盆腔器官脱垂患者阴道组织比较蛋白质组学研究

目的:建立盆腔器官脱垂(pelvic organ prolapse,POP)患者及非脱垂对照者阴道组织蛋白质双向凝胶电泳图谱,并结合质谱技术分析其差异表达蛋白质,寻找POP疾病相关蛋白,以进一步阐明POP的发病机制。方法:收集绝经后年龄相匹配的POP组和对照组尿道周围阴道壁组织标本各10份,提取组织总蛋白,进行双向凝胶电泳(two-dimensional gel electro-phoresis,2-DE)得到凝胶图谱,采用Image Master 5.0 2D图象分析软件进行匹配和差异分析,识别两组间差异表达蛋白。将部分差异蛋白点胶内原位酶解后进行MALDI-TOF-MS分析,获取肽质量指纹图谱并进行生物信息学检索鉴定蛋白质。结果:得到了分辨率高,重复性好的POP组和对照组阴道壁组织蛋白双向凝胶电泳图谱,POP组的平均蛋白质点数为(871±96)个,正常对照组为(856±89)个。通过比较分析,差异表达蛋白质点数为27个。选取其中8个具有明显差异的蛋白点进行质谱鉴定,初步鉴定出5种蛋白质,其中2个蛋白质在POP组表达上调,3个蛋白质表达下调。结论:两者间存在一些差异表达的蛋白质,为阐明POP发病机制打下了坚实的基础。     

Proteomic alterations in heat shock protein 27 and identification of phosphoproteins in ascending aortic aneurysm associated with bicuspid and tricuspid aortic valve

Whether or not there are molecular differences, at the intra- and extracellular level, between aortic dilatation in patients with bicuspid (BAV) and those with a tricuspid aortic valve (TAV) has remained controversial for years. We have performed 2-dimensional gel electrophoresis and mass spectrometry coupled with dephosphorylation and phosphostaining experiments to reveal and define protein alterations and the high abundant structural phosphoproteins in BAV compared to TAV aortic aneurysm samples. 2-D gel patterns showed a high correlation in protein expression between BAV and TAV specimens (n=10). Few proteins showed significant differences, among those a phosphorylated form of heat shock protein (HSP) 27 with significantly lower expression in BAV compared to TAV aortic samples (p=0.02). The phosphoprotein tracing revealed four different phosphoproteins including Rho GDP dissociation inhibitor 1, calponin 3, myosin regulatory light chain 2 and four differentially phosphorylated forms of HSP27. Levels of total HSP27 and dually phosphorylated HSP27 (S78/S82) were investigated in an extended patient cohort (n=15) using ELISA. Total HSP27 was significantly lower in BAV compared to TAV patients (p=0.03), with no correlation in levels of phospho-HSP27 (S78/S82) (p=0.4). Western blots analysis showed a trend towards lower levels of phospho-HSP27 (S78) in BAV patients (p=0.07). Immunohistochemical analysis revealed that differences in HSP27 occur in the cytoplasma of VSMC's and not extracellularly. Alterations in HSP27 may give early evidence for intracellular differences in aortic aneurysm of patients with BAV and TAV. Whether HSP27 and the defined phosphoproteins have a specific role in BAV associated aortic dilatation remains to be elucidated.     

Studies on the venom proteome of Bothrops asper: Perspectives and applications

Bothrops asper is responsible for the vast majority of snakebite accidents in Central America and several studies have demonstrated that specific toxic and enzymatic activities of its venom vary with the geographic origin and age of the specimens. Variability in venom proteins and enzymes between specimens from the Caribbean and the Pacific versants of Costa Rica has been reported since 1964. Recently, we performed a comparative proteomic characterization of the venoms from one population of each versant. Proteins belonging to several families, including disintegrin, phospholipases A₂, serine proteinases, C-type lectins, CRISP, l-amino acid oxidase, and Zn²⁺-dependent metalloproteinases show a variable degree of relative occurrence in the venoms of both populations. The occurrence of prominent differences in the protein profile between venoms from adults and newborns, and among venom samples from individual specimens of the same region or developmental stage, further demonstrated the existence of geographic, ontogenetic and individual variability in the venom proteome of this species. These findings provide new insights towards understanding the biology of B. asper, contribute to a deeper understanding of the pathology induced by its venom and underscore the importance of the use of venoms pooled from specimens from both regions for producing antivenom exhibiting the broadest cross-reactivity. Furthermore, knowledge of the protein composition of B. asper venom paves the way for detailed future structure–function studies of individual toxins as well as for the development of new protocols to study the reactivity of therapeutic antivenoms.     

应用SELDI-TOF-MS技术建立胃癌筛选血清蛋白质指纹图谱模型

目的 应用SELDI蛋白质芯片检测胃癌患者血清蛋白质指纹图谱,筛选候选肿瘤标志物以建立诊断模型,并探讨其诊断早期胃癌的临床意义.方法 表面加强激光解吸电离-飞行时间质谱(SEL-DI-TOF-MS)技术及其配套蛋白质芯片检测40例胃癌患者和30例健康人的血清蛋白质组图谱,运用判别分析处理数据筛选标志物并建立诊断模型.结果 8592m/z、13725m/z、8678m/z等3个蛋白质峰组合所构建的诊断模型能达到鉴别胃癌患者和健康人的最佳诊断效果,灵敏度为90%(36/40),特异性为86.5%(26/30).结论 该方法 在胃癌的诊断尤其是早期诊断、术前分期及候选肿瘤标志物筛选等方面具有一定价值,值得进一步研究.     

Cerebellar vermis proteome of chronic alcoholic individuals

Background: Cerebellar changes are commonly associated with alcoholism and chronic alcohol consumption can produce profound impairments in motor functioning and various aspects of cognition. Although the mechanisms underlying alcohol-induced changes in the cerebellar vermis are poorly understood, observations in the alcoholic vermis are thought to be consequential to common alcohol-related factors, particularly thiamine deficiency. Methods: In the present study, we used a proteomics-based approach to compare protein expression profiles of the cerebellar vermis from human alcoholic individuals (both neurologically uncomplicated and alcoholic individuals complicated with liver cirrhosis) and healthy control brains. This article complements our recent studies performed on alcoholic prefrontal gray and white matter and splenium of the corpus callosum (CC). Results: Like the CC study, several liver cirrhosis-specific proteins were identified in the vermis, perhaps indicating the effects of liver dysfunction in this brain region. Among other protein expression changes observed are disturbances in the levels of thiamine-dependent enzymes. A derangement in energy metabolism perhaps related to thiamine deficiency seems to be important in both alcoholic groups, even where there are no clinical or pathological findings of Wernicke-Korsakoff syndrome. Conclusions: These results suggest that clinically and pathologically uncomplicated alcoholic cases may not in fact be "uncomplicated," as at the proteome level we seem to be isolating the confounding effects of nutritional deficiencies and liver dysfunction and perhaps their role in alcohol-related vermis damage. Together, these results indicate that the alcohol-related pathology of the vermis is more multifactorial than other brain regions examined previously (prefrontal region and CC splenium).     

Antioxidative action of royal jelly in the yeast cell

Royal jelly is a bee product, secreted from the hypopharingeal and mandibular glands of worker bees. There are many reports on pharmacological activities of royal jelly in experimental animals, but there are few about its antioxidative properties connected to aging. The aim of the work was to investigate the antioxidative action of royal jelly in the cell of the yeast Saccharomyces cerevisiae as a model organism. Yeast was cultivated in YEPD medium enriched with different concentrations of royal jelly like 1, 2 and 5 g/L. Yeast growth was monitored by measuring optical density. At different time points cell energy metabolic activity was measured using the cell energy metabolism indicator resazurin, and 2',7'-dichlorofluorescein was applied to estimate intracellular oxidation. Additionally, protein profile of cell extract was analyzed by 2-D electrophoresis. Results showed that royal jelly decreased intracellular oxidation in a dose dependent manner. Additionally it affected growth and cell energy metabolic activity in a growth phase dependent manner. Protein profile analysis showed that royal jelly in the cell does not act only as a scavenger of reactive oxygen species, but it also affects protein expression. Differentially expressed proteins were identified.     

Differences in expression of retinal proteins between diabetic and normal rats

AIM： To compare and identify the differences in expression of retinal proteins between normal and diabetic rats, and to analyze the molecular pathogenetic mechanisms of retinal diseases caused by diabetes.
METHODS： Changes in protein expression of retinal tissues from diabetic and normal rats were observed using 2-dimensional polyacrylamide gel electrophoresis （2-DE）. Some protein spots exhibiting statistically significant variations （P 〈 0.05） were selected randomly and identified by tandem mass spectrometry and analyzed by bioinformatics.
RESULTS： 2-DE showed that the expression was upregulated in 5 retinal proteins, down-regulated in 23retinal proteins, and disappeared in 8 retinal proteins.Eight spots were identified from the 36 spots by tandem mass spectrometry （MS/MS） and analyzed by bioinformatics. Guanylate kinase 1, triosephosphate isomerase 1, ATP synthase subunit d, albumin and dimethylarginine dimethylaminohydrolase 2 played an important role in signal transduction. Triosephosphate isomerase 1, crystallin alpha B, ATP synthase subunit d and peroxiredoxin 6 were involved in energy metabolism of retinal tissues. Guanylate kinase 1 played an important role in photoexcitation of retinal rod photoreceptor cells.Whether crystallin beta A1 plays a role in diabetic retinas is unknown so far.
CONCLUSION： There are differences in expression of retinal proteins between diabetic and normal rats.These proteins may be involved in the mechanisms and prognosis of retinal diseases caused by diabetes.     

肾母细胞瘤患儿血清蛋白质标记物的筛选及鉴定

目的 筛选肾母细胞瘤患儿特异性血清蛋白质标记物并对其进行鉴定,以确定其作为肾母细胞瘤血清学诊断和预后监测的特异标志物.方法 应用表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)技术检测肾母细胞瘤患儿手术前后及正常小儿的血清蛋白质组,筛选差异蛋白质峰,并对目标蛋白质进行分离纯化、酶解,采用液质联用串联质谱(LC-MS/MS)分析,用SEQUST检索程序查询美国Bioworks公司提供的蛋白质序列数据库.结果 经SELDI-TOF-MS技术筛选出m/z位于6455.5和6984.4的蛋白质标志物在肾母细胞瘤组低表达(表达强度为1029±364、297±126),正常小儿组高表达(2108 ±837、753±226),差异均有统计学意义(均P＜0.01);m/z位于9190.8的蛋白质标志物在肾母细胞瘤术前组低表达(283±154),术后组和正常小儿组高表达(5974 ±657、6231±519)差异均有统计学意义(均P＜0.01).对m/z位于6455.5和9190.8的标志物进行鉴定,结果分别为载脂蛋白CⅢ和触珠蛋白.结论 检测血清中载脂蛋白CⅢ和触珠蛋白含量可能成为肾母细胞瘤的血清学诊断、恶性度分级和预后监测指标,值得进一步研究与应用.     

同卵双胞胎食管癌患者的血清蛋白组学研究

目的 观察河南食管癌高发区同卵双胞胎食管癌患者与高发区散发食管癌患者血清蛋白的表达,筛选食管鳞癌相关血清标记物.方法 收集3对同卵双胞胎、5例散发食管癌患者的血清,采用磁珠蛋白组技术对3对同卵双胞胎中的食管癌患者和10对健康同卵双胞胎(对照组)的血清进行蛋白组对比.结果 在3对同卵双胞胎中的3例食管癌患者中,共同表达的差异蛋白为16个.3对同卵双胞胎中的3例食管癌患者和20对健康同卵双胞胎对比差异蛋白为5905.29、5967.68、1779.04 Da(P＜0.05),5例散发食管癌与10对健康人对比差异蛋白为1779.17、1929.57、1866.43、1881.41、5095.48 Da(P＜0.05).结论 应用磁珠蛋白技术在同卵双胞胎中筛选出的差异蛋白可能成为食管癌筛查的血清蛋白标志物.