Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry

Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round- headed and normal spermatozoa. Two-dimensional （2-D） fluorescence difference gel electrophoresis （DIGE） coupled with mass spectrometry （MS） was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry （MS/MS） exhibited significant changes （paired t-test, P 〈 0.05） in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.

Determination of urinary 8-hydroxy-2′-deoxyguanosine by capillary electrophoresis with molecularly imprinted monolith in-tube solid phase microextraction

Urinary 8-hydroxy-2 -deoxyguanosine(8-OHdG) is an excellent marker of oxidative DNA damage.In this study,employing guanosine as dummy template a novel molecularly imprinted(MIP) monolithic capillary column had been synthesized,and that was used as medium of in-tube solid phase microextraction(SPME).Coupled with capillary electrophoresis-electrochemical detection(CE-ECD),the system of extraction and detection of 8-OHdG in urinary sample had been developed.Because of its greater phase ratio combined with conv...

Dye-Sensitized Solar Cells with An Electrophoresis-Deposited Layer of {001} Exposed Nanosheet-Based Hierarchical TiO_(2) Spheres

Anatase Ti0_(2) nanosheet-based hierarchical spheres(HSs)with nearly 100%exposed{001}facets were synthesized via a facile solvothermal process.Using these hierarchical spheres as a scattering layer on nanocrystaline TiO_(2)film,hi-layered dye-sensitized solar cells(DSSCs)have been fabricated by electrophoresis deposition method,which well preserved the fragile hierarchical structure.Owing to the superior dye adsorption and light scattering effect of HSs,an overall energy conversion efficiency of 7.38%is achieved,which is 26%higher than that of nanoparticle-based photoanode.

Purification of Pseudomonas sp. Lipase by Continuous Elution Electrophoresis Based on Pb^(2+) Precipitation Method

A Pb 2+ precipitation method was designed to get rid of the impure proteins in a lipase. The results show that it was a simple way in the primary treatment of the crude samples and about 20% impure proteins were removed in the precipitation step. Further, continuous elution electrophoresis was also applied as a preparative technique for attaining the highly pure lipase. During the continuous elution electrophoresis, the enzyme was eluted as a single peak and 5.7-fold purification was achieved in a yield of 54.3%. The two steps finally yielded an electrophoretically homogeneous enzyme.

Electrophoresis Analysis on the Protein Expression in Heteromorphic Leaves of Populus euphratica Oliv.

[Objective] This study was to elucidate the cellular and molecular mechanism of the development of heteromorphic leaves of Populus euphratica Oliv. [Method] By employing SDS-PAGE and 2-demensional electrophoresis (2-DE) techniques,proteins in various heteromorphic leaves from the same adult tree of P. euphratica were isolated and separated to the electrophoresis technique suitable for the separation and analysis of proteins in leaves of P. euphratica tree. [Results] There were significant differences in the expressions of proteins in various heteromorphic leaves of P. euphratica tree. SDS-PAGE pattern showed that bands of proteins with molecular weight of 57.2,13.2,30.2,23.9 and 33.3 kDa were remarkably different. 2-D electrophoresis pattern presented that proteins in leaves of P. euphratica tree mainly belong to acidic proteins distributed at pH value of 5.0-6.5 and with molecular weight of 20-40 kDa; totally 73 different protein spots were observed,of which 51 were up expressed and other 22 were down expressed in the serrated ovate leaves. [Conclusion] Based on these results,we speculate that regulated gene expression in leaves of P. euphratica tree results in the generation of different shapes of leaves,in order to adapt to the surroundings better.

Preparation of protein samples for gel electrophoresis by sequential extraction

Since preparation and solubilization of protein samples are crucial factors in proteome research,the authors established a sequential extraction technique to prepare protein samples from the body wall of the 5th instar larvae of silkworm.Bombyx mori.Two kinds of protein samples were obtained from the body wall using the method.Between the two types of samples only about 15% proteins were identical;the majority were different,indicating that more species of proteins could be obtained with the sequential extraction method;which will be useful for preparation of protein samples for proteome study.

Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

Objective To identify the sperm membrane proteins that are associated with antisperm antibody Methods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody. Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5～6.2, 4.6,5.1,5.5～5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher. Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two- dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.

Screening of chemokine receptor CCR4 antagonists by capillary zone electrophoresis

CC chemokine receptor 4(CCR4)is a kind of G-protein-coupled receptor,which plays a pivotal role in allergic inflammation.The interaction between 2-(2-(4-chloro-phenyl)-5-{[(naphthalen-1-ylmethyl)-carbamlyl]-methyl-4-oxo-thiazolidin-3-yl)-N-(3-morpholin-4-yl-propyi)-acetamide(S009)and the N-terminal extracellular tail(ML40)of CCR4 has been validated to be high affinity by capillary zone electrophoresis(CZE).The S009 is a known CCR4 antagonist.Now,a series of new thiourea derivatives have been synthesized.Compared with positive control S009,they were screened using ML40 as target by CZE to find some new drugs for allergic inflammation diseases.The synthesized compounds XJH-5,XJH-4,XJH-17 and XJH-1 displayed the interaction with ML40,but XJH-9,XJH-10,XJH-I 1,XJH-12,XJH-13,XJH-14,XJH-3,XJH-8,XJH-6,XJH-7,XJH-15,XJH-16 and XJH-2 did not bind to ML40.Both qualification and quantification characterizations of the binding were determined.The affinity of the four compounds was valued by the binding constant,which was similar with the results of chemotactic experiments.The established CEZ method is capable of sensitive and fast screening for a series of lactam analogs in the drug discovery for allergic inflammation diseases.

镁合金表面TiO_(2)二维薄膜的制备及其耐腐蚀性能研究

由于镁是一种较为活泼的金属,容易被氧化,且生成的氧化膜不具备保护性,这会进一步加快镁的腐蚀速率。通过在镁合金表面制备一层保护膜来阻断其与腐蚀物质的接触,从而增强镁合金的耐腐蚀性。首先根据已有文献制备TiO_(2)胶体,然后利用电泳沉积法,以镁合金基样为阳极,在其表面制备TiO_(2)薄膜,电泳液为上述TiO_(2)胶体,并探究了制备过程中的各项实验参数(不同电泳时间和不同沉积时间)对样品的影响。采取XRD、SEM等测试方法对制备的TiO_(2)薄膜进行分析,并进一步测试样品的极化曲线并做出分析。通过一系列参数优化可知,最优参数为电泳电压20 V,沉积时间90 s,此时制备的TiO_(2)薄膜的腐蚀电位最正,为-1.26 V,同时腐蚀电流密度也最低,为4.21×10^(-6)A/cm^(2)。

Agronomic Trait and Protein Component of F_2 Hybrid Originated from Intergeneric Somatic Hybridization Between Triticum aestivum and Agropyron elongatum

Protoplasts derived from common wheat (Triticum aestivum L,. cv. Jinan 177) were fused with UV-treated protoplasts of Agropyron elongatum. (Host) Nevski by PEG method, and fertile asymmetric somatic hybrid plants resembling wheat morphology were obtained. The F-2 hybrid plants could be divided into 3 types according to their morphology. Type I hybrids had high and loosely standing stalks with big spikes and grains. Type ii hybrids were dwarf and compact in shape with high tillering ability and smaller spikes. Type III hybrids were similar to type I as a whole but had more compact and erect spikes. All the F-2 hybrid lines were superior to wheat in seed protein content, although some difference existed between themselves. Protein analysis of immature embryos and flag leaves from hybrids by two-dimensional electrophoresis showed that they possessed characteristic proteins of both parents and some new proteins. There existed also some different kinds of proteins in different lines.

Pb^(2+)对鱼肠DNA光谱特性的影响

利用紫外吸收光谱、荧光光谱、电泳手段研究了不同浓度Pb2 + 与鱼肠DNA的相互作用。紫外吸收光谱测试表明 :随着Pb2 + 的加入 ,DNA产生明显的减色效应 ,同时DNA的 2 0 7nm峰发生明显蓝移 ;荧光发射光谱结果表明 ,随着Pb2 + 的加入 ,DNA荧光发射强度逐渐降低 ,Pb2 + 在DNA上的结合位点数为 0 8个 ,Pb2 + 引起DNA的荧光猝灭属于静态猝灭 ,结合位点的结合常数分别为 6 0 8× 10 4和 2 82× 10 4L·mol-1;电泳结果表明 ,不同浓度的外源Pb2 + 处理未引起DNA的断裂。认为外源Pb2 + 对DNA的构象有一定的破坏作用 ,但并不引起DNA的断裂。

北京市大气气溶胶PM_(2.5)中水溶性有机酸的测定

采用毛细管电泳的方法对北京大气PM2 5中低分子量二元羧酸、醛酸、酮酸和甲磺酸等 1 0种水溶性有机酸进行了测定 .方法的定量检测限为 1 1 0～ 2 80 μg/L(提取液 ) ,峰面积的相对标准偏差为 1 7%～ 6 0 % ,标准工作曲线的线性相关系数为0 995～ 0 999.北京夏、秋、冬 3季 1 0种有机酸的总量平均分别为 72 1ng/m3 、36 2ng/m3 、32 5ng/m3 ,浓度水平总体上与国外大城市相当 .其中乙二酸的含量最为丰富 .通过各种有机酸浓度的季节变化规律 。

奶牛乳腺组织蛋白质样品的制备及2-DE图谱分析

本研究以奶牛乳腺组织为对象,采用了普通离心、超速离心、2-D clean-up kit和TCA/丙酮沉淀方法制备蛋白质样品,经二维凝胶电泳(2-DE)获得蛋白表达图谱。根据凝胶图谱上蛋白质斑点判断图谱的质量,并运用PDQuest7.4软件分析图谱。结果显示:普通离心法制备的蛋白样品的凝胶图谱蛋白质斑点较为清晰,但横向条纹较多;超速离心法纯化蛋白样品的图谱,蛋白斑点清晰且可检测到较多的斑点;TCA/丙酮沉淀和2-D clean-up kit方法纯化蛋白样品的凝胶图谱条纹减少,蛋白斑点清晰,但可检测到的斑点数量有所减少。研究表明,超速离心方法纯化的蛋白质样品适合建立奶牛乳腺组织的蛋白质表达图谱。

毛细管电泳免疫化学发光检测鼠血管平滑肌细胞中zmol骨形成蛋白-2

In this study, a capillary electrophoresis immunoassay(CEIA) method based on the enhanced chemiluminescence(CL) detection was developed. A horseradish peroxidase(HRP) label catalyzing the luminol/H 2O 2/p-iodophenol(PIP) reaction was performed, and the HRP was detected with detection limit(S/N=3) of 4.4 pmol/L(53 zmol), which is one of the highest sensitivity of HRP reported yet. The HRP was linked to bone morphogenic protein-2(BMP-2) in rat vascular smooth muscle(VSM) cells in noncompetitive format and first detected by CL. HRP-Ab 2-mAb-BMP-2 complexes were baseline separated from free HRP label in 3 min. The detection limit(S/N=3) of BMP-2 is 6.2 pmol/L(75 zmol). This technique has been applied to arteriosclerosis pathology research. The change of BMP-2 contentin VSM cells which were stimulated by angiotensin Ⅱ(AgⅡ) for different hours was investigated in the concentration range of 1.0-10.0 pmol/L. The results are in accord with that obtained by common used Pathology image analysis system.

针对bcl─2RNA核酶体外切割反应的琼脂糖凝胶电泳

目的：用琼脂糖凝胶电泳检测体外ｂｃｌ┐２ｃＤＮＡ和ＲＺＤＮＡ转录物与鉴定核酸的体外切割活性．方法：琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳．结果：琼脂糖凝胶电泳可见核酶和ｂｃｌ－２ＲＮＡ转录物及其切割ｂｃｌ－２ＲＮＡ后的产物．

2-DE技术中疏水性和碱性蛋白质的研究进展

双向凝胶电泳(2-DE)具有高分辨率、高通量等特点,已被广泛地用于蛋白质组的分离.但是它在分离疏水性蛋白质和碱性蛋白质时却遇到了极大的挑战.然而,疏水性与碱性蛋白质在全蛋白质中占相当大的比例,且具有很重要的生物学意义.因而,近年来,越来越多的研究者将目标瞄准这些蛋白质,并且取得了一些令人鼓舞的进展:用亚细胞预分离技术,顺序提取法等方法来富集疏水性蛋白质,用一些新的有效的增溶剂如硫脲,ASB-14等来改善疏水性蛋白质的溶解,应用这些技术2-DE可分辨出总平均疏水值达0.80的蛋白质;在碱性蛋白质分离方面,通过等电聚焦预处理,使用窄pH梯度胶条等大大地改善了碱性蛋白质在2-DE中的分离,能分辨出等电点达11.7的蛋白质.现对2-DE技术中疏水性和碱性蛋白质分离的研究进展进行综述.

以2-O-单取代羟丙基β-环糊精用于毛细管电泳手性分离添加剂

本文研究了一种新的环糊精衍生物2－O－单取代羟丙基β－环糊精做为毛细管电泳手性分离添加剂时，对3种碱性药物对映体；扑尔敏，麻黄碱，心得安的拆分性能。并与β－环糊精，2．6－O－二甲基β－环糊精，多取代2－O－羟丙基β－环糊精的手性识别性能做了对比。

2型糖尿病患者肠道拟类菌属和双歧杆菌属多样性的PCR-DGGE指纹图谱分析

目的分析2型糖尿病患者肠道拟类菌属和双歧杆菌属的多样性及其与健康个体的差异,探讨糖尿病与肠道优势菌属的相关性。方法收集2型糖尿病患者和健康志愿者粪样,提取样品中菌群总DNA,利用拟类菌和双歧杆菌16SrRNA种属特异性引物,进行PCR扩增,将产物用变性梯度凝胶电泳分离,获得肠道菌群分子指纹图谱,进行多样性和相似性等特征分析。结果糖尿病组和健康人组样品DGGE图谱多样性指数范围分别是:拟类菌属0.89(0.75～0.95),0.95(0.89～0.97);双歧杆菌属:0.85(0.75～0.94),0.87(0.73～0.93)。组内成对相似性系数累积曲线分析,拟类菌属:小于0.5的Cs值在糖尿病组占到总Cs值个数的85.83%,而健康人组只占到35.71%;双歧杆菌属:小于0.4的Cs值在糖尿病组占到总Cs值个数的90%,健康人组占到总Cs值个数的60.7%。结论糖尿病患者和健康人肠道拟类菌属和双歧杆菌属指纹图呈现个体特异性,糖尿病患者两种菌属多样性与健康个体无显著差异,而糖尿病患者个体之间的相似性较健康个体之间低。

2-甲基-β-环糊精作为手性选择剂对5种药物对映体的毛细管电泳拆分研究

目的以2-甲基-β-环糊精(2-O-methylated-β-cyclodextrin,2-O-M-β-CD)为手性添加剂,利用毛细管电泳法(capillary electrophoresis,CE)对消旋体药物甲溴后马托品、氧氟沙星、酮康唑、利阿唑和卡维地洛进行拆分研究。方法考察了背景电解质溶液的pH值、2-O-M-β-CD浓度、缓冲盐浓度对分离的影响,优化了分离条件。结果在最佳分离条件下,甲溴后马托品、氧氟沙星、酮康唑、利阿唑对映体达到完全分离,分离度分别为3.6、2.8、1.7、1.5;卡维地洛对映体分离度达到1.4。结论所建立的毛细管电泳法适于除卡维地洛外其余4种药物的对映体分离。

瑞香狼毒诱导HL-60细胞凋亡和调节SGC-7901细胞bcl-2蛋白表达(英文)

目的 探索瑞香狼素 (SC)抗肿瘤机制。方法 以 HL- 6 0和 SGC- 790 1为靶细胞 ,用 MTT比色法测定细胞增殖抑制 ,荧光显微镜观察凋亡细胞的形态学改变 ,DNA电泳和流式细胞仪检测DNA断裂 ,免疫组化检测 bcl- 2蛋白表达。结果 含 SC药物血清处理细胞 48h后 ,HL- 6 0细胞增殖呈剂量依赖性抑制 ,并表现出典型的凋亡细胞形态学改变及 DNA断裂 :即染色体聚集、核固缩、断裂及阶梯状 DNA电泳条带 ,G1 期前细胞从 11.7%增至 5 7.4% ;而 SGC- 790 1细胞 bcl- 2蛋白表达率从 78.3%下降到 32 .9%。结论 SC可诱导肿瘤细胞凋亡 ,降低 bcl- 2蛋白表达。