受低毒病毒调控的板栗疫病菌总蛋白质组学

【目的】为解析低毒病毒调控板栗疫病菌生理性状和致病性的机制,在总蛋白质组水平上寻找受病毒侵染调控的宿主蛋白及其编码基因。【方法】使用双向电泳方法对野生型菌株EP155和受低毒病毒感染的菌株EP713进行差异蛋白质组分析,同时,对差异表达蛋白质编码基因的mRNA水平进行定量分析。【结果】共找到71个因病毒侵染而发生差异表达的蛋白质点,分别属于58种不同蛋白质。以EP155为对照,表现为上调的19个,下调的52个,主要涉及能量代谢,蛋白质、核酸和碳水化合物代谢,信号传导以及压力应激和氧化还原反应。对10个受病毒调控的蛋白的编码基因进行了mRNA水平定量,其中7个基因受病毒感染后的mRNA水平变化与蛋白水平变化趋势一致,3个基因的mRNA水平变化与蛋白水平变化趋势不相符,表明低毒病毒对板栗疫病菌不同基因的调控可以发生在不同的调控层面上。【结论】低毒病毒的侵染弱化了宿主TCA循环的能量流动,调控了宿主体内的甲基化过程及真菌致病因子的表达。     

利用TMT标记结合2DLC-MS/MS技术对水牛卵母细胞体外成熟前后差异蛋白质组的分析

本试验利用TMT标记并结合二维高效液相色谱/串联质谱联用的研究策略对水牛卵母细胞成熟前后差异蛋白质组进行分析。试验首先收集水牛成熟前卵母细胞和成熟后卵母细胞,分别提取卵母细胞这两个时期的蛋白质,酶解蛋白后进行TMT标记,其中TMT-126标记成熟后的肽段,TMT-129标记成熟前的肽段,标记后采用强阳离子交换柱对酶解得到的肽段进行分离,接着进行nano LC分离,质谱分析采用在线连接电喷雾串联Orbitrap的方法,最后使用SEQUEST软件进行数据库搜索,采用生物信息学方法对鉴定得到的差异蛋白质进行初步分析。根据定量差异倍数≥2即认为蛋白表达存在差异,鉴定出卵母细胞成熟前高表达的蛋白有18种,成熟后高表达的蛋白有26种。对这些差异蛋白进行生物信息学分析表明,利用TMT标记并结合二维高效液相色谱/串联质谱联用的研究策略可以有效地分离和鉴定水牛卵母细胞成熟前后的蛋白质。本试验发现可能与水牛卵母细胞成熟相关的标志性蛋白:调控细胞凋亡蛋白(BCL2L10)、胎球蛋白(AHSG)、伴侣蛋白(Erp29),这可能为今后研究水牛卵母细胞成熟前后的蛋白质表达变化规律提供了试验依据。     

REV感染DF-1细胞分泌外体的生物信息学分析

该研究探讨了REV感染DF-1细胞分泌外体携带表达差异的蛋白质和微RNA(micro RNA,mi RNA),及其基因功能和参与的信号通路,为病毒致病机制研究提供了基础。通过蛋白组学和转录组学检测技术,筛选病毒感染组与对照组DF-1细胞分泌外体的差异蛋白质和mi RNA,并利用在线软件数据库对其进行GO功能富集和KEGG信号通路分析。结果筛选到101个差异表达蛋白质,其中56个表达上调,45个表达下调,共参与155条信号通路,参与蛋白质最多的是肿瘤相关通路;并筛选到3个表达上调的mi RNA,其中mi RNA-155、mi RNA-146a-3p编码的靶蛋白(整合蛋白)与差异蛋白质肌动蛋白相关2/3复合体(actinrelated 2/3 complexs,Arp2/3)共同参与肌动蛋白细胞骨架信号通路;差异蛋白质及mi RNA编码靶基因均含有病毒成分,并参与细胞信号转导、免疫、黏附、运动、生物调节等过程。该研究结果表明,REV感染DF-1细胞来源外体表达差异的蛋白质和mi RNA及其参与的生物学过程和信号通路与肿瘤形成密切相关,外体途径可能在REV致瘤机制中具有重要作用。     

丝状真菌表面展示技术研究进展

丝状真菌表面展示技术是将表达的目的蛋白固定在丝状真菌细胞表面的一项新兴基因工程技术。丝状真菌具有极强的蛋白质分泌能力和良好的蛋白质翻译后加工能力,因而越来越多的丝状真菌表面展示技术得到开发和应用。本文就丝状真菌表面展示系统的研发和应用进展进行综述,并介绍与该系统构建密切相关的丝状真菌的细胞壁组成、锚定蛋白和遗传转化方法等技术。     

板栗疫病菌cpomt基因的功能

板栗疫病菌(Cryphonectria parasitica)是引起板栗疫病的一种丝状子囊菌。UV57是一个不支持病毒复制且致病力丧失的C.parasitica紫外诱变突变株。前期蛋白质组研究结果显示,蛋白86233(一种甲基转移酶)只在UV57中出现,而在野生型对照株EP155中没有检测到。为研究编码86233蛋白的cpomt基因的功能,本研究通过同源重组方法成功构建了缺失突变体Δcpomt及其互补转化株。与野生型EP155相比,Δcpomt菌株生长缓慢,色素分泌减少,产孢量降低,菌丝形态异常,对休眠板栗树枝的致病性显著降低。而在互补转化株Δcpomt-com中,这些表型及致病力变化均可以恢复到野生型水平。cpomt基因的缺失对低毒病毒CHV1-EP713的复制累积量没有影响,但导致抗逆相关基因G-α,产孢基因CLS-32,色素合成酶基因PKS转录水平明显下调。本研究为阐明甲基转移酶在病原真菌中的作用提供了新的知识。     

肠道病毒71型感染前后宿主细胞蛋白质组的二维液相色谱分离和比较

以肠道病毒71型及其宿主细胞为研究主体,建立了一种二维液相色谱分离和分析比较病毒感染前后细胞蛋白表达谱的方法。该方法以高效液相色谱(HPLC)为技术平台,对细胞裂解物先后进行一维色谱聚焦分离和二维反相色谱分离。利用ProteoVue软件将二维色谱数据转换成模拟胶图,再利用DeltaVue软件对感染前后的宿主蛋白表达谱进行比较和分析,找出差异蛋白。二维液相色谱分离法能够根据蛋白的等电点和疏水性建立精确的细胞蛋白表达图谱,每0．2个pH为一个收集区段,在pH8．5～3．9的范围内可见蛋白条带约1200条。该方法良好的重现性、自动化以及结果分析的简易化,使之在细胞表达谱差异显示中的应用潜力巨大,并且为研究病毒与宿主相互作用提供了新的方法和思路。     

金鱼雌核发育单倍体发育过程中的比较蛋白质组学研究

前期已有工作发现在金鱼雌核发育单倍体中一些与发育调控相关的重要蛋白质表达受阻导致单倍体的发育畸形。为了进一步阐明单倍体的发育机制,我们共收集了3个不同发育时期金鱼单倍体胚胎（HE-1、HE-2、HE-3）进行雌核发育单倍体的差异蛋白质组研究。研究采用二维凝胶电泳进行分离,利用PDQuest软件进行图谱分析,质谱分析初步鉴定到了15个差异蛋白质。这些蛋白质在金鱼雌核发育单倍体的发育中起着重要作用,为进一步阐明单倍体的发育机制奠定了良好的基础。     

草菇子实体不同成熟阶段的比较蛋白质组学分析

采用iTRAQ标记结合二维液相色谱串联质谱技术对草菇不同成熟阶段的差异蛋白质组进行研究。首先将提取的草菇不同成熟阶段蛋白样品进行SDS-PAGE分析,其次将经二维液相色谱串联质谱技术获取的串联质谱数据通过MASCOT软件搜库,之后对鉴定蛋白质数据进行了KEGG代谢通路分析。试验共计获得2 335个不同肽段, 鉴定到1 039个蛋白质,其中1 030个蛋白质具有定量信息。与蛋形期相比,在伸长期和成熟期阶段显著上调蛋白质85个,下调蛋白质103个。KEGG代谢通路分析结果显示,草菇不同成熟阶段中的68个差异蛋白质可定位于4种伞菌目模式真菌（灰盖鬼伞、双色蜡蘑、可可丛枝病菌和裂褶菌）的45个不同生物代谢途径,全景展示出草菇成熟阶段差异表达蛋白质定位的代谢网络。结果表明,iTRAQ标记技术结合二维液相色谱串联质谱可对不同生长发育时期的草菇蛋白样品进行有效地分离和鉴定。     

应用蛋白质组学技术筛选宫颈癌临床分期相关蛋白

目的：通过比较早期宫颈癌和中一晚期宫颈癌的差异表达蛋白,以发现与宫颈癌临床分期相关蛋白,为临床预后和复发的预测提供新指标。方法：收集早期宫颈癌和中一晚期宫颈癌组织标本,提取组织总蛋白进行二维凝胶电泳,选择部分差异表达蛋白进行MALDI—TOF质谱分析和生物信息学分析进行蛋白质鉴定,进一步应用WestemBlot和免疫组织化学技术检测部分差异表达蛋白的表达情况。结果：建立了早期宫颈癌组和中一晚期宫颈癌组的二维凝胶电泳图谱,其中两组的平均蛋白质点数分别为1098±23、1142±21,通过进行质谱分析和生物信息学查询,与早期宫颈癌组比较,鉴定了在中一晚期组下调的蛋白4个。包括HemoglobinsubunitbetaHB、caspase-14、galectin-7、CK19;中-晚期组上调的蛋白8个,包括NMP238、HSP70、Calmodulin-like5、S100A9。WesternBlot和免疫组化检测结果均显示S100A9在中-晚期组中的表达高于早期组,CK19在早期组中的表达高于中-晚期组。结论：早期宫颈癌和中-晚期宫颈癌中存在着差异表达蛋白,这些蛋白可能成为宫颈癌预后和复发预测的生物标志物。     

无核荔枝胚胎发育时期蛋白质图谱分析

通过二维聚丙烯酰胺凝胶电泳(2DE)以及计算机辅助的图像分析技术,对荔枝开花后20d的正常与败育胚蛋白质图谱进行了初步分析。结果表明,正常胚总蛋白质斑点数为129,败育胚总蛋白质斑点数为130,其中24个蛋白质点在两种胚中的表达丰度没有明显变化,35个蛋白质点在表达丰度上有明显差异,55%的蛋白则发生了蛋白质缺失、增加以及位置改变等变化。这两种蛋白质组的表达差异说明了胚内蛋白质成分在其败育过程中发生了变化,这些蛋白可能参与了胚败育的调节和控制。     

Management of time by the cell and by the organism

Time-dependent regulations of cells and organisms can be analysed at different levels. One of these levels is the periodicity of cell functions such as cell division, metabolic processes (generation of ATP by glycolysis or oxidative mitochondrial processes) and the biosynthesis of cell constituents. Studies carried out on unicellular eukaryotes revealed the periodic, oscillatory nature of most of these processes. Time constants of these reactions vary from nanoseconds to hours-days, necessitating coupling mechanisms. Comparative studies revealed the coupling of the rapid processes (mitochondrial ATP generation) to the slower rhythms of the biosynthetic processes of macromolecules. Adenine nucleotides are involved in the coupling mechanisms between rapid and slow processes ("the slow dance of life to the music of time"). The mechanisms underlying these rhythmic processes involve either key allosteric regulatory enzymes (PFK for glycolysis) or "desensitization" of receptors by phosphorylation-dephosphorylation. At the organismic level the study of rhythmic processes is illustrated by the periodicity of heart beats, shown to exhibit multifractality, following apparently the formalism of deterministic chaos. Another example is the rhythmic oscillatory discharges of neuronal networks. The existence of subrhythmes mostly of epigenetic nature, facilitated probably the progressive adjustment of cells during evolution to the slow increase of day time since the separation of the moon from the earth. We analysed the mechanisms underlying the decline of these processes during aging. Loss of receptors or/and their uncoupling from their transmission pathway appear to be involved in most of these processes of decline. One conclusion of this review is the importance of epigenetic mechanisms both in the genesis and in the decline of these rythmic processes involved in time keeping by the cell.     

Amelioration of the teratogenicity of cadmium by the metallothionein induced by bismuth nitrate

The participation of maternal hepatic metallothionein (MT) in the amelioration of cadmium teratogenicity in mice was examined. Pretreatment with bismuth nitrate (subcutaneously) ameliorated the teratogenicity, including exencephaly and abnormalities of the axial skeleton, caused by a single intraperitoneal injection of cadmium sulfate. Pretreatment with bismuth nitrate for 3 days induced MT drastically in maternal liver and kidney. Six and 24 hr after the injection of cadmium sulfate, the accumulation of maternal hepatic cadmium increased and that in the decidua, including embryos, decreased after pretreatment with bismuth nitrate. Mouse embryos on day 7 of gestation were cultured for 48 hr. Exposure to cadmium sulfate in vitro induced unfused brain fold, which corresponds to exencephaly in vivo. From the in vitro experiment, it was suggested that the teratogenicity of cadmium on day 7 of gestation is a direct action against the mouse embryo. In the present experiment it was suggested that pretreatment with bismuth nitrate induced maternal hepatic and renal MT; cadmium was therefore trapped and detoxicated, and consequently embryos were exposed to a lower concentration of cadmium.     

Inhibition by diphenols of the induction of micronuclei by gamma-radiation

A study was made of the influence of diphenols (for instance, resorcinol, hydroquinone, and pyrocatechin) on gamma-radiation induction of micronuclei (1.5 Gy). The position of the diphenol molecule hydroxyl group (the isomeric effect) was shown to influence their antimutagenic activity. This antimutagenic effect of the diphenols is associated with their ability to produce semiquinone and quinone forms which are peculiar for the process of oxidation of pyrocatechin (ortho-) and hydroquinone (para-) as opposed to resorcinol (meta-position of the hydroxyl group).     

To live or die by the sword: the regulation of apoptosis by the proteasome

The proteasome has been implicated in the control of apoptosis by modulating the levels of both pro- and antiapoptotic molecules. A recent study published in the April 9th issue of Molecular Cell reveals that caspase-dependent inactivation of the proteasome can amplify the activation of apoptosis.     

Interference by Tris buffer in the estimation of protein by the Lowry procedure

Tris buffers were found to distort the measurement of protein by the Lowry method both by decreasing chromophore development with protein and by contributing blank color. Tris at an assay concentration of 0.37 mm markedly affects measured results. Similar Tris effects were observed at all wavelengths between 450 and 800 nm and with diverse protein samples. The distortion due to Tris is not correctable by simple blank correction, but it can be overcome by incorporating the same amount of Tris in the standards used. The distortion at Tris concentrations <0.15 mm appears to be within tolerable limits. No interference or distortion was observed with sodium phosphate buffer to an assay concentration of 40 mm. An automated Lowry procedure is also presented which gives excellent correlation with the manual method and an average coefficient of variation of <4%.