Combining proteomics and lipid analysis to unravel Confidor stress response in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model for studying the influence of different stress factors on eukaryotic cells. In this work we used the pesticide imidacloprid, in the Confidor formulation, as the stress factor and analyzed its influence on the metabolic activity, proteome and lipid content and composition of Saccharomyces cerevisiae yeast. During the cultivation of yeast, the lowest recommended application dose of Confidor (0.025%, v/v) was added to the growth media and its influence on the mitochondria, cytosol with microsomes, and the whole yeast cells was monitored. The results show that under the stress provoked by the toxic effects of Confidor, yeast cells density significantly decreased and the percentage of metabolically disturbed cells significantly increased comparing with untreated control. Also, there was a downregulation of majority of glycolytic, gluconeogenesis, and TCA cycle enzymes (Fba1, Adh1, Hxk2, Tal1, Tdh1,Tdh3, Eno1) thus providing enough acetyl‐CoA for the lipid restructuring and accumulation mechanism since we have found the changes in the cell and mitochondrial lipid content and FA composition. This data suggest that lipids could be the molecules that orchestrate the answer of the cells in the stress response to the Confidor treatment.     

大肠侧向发育型肿瘤细胞株双向电泳技术成功建立

目的:建立侧向发育型肿瘤细胞(LST)株双向电泳技术.方法:提取LST细胞株总蛋白质,采用不同pH范围IPG胶条进行LST细胞株总蛋白质双向电泳,并对实验步骤进行一系列的调整优化.结果:获得了较为清晰的LST细胞株双向电泳图谱,采用pH 3～10线性IPG胶条分离出1356±43个蛋白质斑点;pH 4～7 IPG胶条两种上样量(250μg和150μg)分别分离出了1285±51和989个蛋白质斑点.结论:通过相关条件的调整优化,成功建立了双向电泳技术,为研究LST特殊性生物学行为相关蛋白质奠定了坚实的基础.     

人胃黏膜组织蛋白质组二维电泳图像的获得

目的初步建立并优化人类胃黏膜组织蛋白质组分析所需的双向凝胶电泳技术，提高其分辨率及重复性。方法刮取手术胃黏膜组织，对以固相pH梯度为第一向的双向凝胶电泳的关键因素与环节，如样品处理、上样量、电泳参数、凝胶浓度和SDS凝胶电泳染色方法等进行一系列的优化。以固相pH梯度——IPG胶条(pH=3—10)进行第一向等电聚焦，以SDS均一胶(13％)的垂直电泳为第二向。结果成功地得到了胃黏膜组织的双向凝胶电泳图谱。     

桂枝汤对发热大鼠下丘脑蛋白质组影响初探

目的：初步观察桂枝汤对发热大鼠下丘脑组织中蛋白质的差异表达，为进一步探讨桂枝汤的解热分子机制奠定基础。方法：应用蛋白质组技术，对酵母发热大鼠模型和桂枝汤治疗组下丘脑组织中蛋白质表达进行比较，观察其差异点。结果：发现模型和治疗大鼠下丘脑蛋白表达有明显差异，主要是蛋白表达量的增加和减少以及个别蛋白等电点的改变，其中在给予桂枝汤后有8种蛋白（Mr/pI：28.9kD/4.47，24.4kD/6.28，25.4kD/6.39，25.9kD/6.39,17.6中7.38,17.2kD/7.43，24.9kD/7.39，26.9kD/7.59）表达增强，6种蛋白Mr/pI：14.3kD/4.83，28.5kD/4.39，16.2kD/4.11，15.3kD/6.7，30.5kD/7.09，30.5kD/7.13）表达降低，1种蛋白（Mr/pI：14.8kD/5.4）等电点发生了改变，差异蛋白数量约占可分辨蛋白点的2.3％，本次实验未发现新的差异蛋白点。结论：桂枝汤的解热作用可能与改变下丘脑组织中某些蛋白质的表达及修饰有关。     

桂皮醛对发热大鼠下丘脑蛋白质组双向凝胶电泳分析

目的：观察桂皮醛对酵母致热大鼠的解热作用及其时下丘脑蛋白质组的影响。方法：采用双向凝胶电泳技术和计算机辅助图像分析方法，对蛋白质进行分离，以模型组作为参考胶进行匹配。结果：口服给予桂皮醛后时发热大鼠有解热作用，双向电泳图像分析显示桂皮醛组与模型组匹配率为86％，多个蛋白质点表达量有不同程度的改变。结论：桂皮醛时酵母致热大鼠有解热作用，其差异表达的蛋白质点可能参与了解热过程。     

叶绿酸抑制细胞恶性转化的蛋白表达差异分析

目的：采用蛋白质组学技术分析叶绿酸抑制细胞恶性转化的蛋白差异表达,探讨叶绿酸抗细胞转化的分子机制.方法：一步法分别提取二羟环氧苯并芘（BPDE）诱导的转化细胞B-1和叶绿酸与BPDE共处理的抗转化细胞CB-1总蛋白,应用二维凝胶电泳技术和相应软件ImageMaster 2D3．10分析两种细胞的蛋白质组变化．基质辅助激光解吸电离-飞行时间-质谱（MALDI-TOF-MS）结合数据库检索鉴定部分表达有差异的蛋白斑点。结果：与B-1细胞相比．CB-1细胞中有47个蛋白斑点出现表达差异，其中19个蛋白斑点表达升高．28个蛋白斑点表达降低；质谱分析初步鉴定出5个差异蛋白斑点。结论：叶绿酸抑制细胞恶性转化可引起大量蛋白表达差异．这些差异蛋白可能参与了叶绿酸抗细胞转化的过程。     

Urine proteomes of healthy aging humans reveal extracellular matrix (ECM) alterations and immune system dysfunction

Aging is a complex physiological process that poses considerable conundrums to rapidly aging societies. For example, the risk of dying from cardiovascular diseases and/or cancer steadily declines for people after their 60s, and other causes of death predominate for seniors older than 80 years of age. Thus, physiological aging presents numerous unanswered questions, particularly with regard to changing metabolic patterns. Urine proteomics analysis is becoming a non-invasive and reproducible diagnostic method. We investigated the urine proteomes in healthy elderly people to determine which metabolic processes were weakened or strengthened in aging humans. Urine samples from 37 healthy volunteers aged 19–90 years (19 men, 18 women) were analyzed for protein expression by liquid chromatography–tandem mass spectrometry. This generated a list of 19 proteins that were differentially expressed in different age groups (young, intermediate, and old age). In particular, the oldest group showed protein changes reflective of altered extracellular matrix turnover and declining immune function, in which changes corresponded to reported changes in cardiovascular tissue remodeling and immune disorders in the elderly. Thus, urinary proteome changes in the elderly appear to reflect the physiological processes of aging and are particularly clearly represented in the circulatory and immune systems. Detailed identification of “protein trails” creates a more global picture of metabolic changes that occur in the elderly.     

A clinical study on plasma biomarkers for deciding the use of adjuvant corticosteroid therapy in bronchopulmonary dysplasia of premature infants

Objective: The study was designed to investigate some plasma markers which help us to decide the use of adjuvant corticosteroid therapy in bronchopulmonary dysplasia (BPD) of premature infants.Methods: Thirty BPD infants were treated by dexamethasone. Among these cases, dexamethasone was significant effective in 10 cases, and no significant effective in 20 cases. These patients were divided into two groups as the significant effect (SE) group (n=10) and the non-significant effect (NE) group (n=20) according to the curative effect of dexamethasone. Fifteen non-BPD infants with gestational age and gender matching were selected as the control group. Plasma samples before and after dexamethasone treatment were collected from three infants chosen randomly from SEG for the data-independent acquisition (DIA) analysis. ELISA was further used to detect the levels of differential proteins LRP1 and S100A8 in all individuals, including SE, NE and control groups.Results: DIA analysis results showed that after dexamethasone treatment, there were a total of 52 plasma proteins that showed significant differences, of which 43 proteins were down-regulated and 9 proteins were up-regulated. LRP1 and S100A8 were two plasma proteins that were significantly changed after dexamethasone treatment. Compared with the control group, plasma LRP1 was significantly increased in BPD. Interestingly, the plasma concentration of LRP1 in the NE group was significantly higher than that in the SE group. S100A8, as an indicator of plasma inflammation, was significantly higher in BPD than the control group. Unlike LRP1, there was no significantly difference between the SE and NE group (P=0.279) before dexamethasone treatment.Conclusion: Elevated plasma LRP1 and S100A8 in BPD infants are two indicators that correlated with the efficacy of dexamethasone, and might be used as biomarkers for deciding the use of adjuvant corticosteroids therapy in the BPD.     

缬沙坦治疗原发性高血压患者血浆蛋白质组研究

目的探讨缬沙坦治疗前后原发性高血压(EH)患者血浆蛋白质组差异。方法筛选健康正常血压者及EH患者,设为正常对照组(NT组)、EH患者(EH组)、缬沙坦治疗伴有心肌肥厚EH患者(EHT1组)和缬沙坦治疗不伴有心肌肥厚EH患者(EHT2组),每组21例。分离血浆,去除高丰度蛋白质,制备蛋白质样品,使用二维电泳结合基质辅助激光解析电离飞行时间质谱技术,进行各组血浆蛋白质组的分析和鉴定。结果与NT组比较,EH组和EHT1组的血肌酐显著升高(P<0.05),EH组、EHT1组和EHT2组收缩压显著升高(P<0.05)。与NT组比较,EH组血浆中存在22个差异表达的蛋白质点,其中16个表达量上调,6个下调;EHT1组存在12个,其中10个上调,2个下调;EHT2组存在11个,9个上调,2个下调。与EH组比较,EHT1组存在17个差异表达的蛋白质点,其中4个上调,13个下调;EHT2组存在19个,其中5个上调,15个下调(P<0.05)。结论 EH患者血浆蛋白质组与正常血压者之间存在明显差异,缬沙坦治疗可以缓解但并不能完全恢复血浆蛋白质组的水平。