Differential regulation of collagen types I and III expression in cardiac fibroblasts by AGEs through TRB3/MAPK signaling pathway

Advanced glycation end products (AGEs) play an important role in collagen deposition in diabetic cardiomyopathy. TRB3, a mammalian homolog of Drosophila tribbles, functions to increase glucose intolerance and regulates cell proliferation. We demonstrated that AGEs induce collagen type I expression but inhibit collagen type III expression, accompanied by increased TRB3 expression. Furthermore, the collagen type I induced byAGEs was down-regulated after inhibition of ERK and p38-MAPK, the collagen type III reduced by AGEs was up-regulated after inhibition of ERK. The expression of collagen types I and III regulated by AGEs through MAPK was partly reversed after treatment with TRB3 siRNA. It suggests that the TRB3/MAPK signaling pathway participates in the regulation of collagen types I and III by AGEs and may provide new therapeutic strategies for diabetic cardiomyopathy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 08 May 2008; received after revision 25 June 2008; accepted 22 July 2008 M. Tang, M. Zhong: These two authors contributed equally to this work.     

Ruminations on dietary restriction and aging

Calorie restriction has been known for many decades to extend the life span of rodents. Since the more recent discovery that a long-term reduction in nutrient intake also extends life span in nearly every invertebrate model organism used for aging research, the mechanisms behind the longevity benefits of this intervention have been under intense scrutiny. While models have been developed in yeast, worms, and flies, the molecular mechanisms governing life span extension by calorie restriction remain controversial, resulting in great anticipation of mammalian studies testing these models. Here we discuss the links between nutrient reduction and enhanced longevity with emphasis on evolutionarily conserved nutrient response signaling. Received 1 November 2006; received after revision 15 December 2006; accepted 27 February 2007     

酵母(Saccharomyces cerevisiae )蛋白激酶SCH9激活环磷酸化调控胁迫应答

通过比较野生型、△sch9和△sch9(SCH9-3HA)三种酵母茵种在葡萄糖、半乳糖、蔗糖和麦芽糖作碳源培养基上的生长表型研究SCH9是否参与酵母不同碳源代谢利用调控.结果显示,△sch9细胞茵落大小较野生型细胞小且有明显的生长缺陷,但这种生长缺陷在重新获得sch-3HA基因后得到恢复.通过比较野生型、△sch9和△sch9(SCH9-3HA)三种酵母茵种在渗透压胁迫和热胁迫条件下的生长表型发现sch9可能参与了酵母胁迫应答.利用抗SCH9570位磷酸化苏氨酸特异性抗体(anti-T570-P),通过变性免疫沉淀和免疫印迹方法,研究了生理条件下的△sch9(SCH9-3HA)细胞裂解液中SCH9激活环的磷酸化状态.结果显示,在生理条件下SCH9激活环T570位点发生了明显磷酸化.进一步研究了渗透压胁迫条件下SCH9激活环T570位点的磷酸化水平.结果表明,渗透压胁迫条件下SCH9激活环T570位点磷酸化水平较生理条件下显著增强.结果显示SCH9可能通过增强激活环磷酸化水平来参与调控酵母不同碳源代谢和胁迫应答.     

Characteristics of Two Intermediates Trapped in the Unfolding Pathway of Arginine Kinase Induced by Guanidinium Chloride

Equilibrium guanidinium chloride （GdmCl）-induced unfolding of arginine kinase （AK） was investigated by enzymatic activity, intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonic acid （ANS） fluorescence, circular dichroism （CD） spectrum, and size-exclusion chromatography. The measurements showed that AK unfolded through two equilibrium intermediates： the molten globule state and the partly folded state. Both intermediates have no enzyme activity. The molten globule state exists at 0.4-0.8 mol/L GdmCi, perhaps after the N-terminal domain has unfolded but the C-terminal domain is still intact. The partly folded state occurs at 1.1-1.5 mol/L GdmCI with a hydrodynamic volume no more than 1.6-fold larger than the native state and a pronounced far UV-CD signal. Its ANS fluorescence intensity is about 50% of the molten globule state. This partly folded state shares similarities with the “burst” kinetic intermediate of protein folding.     

Rho激酶抑制剂治疗急性心肌梗死血清炎症因子的变化及疗效观察

目的给予急性心肌梗死患者早期应用Rho激酶抑制剂,测查其血清炎症因子IL-6,TNF-a,NO的变化情况,并以此为依据,观察Rho激酶抑制剂临床治疗效果.方法选择同期收治的未接受冠脉介入治疗的急性心肌梗死患者50例.随机分为治疗组和常规组,各25例.两组均给予硝酸甘油、硫酸氢氯吡格雷、辛伐他丁等常规治疗.治疗组在常规治疗的基础上静脉滴注盐酸法舒地尔30 mg,2次/d,10 d为1个疗程,观察患者血清炎症因子IL-6,TNF-a,NO浓度变化.结果加用法舒地尔的治疗组患者血清炎症因子IL-6,TNF-a浓度下降,NO浓度增高,均比常规组明显,且患者心功能检测治疗组改善更明显.结论 Rho激酶抑制剂治疗急性心肌梗死能够更有效地保护心肌细胞,降低炎症因子对心肌细胞的损伤,对改善患者心功能有显著效果.     

c-Jun氨基末端激酶3结构域突变质粒的构建、鉴定与转染

为构建c-Jun氨基末端激酶3 (c-Jun N-terminal kinase3,JNK3)的p VP16-eGFP-Myc-JNK3活化环(activation-loop,A-loop)结构域突变型重组质粒,通过A-loop突变型JNK3的c DNA序列以及p VP16-eGFP-Myc的酶切位点设计引物,PCR扩增目的基因,使用限制性内切酶Mlu I与Xba I将基因克隆到p VP16-eGFP-Myc真核表达载体中,构建p VP16-eGFP-Myc-JNK3(A-loop)结构域突变型重组质粒。转化后,通过双酶切鉴定与DNA测序判断是否成功构建重组质粒,使用Trans IntroTMEL Transfection Reagent转染人胚肾(human emborynic kidney,HEK) 293T细胞,通过荧光显微镜下观察融合蛋白表达情况。结果表明:构建p VP16-eGFP-Myc-JNK3 (A-loop)结构域突变型重组质粒,双酶切鉴定和测序结果显示构建成功; p VP16-eGFP-MycJNK3 (A-loop)成功转染HEK293T细胞且表达。鼠源JNK3结构域突变型质粒构建成功,对进一步研究JNK3结构域在相关疾病的作用和机制提供实验工具。     

v-src,Ca~(2+),PKC和cAMP对3T3-tsLA90细胞间隙连接通讯的调节

以3T3-tsLA 90细胞为正常和转化细胞模型,采用划痕标记染料示踪技术,初步研究了 v-src 基因及 Ca~(2+)-钙调素,蛋白激酶 C,cAMP 等细胞内信号物质对细胞间隙连接通讯的调节作用.40℃时正常表型的细胞间隙连接通讯正常,33℃时转化表型的细胞间隙连接被阻断.用 v—src 基因产物抑制剂、钙通道抑制剂、钙离子载体、钙调素和蛋白激酶 C 抑制剂及 cAMP研究了对细胞间隙连接的作用.当3T3-tsLA 90细胞由40℃转移到33℃时,v-src 基因活化,其产物 pp 60~(v-s c)通过 Ca~(2+)-CaM 和 DG-PKC 通路及 cAMP 系统相互作用对细胞间隙连接通讯功能起下降调节作用,从而导致细胞呈转化表型状态.