The Human Serum Transferrin-Cisplatin's Characteristics of Mass Spectrometry and Spectrum and the Molecular Mechanism of Targeting and Apoptosis to Hepatoma Cell from the Complex

外科治疗，放射性治疗(放疗)，化学药物治疗(化疗)、和免疫（生物）治疗是目前癌症治疗的四大主要手段，其中化疗法具有能进行全身性癌症治疗、多发性癌症治疗、以及癌症根治率相对较高等优点，成为应用最广泛、发展最快的治疗手段。但是，传统的化疗药物都不能特异性的杀伤癌细胞，化疗效果和严重毒副作用并存。疗效依赖于药物毒性、给药剂量、给药途径、给药次数和疗程的各种化疗药物均具有细胞毒性极强、给药剂量大，并易产生耐药性等缺陷，给化疗患者带来难以忍受的痛苦。近十年来，高效靶向化疗成为肿瘤治疗学的研究热点之一，在组织器官水平、细胞水平和分子水平上的靶向治疗都有大量的研究报道，部分研究成果已应用于临床治疗。 本文...Four therapies including surgery, radiotherapy, chemotherapy,and immunotherapy are the major clinical therapies of cancer. In which chemotherapy is most widely used due to its potential of treating with systemic cancers and recurrent cancers, as well as it’s relatively high cure rate. However, few traditional drugs for chemotherapy can selectively kill cancer cells, which means a lot of normal cel...学位：理学博士院系专业：生命科学学院生物化学与生物技术系_生物化学与分子生物学学号：2162006015332

海洋来源放线菌HN-E31次生代谢产物的分离与鉴定

利用反相硅胶柱色谱、Sephadex LH-20凝胶柱色谱、半制备高效液相色谱等分离方法,对采自中国南海的鸟氨酸微菌属放线菌HN-E31发酵液乙酸乙酯萃取部位的化学成分进行分离研究,通过1H NMR、13C NMR和质谱等波谱技术,并参照相关文献数据,对化合物进行结构鉴定。结果共分离得到了12个化合物,分别鉴定为:环(脯-缬)二肽(1)、环(脯-亮)二肽(2)、环(脯-异亮)二肽(3)、环(脯-苯丙)二肽(4)、环(亮-丙)二肽(5)、环(异亮-丙)二肽(6)、苯丙氨酸(7)、3-hydroxyacetyl-indole(8)、3-吲哚甲醛(9)、水杨酸(10)、尿嘧啶(11)和对羟基苯甲醛(12)。上述化合物均为首次从鸟氨酸微菌属放线菌中分离获得。国家自然科学基金项目(31201969);;厦门市海洋与渔业局科研项目(17GYY008NF04)~

棕色固氮菌细菌铁蛋白捕获能力、稳定性和亚基相互作用的强度

选用柱层析、电泳和反相高效液相色谱(RP-HPLC)技术制备质谱纯棕色固氮菌细菌铁蛋白(Bacteri-al ferritin ofAzotobacter vinelandii,AVBF),并采用释放铁动力学和肽质量指纹图谱(Peptide mass fingerprint-ing,PMF)技术分别鉴定AVBF。基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)和电泳技术揭示AVBF亚基之间相互作用强度、稳定性和聚合态。AVBF可直接捕获有机小分子亚甲蓝(MB),其捕获率为15.0±2.0MB/AVBF,认为介于AVBF亚基单体之间的血红素参与捕获MB。较高浓度(40%~50%)的乙腈和丙酮均能使AVBF和鲨鱼肝铁蛋白(Liver ferritin of shark,SLF)释放不稳定亚基,但在较低浓度(20%~30%)的乙腈条件下,却需要借助来源于质谱仪的激光才能使AVBF或SLF释放不稳定亚基,并供质谱分析。AVBF亚基之间的相互作用强度明显低于SLF。铁蛋白亚基之间的相互作用强度高低与铁蛋白执行释放和储存铁的速率有关

Cloning and prokaryotic expression of the multi-copies of the human proinsulin-like gene

根据大肠杆菌密码子偏好性,优化人胰岛素原基因序列,同时用2个赖氨酸取代C链。重叠PCr扩增法克隆优化的类人胰岛素原基因,PCr扩增引物中引入胰蛋白酶酶切位点和核酸限制性内切酶识别位点。经酶切、拼接获得类人胰岛素原多拷贝基因,并构建人胰岛素原基因多拷贝原核表达载体,转化感受态大肠杆菌诱导表达融合蛋白。表达产物经SdS-PAgE电泳,考马斯亮兰染色和融合蛋白染色条带光密度分析表明,含有4拷贝的类人胰岛素原重复基因序列原核表达载体的诱导表达类人胰岛素原效率最高,是单拷贝类人胰岛素基因原核表达载体表达效率的3倍左右。表达产物经质谱鉴定,确定为优化设计的类人胰岛素原,表明构建的多拷贝类人胰岛素原基因表达载体可应用于后续人胰岛素原的高效表达生产研究。The human pre-insulin gene was redesigned for optimal expression in Escherichia coli through the alteration of its codon bias to reflect were redesigned according to the Codon Preference Parameter of Escherichia coli codon preference,and the C peptide of human preinsulin was replaced with Lys-Lys.Then the redesigned human pre-insulin gene(hINS)was cloned using Polymerase Chain Reaction(PCR),and further performed as a template on which based the multi-copy genes of hINS(hINS-M)was cloned by PCR with several couples of special primers.Besides,sequences of both the Restriction Enzyme and Trypsin cutting sites were included in the primers.Accordingly,restriction Endonucleases were employed to cut the PCR products,and T4DNA Ligase was then employed to tape the cut PCR produces to the differently designed multi-copies of the hINS-M.The prokaryotic expression vectors of these multi-copies genes were constructed and transformed into host Escherichia coli strains for expression.Furthermore,dodecyl sufate,sodium salt-polyacrylamide gel electrophoresis(SDS-PAGE),coomassie brilliant blue staining,photodensitometry,and mass spectrometry were employed to analyze the the expression products of the hINS-M.Our results indicated that the expression efficiency of the 4-copies gene of hINS-M was significantly higher than that of the others.The ratio of the expression product of the 4-copies gene hINS-Mthe human preinsulin to the total expression proteins of the host strains was over 28.0%.The ratio was about 3times of that of the expression product of the 1-copy gene of hINS-M.Moreover,the expression products from the different mutil-copes genes of hINS-M were all indentified to be the re-designed human preinsulin.Therefore,the prokaryotic expression vectors of the multi-copies genes of hINS-M could be used to improve the expression efficiency of the recombinant human preinsulin.国家自然科学基金资助项目(31201969); 厦门市科技计划项目(3502Z20093041

Tolerance of Gill Cells in Primary Cultured Oyster to Zinc Ion

本文以福建牡蛎为实验材料,探索体外原代细胞培养方法,测试体外培养细胞对锌离子的耐受特性。结果表明,以pH7.25、渗透压摩尔浓度为1000mosm、10%透析胎牛血清的L-15优化培养基,能顺利的在体外培养牡蛎鳃细胞且细胞活力在80%以上;原代牡蛎腮细胞对锌离子暴露的耐受特征与牡蛎对锌离子的耐受特征高度相似,24、48、72、96小时的半致死浓度分别为239.47、72.60、12.13和8.19mg·L~（-1）,表明体外培养的牡蛎鳃原代细胞维持了原有的锌耐受特性。In the paper, Fujian oysters were used as experimental materials to explore the primary cell culture methods in vitro and to test the tolerance of cells to zinc ions in vitro. The results showed that the activity of oyster gill cells and the cell viability were more than 80% in the L-15 optimized medium with pH7.25, osmolality of 1000 mosm and 10% dialysis fetal bovine serum. The tolerance to zinc exposure was similar to that of oyster, and the half lethal concentrations at 24, 48, 72 and 96 hours were 239.47, 72.60, 12.13 and 8.19 mg · L-1, respectively. Cultured oyster gill primary cells maintained the original zinc tolerance characteristics.厦门市科技计划项目（3502Z20143027）; 福建省教育厅自然科学项目（451JA13424

用MALDI-TOF质谱和电子光谱研究纳米胰岛素核-铁蛋白的构建技术和特性

小批量制备了电泳纯鲨鱼肝铁蛋白(Liver ferritin of Sphyma zygaena,SZLF).用透射电子显微镜技术研究SZLF的铁核和蛋白壳亚基解离和重组过程.用盐酸解离(pH=1.5)和分离膜透析技术制备铁蛋白亚基,并用酸碱中和方法重组亚基成为脱铁核铁蛋白(apoSZLF),同时将胰岛素(Insulin,INS)包裹于apoSZLF蛋白壳内,构建纳米INS核-SZLF.用电子光谱、MALDI-TOF质谱和SDS-PAGE技术分别揭示了纳米INS核-SZLF分子结构的真实性,提出铁蛋白亚基包裹INS构建为纳米INS核-铁蛋白的途径

养殖叶片山海绵(Mycale phyllophila)次生代谢产物的研究

目的研究养殖叶片山海绵Mycale phyllophila次生代谢产物,为药源海绵规模化养殖和开发提供依据。方法综合利用硅胶柱色谱、Sephadex LH-20凝胶柱色谱、反相ODS柱色谱、半制备液相色谱等分离技术,对化学成分进行分离;结合NMR、MS等光谱方法和文献数据,鉴定化合物的结构。结果从叶片山海绵的低极性萃取物中,分离鉴定了11个单体化合物:3-吲哚甲醛(1)、(1 H-indol-3-yl)oxoacetamide(2)、3-hydroxyacetyl-indole(3)、3-(2-(1 H-indol-3-yl)-2-oxoethyl)-5,6-dihydropyridin-2(1 H)-one(4)、胆固醇(5)、麦角甾-5,24(28)-二烯-3β-醇(6)、胆甾-5,22-二烯-3β-醇(7)、花生酸(8)、1-棕榈酸甘油酯(9)、正十六烷基甘油醚(10)和对羟基苯甲醛(11)。结论所有化合物均为首次从叶片山海绵M.phyllophila中获得。厦门市产学研协同创新及科技合作项目(3502Z20173009);;福建省教育厅中青年教师教育科研项目(JAT170708);;厦门市海洋与渔业局科研项目(17GYY008NF04)资

Metal accumulation and differentially expressed proteins in gill of oyster (Crassostrea hongkongensis) exposed to long-term heavy metal-contaminated estuary

973 Projects [2010CB126403]; NSFC [31201969]; Programme of Introducing Talents of Discipline to Universities [B07034]; PCSIRT [IRT0941]; Earmarked Fund for Modern Agro-industry Technology Research System [CARS-48]Bio-accumulation and bio-transmission of toxic metals and the toxicological responses of organisms exposed to toxic metals have been focused, due to heavy metal contaminations have critically threatened the ecosystem and food security. However, still few investigations focused on the responses of certain organisms exposed to the long term and severe heavy metal contamination in specific environments. In present investigation, the Hong Kong oyster, Crassostrea hongkongensis were obtained from 3 sites which were contaminated by different concentrations of heavy metals (such as zinc, copper, manganese and lead etc.), respectively. Heavy metal concentrations in the sea water samples collected from the 3 sites and the dissected tissues of the oysters with blue visceral mass were determinated to estimate the metal contamination levels in environments and the bio-accumulation ratios of the heavy metals in the different tissues of oysters. Moreover, Proteomic methods were employed to analyze the differentially expressed proteins in the gills of oysters exposed to long-term heavy metal contaminations. Results indicated that the Jiulong River estuary has been severely contaminated by Cu, Zn and slightly with Cr, Ni, Mn, etc, moreover, Zn and Cu were the major metals accumulated by oysters to phenomenally high concentrations (more than 3.0% of Zn and about 2.0% of Cu against what the dry weight of tissues were accumulated), and Cr, Ni, Mn, etc were also significantly accumulated. The differentially expressed proteins in the gills of oysters exposed to heavy metals participate in several cell processes, such as metal binding, transporting and saving, oxidative-reduction balance maintaining, stress response, signal transduction, etc. Significantly up-regulated expression (about 10 folds) of an important metal binding protein, metallothionein (MT) and granular cells was observed in the gills of oysters exposed to long-term and severely heavy-metal-contaminated estuary, it suggested that binding toxic metals with metallothionein-like proteins (MTLP) and storing toxic metals in metal-rich granules (MRG) with insoluble forms were the important strategies of oyster to detoxify the toxic metals and adapt to the high level of metal-contaminated environment. Most of the stress and immunity responsive proteins, such as heat shock proteins (HSP), extracellular superoxide dismutase (ECSOD) and cavortin, and the cellular redox reaction relative proteins such as 20G-Fe (II) oxygenase family oxidoreductase, aldehyde dehydrogenase and retinal dehydrogenase 2, were detected significantly down-regulated in the gills of oysters exposed to long term heavy metal contaminated environments, it indicated that long term exposure different from emergent exposure to heavy metal contamination may significantly suppress the stress and immunity response system of oysters. Moreover, Formin homology 2 domain containing protein (FH2). The only protein domain to directly nucleate actin monomers into unbranched filament polymers, by which will subsequently control gene expression and chromatin remodelling complexes, was also detected greatly up-regulated in the gills of oysters exposed to long-term heavy metal contaminations. It indicated that nuclear activity regulation may also be important for oyster to adapt to the long-term heavy-metal-contaminated environment. (C) 2014 Elsevier Ltd. All rights reserved

Functions of Double Subunits of a Type,Structure of Iron Corn, and Kinetics of Iron Release from Membrane Ferritin of Human Placenta

E-mail: hqhuang@ xmu.edu.cn[中文文摘]以人胎盘组织为实验材料,小批量制备电泳纯人胎盘膜铁蛋白(HPMF),对其结构与功能进行研究。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术揭示,HPMF蛋白壳由分子量分别为15 kDa(MF15)和20 kDa(MF20)的亚基组成,其中MF15蛋白含量约为MF20的3倍。经肽质量指纹图谱(PMF)技术鉴定,发现PHMF的MF15和MF20亚基与人铁蛋白(HF)L亚基均具有较高的同源性,提出HPMF由单类型但分子量不同的双亚基组成的新观点。在选用基质辅助电离激光解吸飞行时间质谱(MALDI-TOF MS)技术直接分析HPMF亚基稳定性过程中,获得5个质荷比(m/z)值为5071.25,9962.51,15131.55(MF15),19936.40(MF20)和20147.61的特征质谱峰,其对应的亚基分子式分别为[MF15]3+,[MF20]2+,[MF15]+,[MF20]+和[MF20+Fe]+。ICP-MS分析发现,HPMF铁核中的铁和无机磷酸盐(Pi)含量仅为85Fe3+/HPMF和15Pi/HPMF,其中Fe3+/Pi比值约为5.72,明显低于绝大多数哺乳动物铁蛋白,但却高于细菌铁蛋白(BF)。在释放铁动力学方面,HPMF以一级反应动力学途径释放完整铁核中的铁量,其释放时间约需750 min,其释放铁的速率慢于马脾铁蛋白(HSF)和猪胰铁蛋白(PPF)(约60 min)。根据HPMF完全不同于大部分哺乳动物、植物和细菌铁蛋白的新颖结构与功能,提出HPMF在母体和胎儿之间中起着释放、储存和转运铁的复合生理功能的铁转运模型。[英文文摘]As an experimental material of human placental tissue，membrane ferritin of human placenta (HPMF) with electrophoresis purity was prepared in batch． SDS-PAGE approach reveals that the protein shell consists of double subunits of a type in HPMF，which the molecular weights of both subunits were calculated to be approximately 15 and 20 kDa respectively，named MF15 and MF20． In addition，the protein content of MF15 is about three times more than that of M20 in SDS-PAGE gel． Using the approach of peptide mass fingerprinting (PMF) ，both MF15and MF20 subunits were identified to show the high homology with reference to L subunit of human ferritin，pointing out that HPMF was a novel ferritin of two subunits involving in single type and different molecular weight． Using a matrix-assisted laser desorption ionization time of flight mass spectrometry to investigate the subunit-stability of HPMF，five mass peaks corresponding ratio (m/z) of mass to charge such as 5071.25,9962.51,15131.55 (MF15),19936.40 (MF20) and 20147.61 were indicated to have five subunit formula of [MF15]3 +,[MF20]2 +，[MF15]+，[MF20]+ and [MF20 + Fe]+ ，respectively． Using inductively coupled plasma-mass spectrometry to analyze the elemental composition of HPMF，only 85 Fe3 + and 15 inorganic phosphate (Pi) in the iron core of per molecular of HPMF were found，which was significantly lower than that of most mammal ferritins，but higher than that of bacterial ferritin ( BF) ． Kinetic studies showed that iron release from the complete HPMF followed the law with first-order reaction，which also differed from that with two different rates in mammal ferritins． In addition，the time of the iron release completely needed about 750 min in human placental membrane ferritin ( HPMF) ，which was longer than 60 min in both horse spleen ferritin ( HSF) and pig pancreatic ferritin ( PPF) , Accordingly,based on the difference of structure and function among HPMF，mammalian ferritins，plant ferritins，and BF，we proposed M15 and M20 subunit play different role in iron transporting from maternal blood to fetal blood．国家自然科学基金(No.30870515);973项目(No.2010CB12640)资

The role of hybridization in improving the immune response and thermal tolerance of abalone

Hi-Tech Research and Development (863) Program of China [2012AA10A412]; National Natural Science Foundation of China [41106120]; Fujian Science and Technology Program [2012N5012]; Guangdong Province - Department of Education of China Joint Project [2012B091100085]; Earmarked Fund for Modern Agro-industry Technology Research System [CARS-48]Recently, frequent death of cultured abalone drew our attention to the stress tolerance of abalone. Hybridization is an effective way of genetic improvement in aquaculture, which can introduce improved traits to the hybrids. In this study, we challenged the hybrids between Haliotis discus hannai and Haliotis gigantea, and their parents with bacteria (vibrio harveyi, vibrio alginolyticus and vibrio parahemolyticus), then held them at 20 degrees C and 28 degrees C, survival rates of the parental populations and hybrid populations were recorded. Then we tested the immune responses and thermal-induced responses of the four populations at different temperatures. Total hemocyte count (THC), respiratory burst, superoxide dismutase activity (SOD), acid phosphatase activity (ACP), alkaline phosphatase activity (AKP), myeloperoxidase activity (MPO), and HSP70 expression were determined on day 1 and day 7 of the temperature exposure. Results showed higher survival rates of the hybrids than their parents against bacteria challenge. For immune parameters, THCs were evaluated at 28 degrees C, while increased THC was also observed in H. discus hannai female x H. gigantea male (DG) and H. discus hannai female x H. discus hannai male (DD) at 12 degrees C (day 7); at 28 degrees C, respiratory burst was activated (day 1 and 7), while SOD activity first rose then fell over 7-days exposure; AKP activity was elevated at 12 degrees C and 28 degrees C (day 1), most notably in DG, and an increased level of ACP was observed in DG at 28 degrees C (day 7); MPO activity was suppressed at 12 degrees C and 28 degrees C on day 1, but recovered on day 7. For HSP70, increased HSP70 levels were observed in all populations at 28 degrees C (day 1), and DD got the lowest HSP70 level after 7-days exposure at 28 degrees C. Overall, the results suggest that temperature changes could significantly affect the physiological status of abalone, and hybrids may be more resistant to disease and thermal stresses than their parents. (C) 2014 Elsevier Ltd. All rights reserved