肝细胞癌患者血清蛋白质组成分的双向凝胶电泳-飞行时间质谱分析

目的　分析肝细胞癌患者与正常人血清双向凝胶电泳的差异蛋白质 ,寻找肝癌早期血清学诊断的可能指标。方法　固相 pH梯度双向凝胶电泳分离肝细胞癌患者及正常人血清总蛋白。银染显色 ,PDQuest 2DE软件分析 ,对差异蛋白质点用基质辅助激光解吸电离飞行时间质谱 (MALDI TOF MS)测定其胶内酶解后的肽质指纹图谱 ,用PepIdent软件查询SWISS PROT数据库。 结果　获得了重复性较好的双向电泳银染图谱。图像分析检测 3块胶平均匹配的点数占平均蛋白点数的匹配率是 70 2 %。等电聚焦方向的平均偏差为 (1 0 2± 0 2 2 )mm ,十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)方向的平均偏差为 (0 97± 0 14 )mm。将 2 3个差异点进行胶内原位酶解 质谱指纹图分析 ,获得 15张肽质指纹图 ,查询数据库进行了初步鉴定。结论　固相 pH梯度双向凝胶电泳分离人血清总蛋白获得重复性较好的结果 ,但仍需进一步探索如何去除高丰度已知蛋白及脂类等物质的方法 ,以便得到分辨率更高的双向电泳银染图谱。MALDI TOF MS肽质指纹图分析鉴定的结果为人肝细胞癌血清学诊断指标的筛选提供了依据     

MPO、MMP-2和MMP-9在兔房颤模型心房肌中的表达变化

目的:观察兔房颤模型心房肌组织髓过氧化物酶(MPO)、基质金属蛋白酶(MMP)-2和MMP-9的表达,并探讨三者与房颤时心房结构重构的关系。方法:20只新西兰大白兔,开胸后于左心房植入起搏电极,随机分为2组:快速心房起搏组(RAP组)以600 min-1的频率快速起搏心房3周;假手术组(sham组)不予起搏。起搏前、后行超声心动图检查评价心房和心室的结构和功能,行心房burst刺激检测房颤诱发率;起搏后采用Masson染色评价心房的间质纤维化程度,采用RT-q PCR和Western blot检测心房MPO、MMP-2和MMP-9 mRNA和蛋白的表达水平。结果:起搏3周后,与sham组相比,RAP组兔左心房明显扩张伴收缩功能障碍,左心室的结构和功能变化不明显;RAP组房颤诱发率和间质纤维化百分比均明显增加,且心房MPO、MMP-2、MMP-9 mRNA和蛋白的表达明显增加。结论:持续快速心房起搏兔房颤模型会出现明显心房结构重构,心房MPO、MMP-2和MMP-9表达上调可能是其潜在的分子机制。     

Identification of proteins differentially expressed in the conventional renal cell carcinoma by proteomic analysis

Renal cell carcinoma (RCC) is one of the most malignant tumors in urology, and due to its insidious onset patients frequently have advanced disease at the time of clinical presentation. Thus, early detection is crucial in management of RCC. To identify tumor specific proteins of RCC, we employed proteomic analysis. We prepared proteins from conventional RCC and the corresponding normal kidney tissues from seven patients with conventional RCC. The expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The overall protein expression patterns in the RCC and the normal kidney tissues were quite similar except some areas. Of 66 differentially expressed protein spots (p<0.05 by Student t-test), 8 different proteins from 11 spots were identified by MALDI-TOF-MS. The expression of the following proteins was repressed (p<0.05); aminoacylase-1, enoyl-CoA hydratase, aldehyde reductase, tropomyosin alpha-4 chain, agmatinase and ketohexokinase. Two proteins, vimentin and alpha-1 antitrypsin precursor, were dominantly expressed in RCC (p<0.05).     

Upregulation and dephosphorylation of cofilin: modulation by CD44 variant isoform in human colon cancer cells

Colon cancer is among the leading causes of cancer death in North America. Dysregulation of the crypt homeostasis is evident in the early stage of colon cancer. Moreover, cytoskeletal rearrangement of actin also plays a crucial role in morphological changes during apoptosis. CD44, an adhesion and anti-apoptotic molecule is overexpressed in colon cancer and is known to interact with certain cytoskeletal proteins. In this study, we used the human colon cancer cell line SW620, which does not express CD44 but stably transfected with standard, 3-10v and 8-10v variant isoforms of CD44. By two-dimensional isoelectric focusing (2DIEF), we found an increasing concentration of a 21-kDa protein in SW620 colon cancer cells transfected with CD44 3-10v, as compared to cells transfected with an empty vector. Mass spectrometry (MS) and proteomic analysis of this protein identified the peptide fragment (YALYDATYETK) of 11 amino acids in length spanning residue 82 to 92 of cofilin, a widely distributed 21-kDa actin-modulating protein. Western blot analysis of lysates from cells expressing CD44 variant isoforms 3-10v had increased level of expression of cofilin compared to vector control consistent with our finding by 2DIEF. Also, immunocytochemistry showed that cofilin expression in colonic epithelial cells was greater in cells transfected with CD44 3-10v, as compared to vector controls. We observed that the phosphorylated form of cofilin is downregulated in cells expressing the 3-10v isoform of CD44 both by Western blot and immunocytochemistry. Cofilin expression is thus mechanistically associated with CD44 expression and its 3-10v isoform. Dephosphorylation of cofilin could bring about directional motility of cells that could have important implications to the proliferation and motility of colonic epithelial cells in cancer.     

The role of lysosomal rupture in neuronal death

Apoptosis research in the past two decades has provided an enormous insight into its role in regulating cell death. However, apoptosis is only part of the story, and inhibition of neuronal necrosis may have greater impact than apoptosis, on the treatment of stroke, traumatic brain injury, and neurodegenerative diseases. Since the “calpain–cathepsin hypothesis” was first formulated, the calpain- and cathepsin-mediated regulation of necrotic cascades observed in monkeys, has been demonstrated to be a common neuronal death mechanism occurring from simpler organisms to humans. However, the detailed mechanism inducing lysosomal destabilization still remains poorly understood. Heat-shock protein-70 (Hsp70) is known to stabilize lysosomal membrane and protect cells from oxidative stress and apoptotic stimuli in many cell death pathways. Recent proteomics approach comparing pre- and post-ischemic hippocampal CA1 neurons as well as normal and glaucoma-suffered retina of primates, suggested that the substrate protein upon which activated calpain acts at the lysosomal membrane of neurons might be Hsp70. Understanding the interaction between activated calpains and Hsp70 will help to unravel the mechanism that destabilizes the lysosomal membrane, and will provide new insights into clarifying the whole cascade of neuronal necrosis. Although available evidence is circumferential, it is hypothesized that activated calpain cleaves oxidative stress-induced carbonylated Hsp70.1 (a major human Hsp70) at the lysosomal membrane, which result in lysosomal rupture/permeabilization. This review aims at highlighting the possible mechanism of lysosomal rupture in neuronal death by a modified “calpain–cathepsin hypothesis”. As the autophagy–lysosomal degradation pathway is a target of oxidative stress, the implication of autophagy is also discussed.     

Identification of 11 new exoproteins in Corynebacterium pseudotuberculosis by comparative analysis of the exoproteome

This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS^E), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellular proteins for C. pseudotuberculosis, were identified. In the present work, we have used 2D gel electrophoresis to resolve the extracellular proteins of both strains, which were then digested with trypsin, analyzed by MALDI-TOF/TOF and identified with the software MASCOT^®. A total of 45 extracellular proteins of C. pseudotuberculosis were identified by this approach. The comparative analysis between the strains 1002 and C231 identified 13 and 3 strain-specific proteins, respectively, 11 of which are novel. These newly identified proteins may play an important role in the physiology and virulence of C. pseudotuberculosis.