硫化氢预处理延迟相对大鼠心肌缺血再灌注时谷胱甘肽S转移酶表达的影响

目的:探讨硫化氢预处理延迟相对大鼠心肌缺血再灌注时谷胱甘肽S转移酶表达的影响.方法:健康成年Sprague-Dawley雄性大鼠40只,随机分成4组:假手术组(S组)仅开胸、分离冠状动脉左前降支,不阻断血流150 min;缺血再灌注组(IR组)行冠状动脉左前降支阻断30 min,再灌注120 min;硫化氢预处理延迟相组(H组)静脉注射硫化氢(0.05 mg/kg),给药后24h同IR组处理;硫化氢预处理延迟相+线粒体KATP通道阻断剂(5-羟葵酸,5-HD)组(D组)缺血前15min静脉注射5-HD 5 mg/kg,其它同H组处理.再灌注结束后测心肌谷胱甘肽S转移酶(GST)的表达和心梗面积,观察心肌细胞超微结构.结果:与IR组(38.27±5.64)%比,H组(25.40±3.54)%心肌梗死面积减小(P＜0.05),D组(40.53±5.24)%无明显差异(P＞0.05).与S组比,IR组、H组和D组GST均升高(P＜0.05),与IR组比,H组GST增高(P＜0.05),D组无明显差异(P＞0.05).与IR组比,电镜下H组心肌细胞损伤程度减轻,D组无明显差异.结论:硫化氢预处理延迟相对大鼠心肌的保护作用可能与上调心肌谷胱甘肽S转移酶表达有关.     

Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase

Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow, for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism.     

心肌肌钙蛋白Ⅰ对体外循环期心肌损伤的判定价值

目的 探讨心肌肌钙蛋白Ｉ（ｃＴｎＩ）对体外循环期心肌损伤的判定价值。方法 ２０例心内直视手术病人随机分为两组,冷晶体停跳组（组Ｉ）,冷氧合血停跳组（组Ⅱ）,每组１０例,分别于围术期多外时点采取中心静脉血,测定血清ｃＴｎＩ、ＣＫ及ＣＫ－ＭＢ的水平,并观察缺血前后心肌超微结构变化。结果 术前两组的ｃＴｎＩ、ＣＫ及ＣＫ－ＭＢ水平均在正常范围,开放主动脉后１ｈ至术后２４ｈ达峰值,其后缓慢下降。术后２４ｈ     

Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles

Enzymatic inhibition by product molecules is an important and widespread phenomenon. We describe an approach to study product inhibition at the single-molecule level. Individual HRP molecules are trapped within surface-tethered lipid vesicles, and their reaction with a fluorogenic substrate is probed. While the substrate readily penetrates into the vesicles, the charged product (resorufin) gets trapped and accumulates inside the vesicles. Surprisingly, individual enzyme molecules are found to stall when a few tens of product molecules accumulate. Bulk enzymology experiments verify that the enzyme is noncompetitively inhibited by resorufin. The initial reaction velocity of individual enzyme molecules and the number of product molecules required for their complete inhibition are broadly distributed and dynamically disordered. The two seemingly unrelated parameters, however, are found to be substantially correlated with each other in each enzyme molecule and over long times. These results suggest that, as a way to counter disorder, enzymes have evolved the means to correlate fluctuations at structurally distinct functional sites.     

Global kinetic analysis of proteolysis via quantitative targeted proteomics

Mass spectrometry-based proteomics is a powerful tool for identifying hundreds to thousands of posttranslational modifications in complex mixtures. However, it remains enormously challenging to simultaneously assess the intrinsic catalytic efficiencies (k(cat)/K(M)) of these modifications in the context of their natural interactors. Such fundamental enzymological constants are key to determining substrate specificity and for establishing the timing and importance of cellular signaling. Here, we report the use of selected reaction monitoring (SRM) for tracking proteolysis induced by human apoptotic caspases-3, -7, -8, and -9 in lysates and living cells. By following the appearance of the cleaved peptides in lysate as a function of time, we were able to determine hundreds of catalytic efficiencies in parallel. Remarkably, we find the rates of substrate hydrolysis for individual caspases vary greater than 500-fold indicating a sequential process. Moreover, the rank-order of substrate cutting is similar in apoptotic cells, suggesting that cellular structures do not dramatically alter substrate accessibility. Comparisons of extrinsic (TRAIL) and intrinsic (staurosporine) inducers of apoptosis revealed similar substrate profiles, suggesting the final proteolytic demolitions proceed by similarly ordered plans. Certain biological processes were rapidly targeted by the caspases, including multiple components of the endocyotic pathway and miRNA processing machinery. We believe this massively parallel and quantitative label-free approach to obtaining basic enzymological constants will facilitate the study of proteolysis and other posttranslational modifications in complex mixtures.     

Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily

The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis, based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: (i) a new group of presumed Ala-Glu epimerases; (ii) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and (iii) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion.     

Inhibition of Adhesion Molecules Markedly Ameliorates Cytokine-Induced Sustained Myocardial Dysfunction in Dogsin vivo

Adhesion molecules are key molecules for inflammatory cardiovascular diseases and are known to be up-regulated by inflammatory cytokines. However, the role of adhesion molecules in the cytokine-induced myocardial dysfunctionin vivoremains unclear. This role was examined in our novel canine model, in which chronic treatment of the heart with IL-1β-bound microspheres (MS), but not control MS, causes sustained myocardial dysfunctionin vivo. The expression of P-selectin (mRNA and immunoreactivity) was more prominent in the IL-1βgroup than in the control group (treated with control MS alone) after MS injection. The extent of neutrophil infiltration and myocardial myeloperoxidase (MPO) activity were significantly increased in the IL-1βgroup (P<0.01). Pre-treatment with SLeX-OS (a novel oligosaccharide analog of sialyl Lewis^X) or PB1.3 (a monoclonal antibody to P-selectin) prevented the myocardial dysfunction and significantly suppressed the neutrophil infiltration and the increase in myocardial MPO activity induced by IL-1β(P<0.01 each). These results indicate that adhesion molecules play an important role in the pathogenesis of the cytokine-induced sustained myocardial dysfunction in dogsin vivo.     

烫伤大鼠心肌细胞丝裂素活化蛋白激酶的活化及胞内分布规律的研究

目的　观察大鼠严重烫伤后早期心肌细胞中丝裂素活化蛋白激酶 [MAPKs,包括 p38激酶、细胞外信号调节激酶 (ERKs)和应激活化蛋白激酶 (JNK) ]的活化及胞内分布规律 ,探讨其与心肌损伤的关系。　方法　制作严重烫伤大鼠模型 ,于伤后 1、3、6、12、2 4h取其全血及心肌组织标本 ,并取正常大鼠的相应标本为对照。常规检测各血清标本中肌酸激酶同工酶 MB(CK MB)的活力 ;采用Western印迹法 ,检测各心肌组织标本中MAPKs各成员的活化情况 ,并对其组织切片行免疫组化染色。　结果　伤后 p38激酶、ERK均发生活化并伴核转位 ,以 1、3、6h最明显 (P <0 .0 1) ;伤后 1～ 2 4hJNK均未见活化。血清CK MB含量于伤后 3h升高 ,12h达高峰 (P <0 .0 5～ 0 .0 1)。　结论　p38激酶和ERK信号通路可能在严重烧伤后早期心肌细胞发生的损伤性反应中起重要作用 ,而前者可能是烧伤引发心肌损伤的主要信号转导通路之一。