An Overview of the Medical Applications of Marine Skeletal Matrix Proteins

In recent years, the medicinal potential of marine organisms has attracted increasing attention. This is due to their immense diversity and adaptation to unique ecological niches that has led to vast physiological and biochemical diversification. Among these organisms, marine calcifiers are an abundant source of novel proteins and chemical entities that can be used for drug discovery. Studies of the skeletal organic matrix proteins of marine calcifiers have focused on biomedical applications such as the identification of growth inducing proteins that can be used for bone regeneration, for example, 2/4 bone morphogenic proteins (BMP). Although a few reports on the functions of proteins derived from marine calcifiers can be found in the literature, marine calcifiers themselves remain an untapped source of proteins for the development of innovative pharmaceuticals. Following an overview of the current knowledge of skeletal organic matrix proteins from marine calcifiers, this review will focus on various aspects of marine skeletal protein research including sources, biosynthesis, structures, and possible strategies for chemical or physical modification. Special attention will be given to potential medical applications and recent discoveries of skeletal proteins and polysaccharides with biologically appealing characteristics. In addition, I will introduce an effective protocol for sample preparation and protein purification that includes isolation technology for biopolymers (of both soluble and insoluble organic matrices) from coralline algae. These algae are a widespread but poorly studied group of shallow marine calcifiers that have great potential for marine drug discovery.     

应用SELDI-TOF-MS技术对患脂肪肝奶牛血浆差异蛋白的分离鉴定及生物学分析

【目的】脂肪肝是围产期奶牛主要代谢性疾病之一,50%产后奶牛受其影响。产后奶牛生产力降低、繁殖性能低下、免疫功能减弱、肝功能衰竭和过早死亡都与脂肪肝有关。表面增强激光解析/电离飞行时间质谱（SELDI-TOF-MS）是一种对生物样本蛋白质研究的新型高敏感性蛋白质组学方法,文章旨在探讨脂肪肝奶牛血浆蛋白质组学特征。【方法】于黑龙江某集约化养牛场选择产后7-28d,1-2胎次荷斯坦奶牛40头。清晨空腹尾静脉采血10mL,应用肝素抗凝（150U）迅速离心（3 000 r/min,5 min）分离血浆,置于-80℃冰箱冷冻保存。依据血浆甘油三酯（TG＞0.20 mmol·L^-1）, 血浆β-羟丁酸（BHBA＞1.2mmol·L^-1）浓度和临床症状,将其分为试验组（T）20头和健康对照组（C）20头。应用SELDI-TOF-MS技术测得血浆蛋白质谱,利用Ciphergen Protein Chip Software (Version 3.1.1)对在试验组和对照组所有样品中得到的峰图进行分析,得到原始数据。将两组之间的数据峰值做wilcoxon sum rank test统计检验,用计算出的P值判断峰在两组中是否有显著差异。以0.01作为P值的阈值,筛选出P值小于0.01的差异峰。在得到的差异峰中,将差异峰的m/z值与Swissport蛋白数据库中多肽的理论荷质比 (mass to charge ratio,m/z)进行比较,找到最相似的蛋白作为差异峰可能检测蛋白的预测结果。【结果】与对照组相比,试验组奶牛血浆蛋白质谱存在39个差异峰,在得到的39个差异峰中,将差异峰的m/z值与Swissport蛋白数据库中多肽的理论荷质比值进行比较,最终获得26个可预测的差异峰,预测蛋白11种。结果显示,相对于C组,11种预测蛋白在T组中均表达下调。通过生物信息学（Network,GO,Pathway）分析,应用Cytoscape软件对获得的11种蛋白进行牛类基因网络搜索,得到Networks分析结果图。在数据库中搜索到了试验结果中的淀粉样肽前体蛋白(amyloid precursor protein, APP)和纤维蛋白原α链(fibrinogen alpha chain, FGA)两种差异蛋白,并得到其相关的Networks分析结果。应用Cytoscape软件中BinGO插件对11种蛋白进行GO分析,并得到GO分析结果。对这些蛋白质经软件搜库得到已经注释的差异蛋白有9种,分别为FGA、血清淀粉样蛋白A (serum amyloid A protein, SAA)、血浆蛋白酶c1抑制剂（plasma protease c1 inhibitor, SERPING1 or C1INH）、血清载脂蛋白-CⅢ（apolipoprotein c-Ⅲ,ApoCⅢ）、海帕西啶（hepcidin, HAMP）、骨桥蛋白（osteopontin, OPN or SPP1）、 甲状腺素运转蛋白（transthyretin, TTR）、胱蛋白酶抑制剂C （cystatin-c, CysC or CST）、神经分泌血管生长因子蛋白（neurosecretory protein, VGF）。应用KEGG（Kyoto Encyclopedia of Genes and Genomes）pathway数据库搜索。搜索结果显示只有6种蛋白质可在数据库中搜到,分别是FGA、SERPING1、APO-CⅢ、APP、CysC、SPP1。并得到其相关的Pathway分析结果。这些蛋白可能是与奶牛脂肪肝发病机制相关的物质。【结论】应用SELDI-TOF-MS技术有效的分离了健康牛与患病牛之间的血浆差异表达蛋白,均在肝脏代谢或脂肪肝疾病发生发展过程中起重要的调节作用,对探究奶牛脂肪肝发病机理及其对机体生物学功能的影响具有重要的理论价值。差异蛋白对奶牛脂肪肝的发病机理的影响尚待进一步研究。     

iTRAQ技术及其在水稻蛋白质组学中的应用研究进展

同位素标记相对和绝对定量技术(iTRAQ)是一种新型的高通量的蛋白质测序技术,其通过与高精度的质谱仪串联,可同时对多达8个样品进行定性与定量分析,测定蛋白范围广泛、检测限低、分析结果可靠、精度高;目前已广泛应用于植物体的结构、功能、表达等方面的研究。水稻是人类的主要粮食作物,也是首个完成基因组测序的谷类模式植物,在蛋白质组水平上研究水稻的生长发育、调控机制具有重要意义。综述了iTRAQ的原理、操作流程和优缺点,重点就iTRAQ技术在水稻组织器官、亚细胞水平、逆境胁迫、激素诱导、突变体等方面的蛋白质组学研究进行了分析,并对iTRAQ技术在水稻蛋白质组学上的应用研究进行了展望。     

蛋白质组学在油料作物中的应用研究进展

为蛋白质组学技术在油料作物中的深入应用提供参考,对蛋白质组学在花生、大豆、油菜、向日葵、棉籽、芝麻、橄榄、蓖麻等常见油料作物器官与组织、亚细胞、响应逆境及其他条件下的应用研究进行了综述,并对油料作物蛋白质组学在植物生长发育、代谢调控等生命活动规律方面的应用研究进行了展望。     

奶牛乳房炎相关功能基因组学与蛋白质组学研究进展

功能基因组学与蛋白质组学是在基因组和蛋白质整体水平上分析基因功能,研究技术包括差异显示反转录PCR、表达序列标签、基因表达系列分析、生物芯片、RNA干涉、生物信息学、二维凝胶电泳(2-DE)、质谱(MS)等.综述了功能基因组学和蛋白质组学及其研究技术在奶牛乳房炎相关的基因表达、抗性候选基因鉴定、病原微生物蛋白质组学、基因治疗、新诊断靶标筛选等的应用研究进展及展望.     

龙眼胚性培养物高分辨率蛋白质双向电泳技术

试验以龙眼胚性培养物为材料,通过对小型垂直板双向电泳和固相pH梯度凝胶双向电泳比较分析,进行龙眼胚性培养物高分辨率蛋白质双向电泳技术方法的研究.结果表明,采用固相pH梯度凝胶双向电泳可以极大地提高分辨率,相同龙眼胚性培养物的蛋白样品染色后所得的蛋白质谱点数从334左右增加到810左右;由于龙眼胚性培养物的蛋白等电点集中在3.5-7.0之间,采用13 cm的IPG胶条时,以pH 4-7的IPG胶条分离蛋白的效果佳.     

Protein markers of Marteilia sydneyi infection in Sydney rock oysters, Saccostrea glomerata

Marteilia sydneyi is the causative agent of QX disease in Sydney rock oyster, Saccostrea glomerata . It is responsible for disease outbreaks among oysters that occur during summer and can result in up to 95% mortality. QX disease has significantly decreased S. glomerata production in some areas of Australia's eastern seaboard over the past 30 years. Marteilia sydneyi sporulates in the digestive gland of oysters leading to complete disorganization of the infected tissues. The current study used proteomics to identify potential molecular markers of sporulating M. sydneyi infection during a field trial undertaken in the Georges River, Sydney, between December 2006 and May 2007. Early stages of M. sydneyi infection were detected by polymerase chain reaction, whilst cytological examination was used to identify sporulating M. sydneyi in the gut. Protein expression in oyster haemolymph was assessed during the M. sydneyi infection period by two dimensional electrophoresis. Proteome maps identified significant differences in the expression of four proteins in oysters with sporulating M. sydneyi infections.     

Salt‐resistant and salt‐sensitive wheat genotypes show similar biochemical reaction at protein level in the first phase of salt stress

Salinity has a two‐phase effect on plant growth, an osmotic effect due to salts in the outside solution and ion toxicity in a second phase due to salt build‐up in transpiring leaves. To elucidate salt‐resistance mechanisms in the first phase of salt stress, we studied the biochemical reaction of salt‐resistant and salt‐sensitive wheat (Triticum aestivum L.) genotypes at protein level after 10 d exposure to 125 mM–NaCl salinity (first phase of salt stress) and the variation of salt resistance among the genotypes after 30 d exposure to 125 mM–NaCl salinity (second phase of salt stress) in solution culture experiments in a growth chamber. The three genotypes differed significantly in absolute and relative shoot and root dry weights after 30 d exposure to NaCl salinity. SARC‐1 produced the maximum and 7‐Cerros the minimum shoot dry weights under salinity relative to control. A highly significant negative correlation (r² = –0.99) was observed between salt resistance (% shoot dry weight under salinity relative to control) and shoot Na⁺ concentration of the wheat genotypes studied. However, the salt‐resistant and salt‐sensitive genotypes showed a similar biochemical reaction at the level of proteins after 10 d exposure to 125 mM NaCl. In both genotypes, the expression of more than 50% proteins was changed, but the difference between the genotypes in various categories of protein change (up‐regulated, down‐regulated, disappeared, and new‐appeared) was only 1%–8%. It is concluded that the initial biochemical reaction to salinity at protein level in wheat is an unspecific response and not a specific adaptation to salinity.     

Identification of regulated proteins by resveratrol in glutamate-induced cortical injury of newborn rats

Glutamate induces neuronal damage by generating oxidative stress and neurotoxicities. The neurological damage caused by glutamate is more severe during brain development in newborns than in adults. Resveratrol is naturally present in a variety of fruits and medicinal plants and exerts a neuroprotective effect against brain damage. The goal of this study was to evaluate the neuroprotective effects of resveratrol and to identify changed proteins in response to resveratrol treatment during glutamate-induced neonatal cortical damage. Sprague-Dawley rat pups (7 days old) were randomly divided into vehicle, resveratrol, glutamate, and glutamate and resveratrol groups. The animals were intraperitoneally injected with glutamate (10 mg/kg) and/or resveratrol (20 mg/kg) and their brain tissue was collected 4 hr after drug administration. Glutamate exposure caused severe histopathological changes, while resveratrol attenuated this damage. We identified regulated proteins by resveratrol in glutamate-induced cortical damaged tissue using two-dimensional gel electrophoresis and mass spectrometry. Among identified proteins, we focused on eukaryotic initiation factor 4A2, γ-enolase, protein phosphatase 2A subunit B, and isocitrate dehydrogenase. These proteins decreased in the glutamate-treated group, whereas the combination treatment of glutamate and resveratrol attenuated these protein reductions. These proteins are anti-oxidant proteins and anti-apoptotic proteins. These results suggest that glutamate induces brain cortical damage in newborns; resveratrol exerts a neuroprotective effect by controlling expression of various proteins with anti-oxidant and anti-apoptotic functions.     

Charting the proteome of Cryptosporidium parvum sporozoites using sequence similarity-based BLAST searching

Cryptosporidium (C.) spp. are important zoonotic parasites causing widespread diarrhoeal disease in man and animals. The recent release of the complete genome sequences for C. parvum and C. hominis has facilitated the comprehensive global proteome analysis of these opportunistic pathogens. The well-known approach for mass spectrometry (MS) based data analysis using the BLAST tool (MS BLAST) is a database search protocol for identifying unknown proteins by sequence similarity to homologous proteins using peptide sequences produced by mass spectrometry. We have used several complementary approaches to explore the global sporozoite proteome of C. parvum with available proteomic tools. To optimize the output of the MS data, a sequence similarity-based MS BLAST strategy was employed for bioinformatic analysis. Most significantly, almost all the constituents of glycolysis and several mitochondrion-related proteins were identified. In addition, many hypothetical Cryptosporidium proteins were validated by the identification of their constituent peptides. The MS BLAST approach was found to be useful during the study and could provide valuable information towards a complete understanding of the unique biology of Cryptosporidium.