Comparative proteomics analysis of human lung squamous carcinoma

Two-dimensional polyacrylamide gel electrophoresis (2-DE) profiles of human lung squamous carcinoma tissue and paired surrounding normal bronchial epithelial tissue were compared. Selected differential protein-spots were identified with peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. Well-resolved and reproducible 2-DE patterns of both the tumor and the normal tissues were acquired. The average deviations of spot position were 0.873+/-0.125mm in IEF direction and 1.025+/-0.213mm in SDS-PAGE direction, respectively. For the tumor tissues, a total of 1349+/-67 spots were detected and 1235+/-48 spots were matched with an average matching rate of 91.5%. For the corresponding normal tissues, a total of 1297+/-73 spots were detected and 1183+/-56 spots were matched with an average matching rate of 91.2%. A total of 1069+/-45 spots were matched between the tumor and the normal tissues. Forty differential proteins between tumor and normal tissues were characterized. Some proteins were the products of oncogenes and others were involved in the regulation of cell cycle and signal transduction. These data are valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis, establishing human lung cancer proteome database and screening molecular marker to further study human lung squamous carcinoma.     

Tegumental glucose permeability in male and female Schistosoma mansoni

Tegumental hexose transporters have been kinetically characterized in mated and separated male and female Schistosoma mansoni 8-12 wk postinfection. Significant gender-specific differences in Km and Vmax were observed. In mated males, the estimated constants (mean +/- SE) were: Km = 0.63 +/- 0.31 mM, Vmax = 0.93 +/- 0.44 nmol/mg worm water/min, and the Kd = 0.25 +/- 0.09 microliter/mg worm water/min. In mated females the kinetics were: Km = 0.99 +/- 0.40 mM, Vmax = 1.22 +/- 0.42 nmol/mg worm water/min, and Kd = 0.60 +/- 0.14 microliter/mg worm water/min. The influx of 2-deoxy-D-glucose and 3-O-methylglucose has been similarly characterized; these analogs share the same glucose transporter in male and female schistosomes. 2-Deoxy-D-glucose has a higher affinity, and 3-O-methylglucose a lower affinity, than does glucose. Because mated male schistosomes supply glucose to female partners, similarities between the free glucose concentration of the male and the affinity of the transporter determined for mated female schistosomes suggest that male-to-female transfer may be a potentially rate-limiting step in glucose utilization by the female. Permeability x surface are (PS) products and Vmax/Km ratios were significantly elevated in mated schistosomes, suggesting that the transporter is primarily localized to the dorsal surface of the male. Gender- and mating-specific analyses of PS products indicate that tegumental permeability to glucose is significantly increased in mated schistosomes, and compares very favorably to that of the host liver.     

Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments

Maturation and maintenance of normal reproductive function in female Schistosoma mansoni require a permanent association with the male, but the nature of this relationship is not well understood. The regional localization of a stimulatory factor in the male and its target in the female were investigated. Unisexual female and mature male worms were transected into segments of various lengths. Various combinations of transected male and female segments and intact worms were transferred to the mesenteric veins of recipient hamsters and were also maintained in vitro. In hamsters and in vitro, pairing took place between intact worms of each sex and segments of the other, and between segments of both sexes. The majority of female worms and segments so paired showed some reproductive development, as assessed by vitelline gland differentiation. In intact unisexual females paired with small male segments, vitelline gland development was limited to that portion of the worm that had been held by the male. Worm segments continued to display normal body contractions throughout 24 days of in vitro maintenance and morphological integrity was retained. It is concluded that 1) in the absence of a functioning gut, worm segments can survive for prolonged periods on nutrients absorbed through the tegument; 2) worm pairing, male stimulation, and the female developmental response are independent of central nervous control by the cerebral ganglia; 3) males have no centralized localization for the female-stimulating factor; 4) vitelline gland differentiation in the female requires local stimulation through male contact, and this is not propagated throughout the worm.     

Schistosoma japonicum translationally controlled tumour protein,which is associated with the development of female worms,as a target for control of schistosomiasis

Schistosomiasis is a globally important helminthic disease of both humans and animals, and is the second most common parasitic disease after malaria. Although praziquantel is extensively used for treatment of parasitic diseases, drug resistance has been reported. Therefore, new drugs and effective vaccines are needed for continuous control of schistosomiasis. Eggs produced by schistosomes are responsible for the occurrence and spread of schistosomiasis. Revealing the reproductive mechanism of schistosomes will help to control this disease. In this study, the proteomic profiles of single-sex infected female worms and bisexual infected mature female worms of Schistosoma japonicum at 18, 21, 23 and 25 days p.i. were identified with isobaric tags for relative quantitation-coupled liquid chromatography–tandem mass spectrometry. Differentially expressed proteins were subsequently used for bioinformatic analysis. Six highly expressed differentially expressed proteins in mature female worms were selected and long-term interference with small interfering RNA (siRNA) was conducted to determine biological functions. SiRNA against S. japonicum translationally controlled tumour protein (SjTCTP) resulted in the most significant effect on the growth and development of MF worms. Sjtctp mRNA expression gradually increased over time with a high level of expression maintained at 25–42 days p.i., while levels were significantly higher in mature female worms than male and SF worms. The subsequent animal immune protection experiments showed that recombinant SjTCTP (rSjTCTP) reduced the number of adults by 44.7% (P < 0.01), average egg burden per gram of liver by 57.94% (P < 0.01), egg hatching rate by 47.57% (P < 0.01), and oviposition of individual females by 43.16%. rSjTCTP induced higher levels of serum IgG, IL-2, and IL-10 in mice. Collectively, these results show that SjTCTP is vital to reproduction of female worms and, thus, is a candidate antigen for immune protection.     

Seeking transformation markers: an analysis of differential tissue proteomes on the rice germplasm generated from transformation of Echinochloa crusgalli genomic DNA

The transformation of distally related genomic DNAs into plant was proposed as a novel technique to breed new cultivars. For example, a restorer rice line, RB207, was successfully developed and stabilized through the transformation of genomic DNAs of Echinochloa crusgalli (E. crusgalli) into a rice line, R207. Although the phenotypes of this variant line are apparently different from its receptor, the molecular bases are not elucidated yet. Herein, we have systematically studied the differential proteomes from the tissues of E. crusgalli, R207, and RB207 in an attempt to find an explanation regarding the phenotypic changes of RB207. The 2-DE method was employed to separate the leaf and embryo proteins of these plants followed by protein identification with mass spectrometry. In the leaf, 953 +/- 15, 1084 +/- 11, and 1091 +/- 11 silver-stained spots were detected, whereas in the embryo, 986 +/- 3, 884 +/- 10, and 892 +/- 14 spots were found from E. crusgalli, R207, and RB207, respectively. In comparison to the 2-DE images of the two rice lines, which showed many similarities, the ones of the E. crusgalli and rice were found to be so different that they were incomparable. There were some differentially expressed 2-DE spots between the two rice cultivars, 72 in leaf and 53 in embryo, respectively. The results of protein identification suggested that, regardless of leaves or embryos, none of the E. crusgalli genes were encoded in the new rice cultivar, RB207. The fact that 60% of the differentially expressed spots between R207 and RB207, however, were verified as the proteins involved in metabolism and photosynthesis makes a rather convincing argument that the DNA fragments transferred from E. crusgalli to rice are responsible for exerting the unknown influence to the expression of rice genes.     

Proteomic comparison of two-dimensional gel electrophoresis profiles from human lung squamous carcinoma and normal bronchial epithelial tissues

Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733+/-0.101 mm in IEF direction, and 0.925+/-0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241+/-88 spots were detected, 987+/-65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190+/-72 spots were detected, and 875+/-48 spots were matched with an average matching rate of 73.5%. A total of 864+/-34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduction. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.     

Failure of in vitro-cultured schistosomes to produce eggs: how does the parasite meet its needs for host-derived cytokines such as TGF-β?

When adult schistosome worm pairs are transferred from experimental hosts to in vitro culture they cease producing viable eggs within a few days. Female worms in unisexual infections fail to mature, and when mature adult females are separated from male partners they regress sexually. Worms cultured from the larval stage are also permanently reproductively defective. The cytokine transforming growth factor beta derived from the mammalian host is considered important in stimulating schistosome female worm maturation and maintenance of fecundity. The means by which schistosomes acquire TGF-β have not been elucidated, but direct uptake in vivo seems unlikely as the concentration of free, biologically active cytokine in host blood is very low. Here we review the complexities of schistosome development and male–female interactions, and we speculate about two possibilities on how worms obtain the TGF-β they are assumed to need: (i) worms may have mechanisms to free active cytokine from the latency-inducing complex of proteins in which it is associated, and/or (ii) they may obtain the cytokine from alpha 2-macroglobulin, a blood-borne protease inhibitor to which TGF-β can bind. These ideas are experimentally testable.     

Differential proteomics analysis of female and male adults of Angiostrongylus cantonensis

In this study, we identified the differentially expressed proteins of female and male adults of Angiostrongylus cantonensis through differential proteomics. We extracted and purified total proteins from male and female adults, separated proteins by two-dimensional difference gel electrophoresis (2D-DIGE) in pH 4-7, analyzed the gel images by DeCyder 7.0 software, and sacrificed the infected rats to count the number of male and female adults. It was found 28 protein spots that were differentially expressed; seven protein spots were then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Five proteins were up-regulated and two proteins down-regulated in male adults compared with female adults. Three of the five up-regulated proteins with known functions ascribed to them were identified as galectin-1, proteasome alpha subunit and peroxiredoxin. The two down-regulated proteins were identified as indoleamine dioxygenase like-myoglobin and galectin. Furthermore, the female was significantly greater than male adults (P<0.01) in the rats. The findings demonstrate the differences in protein expression profiles and the ability to survive in the final host between female and male adults of A. cantonensis, and may provide a theoretical basis to study their developmental biology further.     

Extended survival and reproductive potential of single-sex male and female Schistosoma japonicum within definitive hosts

Schistosomiasis is caused by dioecious helminths of the genus Schistosoma. Recent work indicated that unpaired female and male schistosomes can survive within their definitive host for at least 1 year, although the viability or fertility of these worms after subsequent pairing remained untested. We performed two experiments on laboratory mice, one with female Schistosoma japonicum exposure first and male schistosomes second and another vice versa. After surviving as single-sex unpaired forms for up to 1 year, 58.5% of male and 70% of female schistosomes were able to mate and produce viable eggs. This highlights an additional biological challenge in achieving elimination of schistosomiasis.     

Molecular and enzymatic characterization of Schistosoma mansoni thioredoxin peroxidase

The ability of Schistosoma mansoni to escape oxidative damage from immune system-generated reactive oxygen intermediates has been extensively documented. The limiting step in the parasite's detoxification process appears to be at the level of hydrogen peroxide neutralization. In the present study, the possible role of a novel class of antioxidant enzymes, thioredoxin peroxidase (TPx), in hydrogen peroxide neutralization by schistosomes was investigated. An expressed sequence tag was characterized from the Schistosoma Genome Initiative with high similarity to TPx from other organisms. The gene encodes a polypeptide containing 2 conserved active-site cysteines and flanking amino acids, and 60-70% identity with previously characterized TPx proteins. Recombinant schistosome TPx was enzymatically active and found to have thioredoxin-dependent hydrogen peroxide reducing activity of 4500 nmol hydrogen peroxide/min/mg protein. Native TPx activity was determined to be 48.1 nmol hydrogen peroxide/min/mg protein in adult worm homogenates compared with 46.9 for glutathione peroxidase. TPx activity was precipitated from adult worm homogenates with antibodies prepared against the recombinant protein. Western blotting with antibodies made against recombinant protein showed that TPx was expressed in both male and female adult worms. This is the first demonstration of a TPx activity in schistosomes and our results suggest that TPx plays a significant role in schistosome-host interactions.     

短乳杆菌DM9218核苷酸代谢过程中的蛋白组学分析

目的探讨短乳杆菌DM9218在核苷酸代谢过程中的蛋白表达差异。方法分别提取DM9218菌株与底物(肌苷+鸟苷)反应前后的菌体蛋白,利用蛋白双向凝胶电泳(2-DE)技术,找出该菌株与底物反应前后的差异蛋白质点,选取其中差异变化较大的蛋白点进一步做蛋白质谱分析。结果 2-DE分析显示两样品蛋白点主要分布在等电点4~9和分子量11~90 kD范围内,将所得的蛋白点结合其蛋白得率、浓度、储存蛋白含量进行比较,得到匹配的蛋白点数为732个。从中选取14个差异显著的蛋白点进行质谱分析,质谱结果显示所选取蛋白质点主要与物质代谢、能量转换及基因水平转录和翻译等生物学功能密切相关。结论本研究为后期分析研究短乳杆菌DM9218在核苷酸代谢过程中蛋白的表达奠定了基础。     

大豆雄性不育系与其保持系不同器官蛋白质比较

采用双向凝胶电泳技术对大豆质核互作雄性不育系NJCMS2A及其保持系NJCMS2B的种子、叶片和花药等不同器官蛋白质进行比较分析.结果显示,不育系NJCMS2A与其保持系NJCMS2B的花药2-DE图谱间存在较多差异表达蛋白点,种子2-DE图谱间仅有少量差异表达蛋白点,而叶片2-DE图谱间基本没有差异表达蛋白点.结果表明,不育基因表达具有时空性和器官特异性,与育性有关的蛋白主要在花药中表达.     

柔嫩艾美耳球虫地克株利抗药株与敏感株孢子化卵囊的蛋白质差异分析

利用双向电泳技术,对本实验室诱导保存的柔嫩艾美耳球虫地克株利抗药株与敏感株的蛋白质表达图谱进行差异比较和分析,发现两者之间差异有5个蛋白质斑点,利用MALDI_TOF_TOF质谱技术对其中4个差异明显的蛋白质斑点进行分析鉴定,获得4个明确的肽质量指纹图谱,通过在NCBInr数据库中检索分析,确定了其中2个蛋白质分别为球虫子孢子表面抗原TA4和热休克蛋白Hsp70 ,另外两种为真核细胞的功能蛋白。上述蛋白的鉴定将对球虫的抗药性产生机理和柔嫩艾美耳球虫地克株利抗药株的分子标志物提供了研究方向。     

The defense response of germinating maize embryos against fungal infection: a proteomics approach

Pathogen attack on plants results in numerous host-specific biochemical responses, the activation of some of them being critical for the ability of the plant to withstand disease. We have used high-resolution two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify proteins that are differentially expressed in response to fungal infection in maize embryos. Differential spots corresponding to induced or repressed proteins were apparent in silver stained 2-DE gels of proteins extracted from sterile and fungal-infected germinating embryos. Selected spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and nanospray ion-trap tandem mass spectrometry. Among the proteins induced in response to infection are proteins involved in protein synthesis, or in protein folding and stabilization, as well as proteins involved in oxidative stress tolerance. Additionally, the accumulation of specific pathogenesis-related proteins in tissues of the fungal-infected germinating embryos was studied by 2-DE and immunoblotting.     

Proteomics Associated with Virulence Differentiation of Curvularia lunata in Maize in China

One-dimensional electrophoresis (1-DE) of proteins, two-dimensional electrophoresis (2-DE) of proteins and cloning of cDNA sequence were used to study the virulence differentiation of Curvularia lunata (Wakker) Boed. isolated from maize (Zea maydis L.) in China. From 1-DE gel profiles of proteins, 110 reproducible bands were separated from six isolates of C. lunata CX-3, SD-6, C-152, C107-1, DD-60 and W-18. Sixty-eight bands (61.82%) were polymorphic,suggesting huge biodiversities among the isolates. All isolates for the experiment were clustered into three groups consisting of different virulent types by coefficient value of 0.605. Group 1, consisting of CX-3, SD-6 and C-152 with high virulence displayed more protein bands than Groups 2 and 3, consisting of C107-1 and DD-60 with low virulence. Proteomics approaches based on 2-DE techniques were applied to identify specific proteins associated with the virulence differentiation in CX-3 and DD-60. A total of 423 protein spots were separated. Out of them 75 specific protein spots were displayed in 2-DE gels. Among them 28 protein spots were unique in CX-3 and eight in DD-60, and 39 protein spots were shown on both 2-DE gels but expressed differently in intensity. Twenty protein spots including three unique protein spots and 17 differentially expressed protein spots (more than two-fold DD-60) in CX-3 were further identified with MALDI-TOF MS/MS. Results indicated that most of the identified proteins were found to be associated with virulence differentiation, metabolisms, stress response and signal transduction.One of them was identified as Brn1 protein, which had been reported to be related to melanin biosynthesis and the virulence differentiation in fungi. Combined with our previous findings, we assumed that Brn1 protein and its regulating products might be involved in the virulence differentiation of C. lunata. Consequently, we cloned a Brn1 cDNA fragment and aligned it with the fragments in other fungi. Results indicated that the 633-bp sequence of Brn1 cloned in C. lunata was highly homological with the compared fungi. Further work for the exact gene roles of Brn1 in our case is underway.     

家蚕蛹期雌雄生殖腺蛋白质双向电泳比较分析

分析家蚕Bombyx mori雌雄生殖腺细胞蛋白质,有利于发现性别分化相关的功能蛋白质,探讨生殖腺发育相关基因的表达调控机理。本研究利用蛋白质双向凝胶电泳和图像分析技术,分析家蚕蛹期第2天的雌雄生殖腺细胞蛋白质。结果表明: 在雄蚕生殖腺蛋白质电泳图谱中共检测到435个蛋白斑点,其中特异性蛋白斑点73个,占总蛋白斑点数的16.8％;雌蚕生殖腺的电泳图谱中有417个蛋白斑点,其中特异性蛋白斑点55个,占总蛋白斑点数的13.2％。雌雄能匹配的蛋白斑点有362对,匹配率达85.0％。     

Comparative proteomics analysis of serum proteins in ulcerative colitis patients

In the present study, we investigated the differentially expressed proteins associated with ulcerative colitis (UC) using proteomic methods. Two-dimensional electrophoresis (2-DE) technology was performed to separate the total proteins of ulcerative tissues from those of the normal tissues of UC patients. PDQuest software was applied to analyze the obtained 2-DE images. Candidate protein spots between the two groups were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and bioinformatics analysis. The well resolution and reproducible 2-DE patterns of UC and normal tissues were established. Of the 12 differentially expressed proteins, 9 were successfully identified, of which 6 proteins were up-regulated including apolipoprotein C-III, haptoglobin, receptor tyrosine kinase, aldehyde reductase, pericentriolar material 1, and heat shock factor protein 2, and 3 were down-regulated including keratin, filamin A-interacting protein 1, and tropomyosin 3. These identified proteins were related to hormonal modulation, immune response, oxidative stress, and signal conduction. The 2-DE protein expression profile of the UC tissues displays an obvious difference from that of the normal controls. Various proteins may be involved in the occurrence of UC.