activity. TnI R21C was also found to be more susceptible to proteolysis by calpain II than wild-type TnI. These results suggest that this R21C mutation in TnI affects the Ca(2+)-sensitizing effect of Tn, the ability of TnI to be readily phosphorylated by PKA and the stability of TnI to calpain. The results also suggest that the N-terminal region may have important roles such as modulating the Ca(2+)-sensitivity of force-development.     

钙蛋白酶活化在小鼠心肌缺血再灌注损伤中的作用

目的:探讨钙蛋白酶在小鼠心肌缺血/再灌注(Ischemia/Reperfusion,I/R)损伤中的作用。方法:采用结扎/松解左冠状动脉前降支的方法建立小鼠心肌I/R模型。将实验动物随机分为假手术组、I/R组(冠状动脉结扎45 min,再灌注3h)及PD+I/R组(I/R前30 min静脉注射钙蛋白酶抑制剂-PD150606 1 mg/kg)。再灌注结束后,测定各组心肌梗死面积、细胞色素C含量、caspase-3活性。结果:与I/R组相比,PD150606预处理组心肌梗死区域面积明显减小,且PD150606能明显抑制由I/R引起的细胞色素C含量及caspase-3活性增加。结论:钙蛋白酶活化介导的心肌细胞凋亡在小鼠心肌I/R损伤中发挥了重要的作用。     

Alterations of Ca-responsive proteins within cholinergic neurons in aging and Alzheimer's disease

The molecular basis of selective neuronal vulnerability in Alzheimer's disease (AD) remains poorly understood. Using basal forebrain cholinergic neurons (BFCNs) as a model and immunohistochemistry, we have demonstrated significant age-related loss of the calcium-binding protein calbindin-D_28K (CB) from BFCN, which was associated with tangle formation and degeneration in AD. Here, we determined alterations in RNA and protein for CB and the Ca²⁺-responsive proteins Ca²⁺/calmodulin-dependent protein kinase I (CaMKI), growth-associated protein-43 (GAP43), and calpain in the BF. We observed progressive downregulation of CB and CaMKI RNA in laser-captured BFCN in the normal-aged-AD continuum. We also detected progressive loss of CB, CaMKIδ, and GAP43 proteins in BF homogenates in aging and AD. Activated μ-calpain, a calcium-sensitive protease that degrades CaMKI and GAP43, was significantly increased in the normal aged BF and was 10 times higher in AD BF. Overactivation of μ-calpain was confirmed using proteolytic fragments of its substrate spectrin. Substantial age- and AD-related alterations in Ca²⁺-sensing proteins most likely contribute to selective vulnerability of BFCN to degeneration in AD.     

Calpain inhIbitorⅠ对烧伤小鼠肝脏NF-κB转录及炎性细胞因子分泌的影响

探讨Calpain inhibitor I(CI-I)对严重烧伤小鼠肝脏IκBa表达,NF-κB转录及炎性细胞因子分泌的影响。将CI-Ⅰ腹腔给药预处理小鼠1 h后背部行20%全身体表面积(TBSA)Ⅲ度烧伤,收集肝组织,检测其IκBa表达,NF-κB转录和血清TNF-a、IL-1B、IL-6分泌水平。与烧伤对照组比较,CI-Ⅰ预处理后肝脏NF-κB转录活性和炎性细胞因子分泌有所降低。提示CI-Ⅰ预处理可有效抑制严重烧伤小鼠肝细胞NF-κB活化和血清炎性细胞因子分泌,从而有利于烧伤后的炎症调节。     

Zinc Increases ABCA1 by Attenuating Its Clearance Through the Modulation of Calmodulin Activity

Aim: We previously revealed that Ca⁺⁺-activated calmodulin binds to ABCA1 by the region near the PEST sequence and retards its calpain-mediated degradation to increase HDL biogenesis. Calmodulin activity is reportedly modulated also by other nutritional divalent cations; thus, we attempted to determine whether Zn⁺⁺ is involved in the regulation of ABCA1 stability through the modulation of calmodulin activity.Methods: The effects of Zn⁺⁺ on ABCA1 expression was investigated in J774 mouse macrophage cell-line cells and HepG2 human hepatoma cell-line cells.Results: Zn⁺⁺ increased ABCA1 expression, not by increasing the mRNA but by attenuating its decay rate, more prominently in the presence of cAMP. Accordingly, it enhanced cell cholesterol release with extracellular apolipo-protein A-I. Calmodulin binding to ABCA1 was increased by Zn⁺⁺ and Ca⁺⁺. Zn⁺⁺ suppressed calpain-mediated hydrolysis of the peptide of ABCA1 cytosolic loop, including the PEST sequence and the calmodulin-binding site, in a calmodulin-dependent fashion, in the presence of the minimum amount of Ca⁺⁺ to activate calpain, but not calmodulin. Calpain activity was not directly inhibited by Zn⁺⁺ at the concentration for enhancing calmodulin binding to ABCA1.Conclusion: Nutritional divalent cation Zn⁺⁺ is involved in the regulation of ABCA1 activity and biogenesis of HDL through the modulation of calmodulin activity. The results were consistent with previous clinical findings that Zn⁺⁺ increased plasma HDL in the conditions of sympathetic activation, such as type 2 diabetes and chronic hemodialysis.