人源化甲状腺素脱碘酶单链抗体的结构模拟

以四碘甲状腺素原氨酸为半抗原 ,从人噬菌体展示半合成单链抗体库中筛选与半抗原有特异结合性的单链抗体 E3 ,并测定了 E3的一级结构 ,同时用计算机模拟了其空间结构

去甲肾上腺素电极过程的圆二色谱电化学研究

现场圆二色薄层光谱电化学研究去甲肾上腺素的电化学氧化还原过程 .研究表明去甲肾上腺素 ( pH =7.0磷酸缓冲溶液中 )在玻碳电极上经历了不可逆的电化学氧化 ,且遵从后行化学反应 (EC)机理 ,去甲肾上腺素醌和去甲肾上腺素红的再还原遵从简单电子转移 (E)机理 .由双对数法获得去甲肾上腺素电化学氧化的式电位为E10’=0 .2 0V ,电子转移系数和电子转移数之积为αn =0 .38,标准复相电极反应常数k10 =1 .2× 1 0 -4 cm·s-1.去甲肾上腺素醌和去甲肾上腺素红的电化学还原反应参数分别为E2 0’=0 .2 5V ,αn =0 .37,k2 0 =4.4× 1 0 -5 cm·s-1和E3 0’=- 0 .2 5V ,αn =0 .33,k3 0 =1 .1× 1 0 -4 cm·s-1.

琼东南盆地天然气水合物远景区构造复原与成矿构造分析

南海是西太平洋最著名边缘海之一，在大地构造上位于太平洋板块、欧亚板块和印澳板块的交界部位。琼东南盆地为发育于南海北部大陆边缘、呈北东向伸展的新生代盆地，北以断裂与海南岛隆起相隔、西以断裂与莺歌海盆地分界、东与神狐－暗沙隆起和西沙海槽盆地相接、南界西沙群岛和中建南盆地，是我国重要的常规油气富集区，也是重要的天然气水合物远景区。可划分为陆架区、斜坡区、中央坳陷区和南部断阶带几个次级构造单元。 本文通过运用2DMove软件，对琼东南盆地研究区四条测线的部分剖面进行构造复原，计算出了各条剖面每个时期的伸展参数，对研究区构造活动强度进行了初步的定量分析，进一步明确了琼东南盆地构造演化史，初步探讨了与天然气水合物形成的联系。同时结合研究区天然气水合物成矿地质条件分析和高分辨率地震资料解释成果，探讨了研究区不同位置处天然气水合物主要的构造聚集模式。通过分析得到以下主要初步结论： 研究区内共识别了27处泥底辟构造，其中12处发育在BSR范围区，与BSR相伴生。发育层位多数集中在T5反射界面以下，分布范围较广，遍布于各个构造单元。从平衡剖面的总体伸展参数来看，渐新世（E3）是琼东南盆地构造活跃期，自中－早中新世以来构造活动相对趋缓。琼东南盆地的演化也经历了裂陷和裂后两大构造演化阶段，具有明显的“下断上坳”的双层构造格架，以破裂不整合面T5为界，可分为下、上两大构造层，分别代表早期裂陷作用和晚期裂后坳陷和热沉降作用的产物。结合琼东南盆地天然气水合物成矿构造条件分析，我们探讨了在中央坳陷带处天然气水合物的构造聚集模式主要以泥底辟构造为主；在南部断阶带处天然气水合物的构造聚集模式主要以断裂为主。

洱海沉积物间隙水中溶解有机质的地球化学特性

对洱海沉积物间隙水中溶解有机质(DOM)含量、紫外一可见吸收、荧光以及分子量等的垂直分布特征进行了研究。结果表明，DOC含量在沉积物一水界面明显富集，随后急剧下降，6cm处达到最小值，随后呈上升趋势。DOC与吸光度值、荧光发射光谱强度之间具有一定的线性相关关系。DOM的E3／EAL值范围在1-6之间，绝大多数在1—3．5之间。表征DOM中腐殖质来源的指标荧光指数值处于1．48—1．59之间，说明DOM以陆源输入为主。此外，洱海沉积物间隙水DOM分子量分布呈多峰分布模式，重均分子量(Mw)值在1462—1953Da之间，数均分子量(Mn)值在547—900Da之间，多分散性系数ρ值在2．02—3．05之间。随着沉积深度增大，Mw和Mn有略微的增大趋势，但变化不大。沉积物的氧化还原条件、微生物活动以及铁、锰氧化物等在沉积剖面的差异是控制间隙水中DOM各种地球化学特性的主要因素。

红枫湖溶解有机质的分离富集、地球化学特征和环境效应

作为全球碳循环的主要组成部分，溶解有机质在水生生态系统的各种物理、化学和生物过程中起着十分重要的作用：它是湖泊异养型生物所需能量的主要提供者；是湖泊水体pH的调节剂和控制因素；能够与金属离子或有机污染物相互作用，从而影响它们的迁移转化、毒性和生物可利用性；溶解有机质也是饮用水氯化工艺中生成具有致癌作用消毒副产品的主要前驱物质。溶解有机质组成和结构十分复杂，一般由腐殖质和非腐殖质物质组成，相应的环境地球化学特征和行为也各不相同。因此，研究溶解有机质各分离组分的结构特征和环境效应，将有助于我们对溶解有机质复杂化学结构、循环特征及行为特征的深入理解；有助于揭示溶解有机质对环境中微量污染物的迁移、转化、毒性和生物有效性的影响机理和贡献因素；为水环境评价、预测、控制和管理提供科学依据。 本论文选择云贵高原山区湖泊—红枫湖为研究对象，利用XAD树脂分离技术，把湖泊水体中溶解有机质按极性的不同分成了疏水性酸、碱、中性物质和亲水性酸、碱、中性物质等六种有机组分。主要运用元素分析、有机碳分析仪、傅立叶变换红外光谱、紫外－可见分光光度，高效液相体积排阻色谱、三维荧光光谱和稳定同位素质谱仪等现代分析方法和手段，对各有机组分的地球化学特征及来源的进行了表征和探讨。同时利用荧光猝灭滴定技术研究了溶解有机质分离组分与铜的相互作用，利用顶空气相色谱法测定了各分离组分与氯气反应活性的大小，并确立了生成消毒副产物的主要前驱物质。本论文的研究成果主要有以下几点： 1.改进了分离流程：在XAD－8树脂分离溶解有机质流程的基础上，引入XAD－4树脂，对贵州红枫湖水体中溶解有机质进行了富集分离，得腐殖酸、富里酸、疏水性中性物质、亲水性酸、亲水性碱、亲水性中性物质等六种有机组分，回收率为82%。各分离组分的组成与其它天然水体的组成接近，其中以富里酸为主，占分离组分的51%。 2.溶解有机质分离组分化学结构特征的研究：元素分析表明疏水性组分具有较高的碳和硫含量，而亲水性组分O/C比较高，含有较多的含氧官能团；傅立叶变换红外光谱表明各分离组分在3424－3434 cm-1具有O-H和N-H的吸收峰，腐殖酸组分主要为含氮和聚糖物质，富里酸和亲水性酸表现强的羧基吸收峰。高效体积排阻色谱表明各六种有机组分的分子量较小，重均(Mw)和数均(Mn)分子量分别集中在1688-2355Da和1338-1928Da之间，其中Mw和紫外吸收比值(E2/E3)之间呈负相关关系；三维荧光光谱显示六种有机组分表现了4种不同类型的荧光峰，紫外区类富里酸荧光峰peakA和可见区类富里酸荧光峰peakC；疏水性中性物质还表现了两种类蛋白荧光峰peakB和peakD。荧光强度与紫外吸光系数SUVA254之间呈显著正相关关系，说明了产生荧光和紫外的物质具有某些相似的官能团，其中芳香官能团和共轭双键在这两类光谱的产生中具有重要作用。本研究进一步揭示了溶解有机质不同有机组分的分子量分布、荧光光谱特征和紫外吸收特征之间的一致性，反映了各有机组分之间的亲水、疏水与分子量、光谱特征之间的内在联系，本研究有助于加我们深对溶解有机质复杂性和相似性及化学结构与行为的进一步认识。 3.溶解有机质分离组分来源问题的探讨：我们利用δ13C及C/N比值并结合荧光光谱特征来指示溶解有机质的来源。各分离组分的δ13C及C/N比值表明富里酸主要来自陆源C3植被形成的土壤有机质，而腐殖酸、疏水性中性物质、亲水性酸、亲水性碱、亲水性中性物质属于陆源和内源的混合来源，其中腐殖酸、亲水性酸、亲水性碱接近红枫湖藻类一端，而疏水性中性物质和亲水性中性物质接近陆源一端。荧光光谱表明疏水性中性物质中含有类蛋白荧光峰，可能暗示了该组分还与人类活动有关。该研究加深了对溶解有机质来源的多样性和影响因素复杂性的认识。 4.溶解有机质分离组分与铜离子的相互作用，利用荧光猝灭滴定技术研究了分离组分与铜离子的相互作用，结果表明溶解有机质滴加铜离子后荧光强调显著降低，同时发射波长Em发生蓝移，而激发波长Ex基本不变，说明了分离有机组分在与铜离子相互作用后，π电子减少，共轭性和芳香型降低，直线型分子向非直线型分子转变；溶解有机质分离组分与铜相互作用的滴定曲线与修正的Stern-Volmer模型十分吻合；分离组分的稳定常数logK介于4.73-5.16之间（C峰）和4.64-5.24之间（A峰），logKA与logKC与总酸度以及与酚羟基之间存在显著相关性, 与羧基含量之间也有弱相关关系，说明铜离子优先与配体中强配位点（酚羟基）络合，其次为弱配位基（羧基）。该研究揭示了云贵高原山区湖泊溶解有机质分离组分与金属离子相互作用的差异性和影响因素，为水环境中金属离子迁移转化和毒性的评价提供理论依据。 5.溶解有机质分离组分卤代活性的比较：我们运用顶空气相色谱法测定了红枫湖分离组分与消毒剂反应生成有机卤代物的情况。结果表明红枫湖分离组分氯化消毒副产物主要以三氯甲烷(CHCl3)和二溴一氯甲烷(CHBr2Cl)为主，富里酸是生成三卤甲烷的主要前驱物质，同时富里酸的卤代反应活性也最强。该研究揭示了云贵高原山区湖泊中富里酸是生成消毒副产物的主要前驱物质，该组分以陆源有机质输入为主。因此，该研究对饮用水消毒具有一定的现实意义。

Influence of fat percentage, front thigh skinfold and girth on the maximum radial displacement of the dominant rectus femoris

4.171 JCR (2013) Q1, 6/81 Sport sciences

Bacteremic pneumonia before and after withdrawal of 13-valent pneumococcal conjugate vaccine from a public vaccination program in Spain: a case-control study

The objective of this study is to compare the incidence and epidemiology of bacteremic community-acquired pneumonia (CAP) in the setting of changes in 13-valent pneumococcal conjugate vaccine (PCV13) coverage. In the region of Madrid, universal immunization with the PCV13 started in May 2010. In July 2012, public funding ceased. Vaccination coverage decreased from >95% to 82% in 2013 and to 67% in 2014. We performed a multicenter surveillance and case-control study from 2009-2014. Cases were hospitalized children with bacteremic CAP. Controls were children selected 1:1 from next-admitted with negative blood cultures and typical, presumed bacterial CAP. Annual incidence of bacteremic CAP declined from 7.9/100 000 children (95% CI 5.1-11.1) in 2009 to 2.1/100 000 children (95% CI 1.1-4.1) in 2012. In 2014, 2 years after PCV13 was withdrawn from the universal vaccination program, the incidence of bacteremic CAP increased to 5.4/100 000 children (95% CI 3.5-8.4). We enrolled 113 cases and 113 controls. Streptococcus pneumoniae caused most of bloodstream infections (78%). Empyema was associated with bacteremia (P = .003, OR 3.6; 95% CI 1.4-8.9). Simple parapneumonic effusion was not associated with bacteremia. Incomplete PCV immunization was not a risk factor for bacteremic pneumonia.

Investigation of p75 neurotrophin receptor and the role of its post-translational modifications in tumour cell motility and invasiveness

The p75 neurotrophin receptor (p75NTR) is a member of the tumour necrosis factor superfamily, which relies on the recruitment of cytosolic protein partners - including the TNF receptor associated factor 6 (TRAF6) E3 ubiquitin ligase - to produce cellular responses such as apoptosis, survival, and inhibition of neurite outgrowth. Recently,p75NTR was also shown to undergo γ-secretase-mediated regulated intramembrane proteolysis, and the receptor ICD was found to migrate to the nucleus where it regulates gene transcription. Moreover, γ-secretase-mediated proteolysis was shown to be involved in glioblastoma cell migration and invasion. In this study we report that TRAF6-mediated K63-linked polyubiquitination at multiple or alternative lysine residues influences p75NTR-ICD stability in vitro. In addition, we found that TRAF6-mediated ubiquitination of p75NTR is not influenced by inhibition of dynamin. Moreover, we report beta-transducin repeats-containing protein (β-TrCP) as a novel E3- ligase that ubiquitinates p75NTR, which is independent of serine phosphorylation of the p75NTR destruction motif. In contrast to its influence on other substrates, co-expression of β-TrCP did not reduce p75NTR stability. We created U87-MG glioblastoma cell lines stably expressing wild type, γ-secretaseresistant and constitutively cleaved receptor, as well as the ICD-stabilized mutant K301R. Interestingly, only wild-type p75NTR induces increased glioblastoma cell migration, which could be reversed by application of γ-secretase inhibitor. Microarray and qRT-PCR analysis of mRNA transcripts in these cell lines yielded several promising genes that might be involved in glioblastoma cell migration and invasion, such as cadherin 11 and matrix metalloproteinase 12. Analysis of potential transcription factor binding sites revealed that transcription of these genes might be regulated by well known p75NTR signalling cascades such as NF-κB or JNK signalling, which are independent of γ-secretase-mediated cleavage of the receptor. In contrast, while p75NTR overexpression was confirmed in melanoma cell lines and a patient sample of melanoma metastasis to the brain, inhibition of γ-secretase did not influence melanoma cell migration. Collectively, this study provides several avenues to better understand the physiological importance of posttranslational modifications of p75NTR and the significance of the receptor in glioblastoma cell migration and invasion.

Characterization of the degradation of wild-type and mutant HFE proteins during stress signalling in the endoplasmic reticulum

HFE is a transmembrane protein that becomes N-glycosylated during transport to the cell membrane. It acts to regulate cellular iron uptake by interacting with the Type 1 transferrin receptor and interfering with its ability to bind iron-loaded transferrin. There is also evidence that HFE regulates systemic iron levels by binding to the Type II transferrin receptor although the mechanism by which this occurs is still not well understood. Mutations to HFE that disrupt this function, or physiological conditions that decrease HFE protein levels, are associated with increased iron uptake, and its accumulation in tissues and organs. This is exemplified by the point mutation that results in conversion of cysteine residue 282 to tyrosine (C282Y), and gives rise to the majority of HFE-related hemochromatoses. The C282Y mutation prevents the formation of a disulfide bridge and disrupts the interaction with its co-chaperone β2-microglobulin. The resulting misfolded protein is retained within the endoplasmic reticulum (ER) where it activates the Unfolded Protein Response (UPR) and is subjected to proteasomal degradation. The absence of functional HFE at the cell surface leads to unregulated iron uptake and iron loading. While the E3 ubiquitin ligase involved in the degradation of HFE-C282Y has been identified, the mechanism by which it is targeted for degradation remains relatively obscure. The primary objective of this project was to further our understanding of how the iron regulatory HFE protein is targeted for degradation. Our studies suggest that the glycosylation status, and the active process of deglycosylation, are central to this process. We identified a number of additional factors that can contribute towards degradation and explored their regulation during ER stress conditions.

HDAC6 and Ubp-M BUZ domains recognize specific C-terminal sequences of proteins.

The BUZ/Znf-UBP domain is a protein module found in the cytoplasmic deacetylase HDAC6, E3 ubiquitin ligase BRAP2/IMP, and a subfamily of ubiquitin-specific proteases. Although several BUZ domains have been shown to bind ubiquitin with high affinity by recognizing its C-terminal sequence (RLRGG-COOH), it is currently unknown whether the interaction is sequence-specific or whether the BUZ domains are capable of binding to proteins other than ubiquitin. In this work, the BUZ domains of HDAC6 and Ubp-M were subjected to screening against a one-bead-one-compound (OBOC) peptide library that exhibited random peptide sequences with free C-termini. Sequence analysis of the selected binding peptides as well as alanine scanning studies revealed that the BUZ domains require a C-terminal Gly-Gly motif for binding. At the more N-terminal positions, the two BUZ domains have distinct sequence specificities, allowing them to bind to different peptides and/or proteins. A database search of the human proteome on the basis of the BUZ domain specificities identified 11 and 24 potential partner proteins for Ubp-M and HDAC6 BUZ domains, respectively. Peptides corresponding to the C-terminal sequences of four of the predicted binding partners (FBXO11, histone H4, PTOV1, and FAT10) were synthesized and tested for binding to the BUZ domains by fluorescence polarization. All four peptides bound to the HDAC6 BUZ domain with low micromolar K(D) values and less tightly to the Ubp-M BUZ domain. Finally, in vitro pull-down assays showed that the Ubp-M BUZ domain was capable of binding to the histone H3-histone H4 tetramer protein complex. Our results suggest that BUZ domains are sequence-specific protein-binding modules, with each BUZ domain potentially binding to a different subset of proteins.

Fiber type-specific nitric oxide protects oxidative myofibers against cachectic stimuli.

Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia.

beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1.

beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.

The Trim39 ubiquitin ligase inhibits APC/CCdh1-mediated degradation of the Bax activator MOAP-1.

Proapoptotic Bcl-2 family members, such as Bax, promote release of cytochrome c from mitochondria, leading to caspase activation and cell death. It was previously reported that modulator of apoptosis protein 1 (MOAP-1), an enhancer of Bax activation induced by DNA damage, is stabilized by Trim39, a protein of unknown function. In this paper, we show that MOAP-1 is a novel substrate of the anaphase-promoting complex (APC/C(Cdh1)) ubiquitin ligase. The influence of Trim39 on MOAP-1 levels stems from the ability of Trim39 (a RING domain E3 ligase) to directly inhibit APC/C(Cdh1)-mediated protein ubiquitylation. Accordingly, small interfering ribonucleic acid-mediated knockdown of Cdh1 stabilized MOAP-1, thereby enhancing etoposide-induced Bax activation and apoptosis. These data identify Trim39 as a novel APC/C regulator and provide an unexpected link between the APC/C and apoptotic regulation via MOAP-1.

Ubiquitylation of p53 by the APC/C inhibitor Trim39.

Tripartite motif 39 (Trim39) is a RING domain-containing E3 ubiquitin ligase able to inhibit the anaphase-promoting complex (APC/C) directly. Through analysis of Trim39 function in p53-positive and p53-negative cells, we have found, surprisingly, that p53-positive cells lacking Trim39 could not traverse the G1/S transition. This effect did not result from disinhibition of the APC/C. Moreover, although Trim39 loss inhibited etoposide-induced apoptosis in p53-negative cells, apoptosis was enhanced by Trim39 knockdown in p53-positive cells. Furthermore, we show here that the Trim39 can directly bind and ubiquitylate p53 in vitro and in vivo, leading to p53 degradation. Depletion of Trim39 significantly increased p53 protein levels and cell growth retardation in multiple cell lines. We found that the relative importance of Trim39 and the well-characterized p53-directed E3 ligase, murine double minute 2 (MDM2), varied between cell types. In cells that were relatively insensitive to the MDM2 inhibitor, nutlin-3a, apoptosis could be markedly enhanced by siRNA directed against Trim39. As such, Trim39 may serve as a potential therapeutic target in tumors with WT p53 when MDM2 inhibition is insufficient to elevate p53 levels and apoptosis.