Isoelectric focusing following by electrophoresis of proteins for visualizing their titration curves by zymogram and immunofixation

After isoelectric focusing followed by electrophoresis at right angles in the same gel slab, it is possible to visualize the titration curve of proteins by zymograms or immunofixation even of an unpurified sample. This information can be very useful for the selection of the proper purification strategy by charge-dependent methods, e.g. ion-exchange chromatography, zone and disc electrophoresis and isotachophoresis. The titration curve also gives information on the stability of the protein as a function of the prevailing pH of the medium, in the pH 3-10 range. A region of instability is found for most proteins in acidic conditions, below pH 4.5, while most proteins are stable in the alkaline pH region, at least up to pH 10. The best method for developing zymograms and immunoprints appears to pH 10. The best method for developing zymograms and immunoprints appears to be the 'sandwich technique', by which a thin agarose slab, cast on an hydrophilic polyester sheet, and impregnated with appropriate reagents, is left in contact with a polyacrylamide gel thin layer used to generate the titration curves.     

Charakterisierung extrazellulärer proteine und enzyme aus pektinkulturfiltraten von Botrytis cinerea

Extracellular proteins from Botrytis cinerea CBS 144.55 were separated by use of isoelectric focusing in polyacrylamide gel and thin layer isoelectric focusing. In addition to the protein pattern, the activities of protease, amylase, cellulase, polygalacturonase, pectinesterase and glycosidases were determined.     

17β‐Oestradiol facilitates M2 macrophage skewing and ameliorates arrhythmias in ovariectomized female infarcted rats

Epidemiological studies have suggested a lower incidence of arrhythmia‐induced sudden cardiac death in women than in men. 17β‐oestradiol (E2) has been reported to have a post‐myocardial infarction antiarrhythmic effect, although the mechanisms have yet to be elucidated. We investigated whether E2‐mediated antioxidation regulates macrophage polarization and affects cardiac sympathetic reinnervation in rats after MI. Ovariectomized Wistar rats were randomly assigned to placebo pellets, E2 treatment, or E2 treatment +3‐morpholinosydnonimine (a peroxynitrite generator) and followed for 4 weeks. The infarct sizes were similar among the infarcted groups. At Day 3 after infarction, post‐infarction was associated with increased superoxide levels, which were inhibited by administering E2. E2 significantly increased myocardial IL‐10 levels and the percentage of regulatory M2 macrophages compared with the ovariectomized infarcted alone group as assessed by immunohistochemical staining, Western blot and RT‐PCR. Nerve growth factor colocalized with both M1 and M2 macrophages at the magnitude significantly higher in M1 compared with M2. At Day 28 after infarction, E2 was associated with attenuated myocardial norepinephrine levels and sympathetic hyperinnervation. These effects of E2 were functionally translated in inhibiting fatal arrhythmias. The beneficial effect of E2 on macrophage polarization and sympathetic hyperinnervation was abolished by 3‐morpholinosydnonimine. Our results indicated that E2 polarized macrophages into the M2 phenotype by inhibiting the superoxide pathway, leading to attenuated nerve growth factor‐induced sympathetic hyperinnervation after myocardial infarction.     

电针诱导心肌缺血大鼠延髓头端腹外侧区nNOS和iNOS差异表达

许多研究表明,延髓头端腹外侧区(rostral ventrolateml medulla,RVLM)的NO/NOS系统参与心血管活动的中枢调节.本实验以结扎Wistar大鼠左冠状动脉前降支法建立急性心肌缺血(acute myocardial ischemia,AMI)动物模型,观察针刺"内关"穴改善AMI大鼠的心功能作用,同时检测大鼠RVLM区神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)和诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达的变化,进而探讨针刺治疗AMI的中枢机制.实验观察显示,AMI大鼠心功能各项指标减弱,伴随外周血去甲肾上腺素(norepinephrine,NE)和脑钠肽(brain natriuretic peptide,BNP)水平显著升高,同时RVLM区nNOS阳性神经元数和nNOS mRNA表达升高,而iNOS水平则降低.针刺"内关"穴(Pe 6)(每天30 min,连续5天)改善心功能,降低AMI大鼠血清中NE和BNP的水平,同时升高iNOS并降低nNOS在RVLM的表达.以上结果提示,针刺治疗心肌缺血的同时可以调节iNOS/NO和nNOS/NO在RVLM的变化,这可能与针刺通过调节RVLM区的NO含量进而降低交感传出,从而改善AMI大鼠的心功能有关.     

RT-3DE 评估急性心梗PCI术后左室收缩功能及同步性的临床价值

目的:探讨实时三维超声心动图技术(realtime three dimensional echocardiography,RT-3DE)对评价急性心肌梗死(acute myocardial infarction,AMI)经皮冠状动脉介入治疗(percutaneous coronary intervention,PCI)术后左室收缩功能及同步性的临床应用价值。方法:选择30例左室急性前壁及前间壁心梗并进行急诊PCI术的患者和30例正常对照组,应用Philips IE33彩色多普勒超声成像仪对PCI术前及术后1个月的左心室功能指标进行二维常规超声检查及三维超声心动图检查,应用Q-lab6.0软件进行分析。结果:二维超声心动图显示AMI组术前左心室收缩末容量(ESV)及舒张末容量(EDV)较对照组比较明显增大(P0.01),左心室射血分数(EF)较对照组明显减小(P0.01);急性心肌梗死(AMI)组术后1个月左心室ESV及EDV较术前比较减小(P0.05),左心室EF较术前增大(P0.05);AMI组术后1个月左心室ESV及EDV较对照组比较增大(P0.05),左心室EF较对照组减小(P0.05);三维超声心动图的各参数比较,AMI组PCI术前梗死节段局部收缩末期容量(RESV)及局部舒张末期容量(REDV)较对照组增大(P0.05),左心室梗死节段局部射血分数(REF)较对照组减小(P0.05);AMI组患者梗死节段RESV及REDV术后1个月较术前比较减小(P0.05),梗死节段REF较术前比较有所增大(P0.05),AMI组术后1个月梗死节段RESV及REDV较对照组增大(P0.05),梗死节段REF较对照组减小(P0.05);左室16节段从QRS波起点到最小收缩容积时间的标准差和最大差值(Tmsvl6-SD、Tmsvl6-Dif)以及用R-R间期校正后的Tmsvl6-SD%(左室收缩不同步指数systolic dyssynchrony index,SDI)和Tmsvl6-Dif%较术前比较减小(p0.05)。结论:PCI手术前、后应用RT-3DE能够准确评价左心室17节段的局部收缩功能及运动同步性,对AMI患者心功能的研究具有重要意义。     

细胞核CaMK和Calcineurin 对大鼠心肌肥厚发生的作用

目的:研究大鼠心肌肥厚时,钙依赖的蛋白激酶和蛋白磷酸酶在心肌细胞膜、细胞浆和细胞核的分布规律,以探讨核钙信号与核反应在心肌肥厚发生过程中的病理生理意义.方法:制备腹主动脉缩窄大鼠心肌肥厚模型,同位素32P掺入法分别测定心肌细胞核、细胞浆和细胞膜的蛋白激酶活性及用无机磷生成显色法测定其蛋白磷酸酶活性.结果:腹主动脉缩窄术后4周大鼠心肌显著肥厚,伴有明显的血液动力学异常.与正常对照组相比较,腹主动脉缩窄心肌肥厚组心肌细胞核钙调素蛋白激酶(CaMK)活性增加101.1%(P＜0.01),其膜的酶活性升高40.2%(P＜0.01),而胞浆的酶活性不变(P＞0.05);心肌细胞核钙调神经磷酸酶(Calcineurin)活性增加43.6%(P＜0.05),膜和胞浆中其活性增加无显著性(P＞0.05).正常组和腹主动脉缩窄心肌肥厚组心肌细胞CaMK和Calcineurin活性分布为核＞膜＞胞浆(P＜0.01).结论:腹主动脉缩窄心肌肥厚时核内钙依赖的CaMK和Calcineurin活性增加,提示压力超负荷时细胞核内钙调节的蛋白磷酸化和去磷酸化水平增高,可能在介导心肌肥厚的发生中起重要作用.     

Mesenchymal stem cells modified with angiopoietin-1 improve remodeling in a rat model of acute myocardial infarction

We used human angiopoietin-1 (hAng1)-modified mesenchymal stem cells (MSCs) to treat acute myocardial infarction (AMI) in rats. The hAng1 gene was transfected into cultured rat MSCs using an adenoviral vector. Five million hAng-transfected MSCs (MSC(Ang1)) or green fluorescent protein transfected MSCs (MSC(GFP)) or PBS only (PBS group) were injected intramyocardially into the inbred Lewis rat hearts immediately after myocardial infarction. MSC(Ang1) survived in the infarcted myocardium, and expressed hAng1 at both mRNA and protein levels. The vascular density was higher in the MSC(Ang1) and MSC(GFP) groups than in the PBS group. The measurements of infarcted ventricular wall thickness, infarction area, and left ventricular diameter indicated that heart remodeling was inhibited and heart function was improved in both the MSC(Ang1) and MSC(GFP) groups. However, in contrast to the MSC(GFP) group, the MSC(Ang1) group showed enhanced angiogenesis and arteriogenesis (by 11-35%), infarction area was reduced by 30% and the left ventricular wall was 46% thicker (P<0.05). The results indicated that hAng1-modified MSCs improved heart function, followed by angiogenic effects in salvaging ischemic myocardium and reduced cardiac remodeling.     

哌替啶对心室肌收缩的抑制作用及其机制

为明确哌替啶对心脏收缩的直接效应 ,并探讨其相关机制。采用Langendorff灌流心脏模型 ,观察了哌替啶对大鼠心室收缩功能的影响 ,并用荧光测钙技术和膜片钳技术探讨了哌替啶作用的钙离子机制。结果显示 ,哌替啶剂量依赖性地降低离体灌流心脏的LVDP×HR、 +dP/dt和 -dP/dt,而升高LVEDP。在酶解分离的心室肌细胞上 ,哌替啶剂量依赖性地降低细胞收缩时的钙瞬变幅度 ,并升高舒张末期的钙水平。哌替啶不影响高浓度咖啡因诱导的内钙释放。哌替啶使L 型钙电流强度降低到给药前的 67 4± 10 1% ,而不改变钙通道的激活和失活电位。哌替啶减弱钙电流的作用并不能被阿片受体阻断剂纳洛酮所阻断。以上结果表明 ,哌替啶能通过非阿片受体介导的途径阻断细胞外钙离子的内流 ,对心室收缩产生直接的抑制作用     

Kappa 阿片受体的抗缺血性心脏保护作用--信息机制

有证据表明,心脏细胞产生强腓肽和强腓肽类多肽,它们是kappa阿片受体(κ-0R)的激动剂。κ-0R是心脏一种优势的阿片受体,其激活可改变在体和离体心脏的功能。在正常和病理情况下,内源性κ-阿片肽可能通过自分泌或旁分泌的方式调节心脏功能。心肌缺血是导致心脏功能紊乱的一个常见原因,主要表现为心肌功能减弱,心律失常及心肌梗塞等。心肌缺血时,交感神经发放增强,从而增加作功负荷及氧消耗量;而这又使缺血引发的状况更为恶化。机体抵抗缺血引发心肌损害／心律失常的保护机制之一是抑制β-肾上腺素受体(β—AR)的兴奋。κ-0R确实能抑制β-AR的激动。这种抑制主要是由于GS蛋白受到抑制,也在较小程度上由于信息通路的腺苷酸环化酶的抑制。因为该种酶能通过对百日咳毒素敏感的G蛋白转导β—AR的激动。另一保护心肌对抗缺血性损害的机制是预处理。预处理是指预先受到缺血等损伤使心脏对随后更严重的损伤产生较强的耐受能力。这种保护作用可以在预处理后即时产生,也可延至预处理后1—3天。在采用缺血或其产生的后果之一——代谢抑制作为预处理而致的心脏保护中,κ-OR参与媒介预处理的作用。用κ—OR的特异性激动剂U50488H激活κ—OR(U50488H药理性预处理,UP)可激活蛋白激酶C(PKC),开放ATY敏感的钾通道(KATP channels)及增加热休克蛋白(HSP)的产生。阻断PKC的作用,关闭KATP通道或抑制HSP的合成,均可消除UP的心脏保护作用。这些发现表明,PKC、KATP通道和HSP在UP的心脏保护中均具重要作用。此外,UP也能减低缺血造成心肌损害的因素之一,即Ca^2 的超负荷。这个事实表明UP发挥心脏保护作用至少部分地是通过减低Ca^2 的超负荷。最有趣的是,以阻断剂阻塞KATP通道,在消除UP的延迟性心脏保护作用的同时也降低了UP对Ca^2 超负荷的抑制作用。这个事实揭示了KATP通道开放所致的心脏保护作用至少部分地可能是由于防止或减低了Ca^2 的超负荷。     

曲美他嗪对大鼠放射性心肌损伤后TNF-α表达的影响

目的:放射性心肌损伤是纵膈及胸腔等部位恶性肿瘤放射治疗后发生的严重并发症,目前尚无有效防治方法,而本实验拟探索曲美他嗪对放射性心肌损伤的保护作用。方法:将45只SD大鼠随机分为正常对照组(C组)、单纯照射组(X组)、曲美他嗪治疗组(T组),每组15只,其中X组、T组予以10 Gy/d,共3天。大鼠照射完毕处死后测定血清cTnI浓度,心肌组织TNF-αmRNA及蛋白的表达。结果:1.X组与C组相比,血清cTnI浓度,心肌组织TNF-αmRNA及蛋白的表达均明显升高(P0.05);2.T组与C组相比,血清cTnI浓度,心肌组织TNF-αmRNA及蛋白的表达均明显升高(P0.05);3.T组与X组相比,血清cTnI浓度,心肌组织TNF-αmRNA及蛋白的表达均明显降低(P0.05)。结论:曲美他嗪能够通过减少心肌组织TNF-αmRNA及蛋白的表达,从而有效减轻大鼠放射后心脏炎症反应,以减轻心肌损伤。