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1.
  总被引:3,自引:0,他引:3  
Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an \"open-source\" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.  相似文献   
2.
    
Identification and evaluation of factors important for thermostability in proteins is a growing research field with many industrial applications. This study investigates the effects of introducing a novel disulfide bond and engineered electrostatic interactions with respect to the thermostability of holo azurin from Pseudomonas aeruginosa. Four mutants were selected on the basis of rational design and novel temperature-dependent atomic displacement factors from crystal data collected at elevated temperatures. The atomic displacement parameters describe the molecular movement at higher temperatures. The thermostability was evaluated by optical spectroscopy as well as by differential scanning calorimetry. Although azurin has a high inherent stability, the introduction of a novel disulfide bond connecting a flexible loop with small alpha-helix (D62C/K74C copper-containing mutant), increased the T(m) by 3.7 degrees C compared with the holo protein. Furthermore, three mutants were designed to introduce electrostatic interactions, K24R, D23E/K128R, and D23E/K128R/K24R. Mutant K24R stabilizes loops between two separate beta-strands and D23E/K128R was selected to stabilize the C-terminus of azurin. Furthermore, D23E/K128R/K24R was selected to reflect the combination of the electrostatic interactions in D23E/K128R and K24R. The mutants involving electrostatic interactions had a minor effect on the thermostability. The crystal structures of the copper-containing mutants D62C/K74C and K24R have been determined to 1.5 and 1.8 A resolution. In addition the crystal structure of the zinc-loaded mutant D62C/K74C has also been completed to 1.8 A resolution. These structures support the selected design and provide valuable information for evaluating effects of the modifications on the thermostability of holo azurin.  相似文献   
3.
Divergence of the hyperthermophilic Archaea, Pyrococcus furiosus and Pyrococcus horikoshii, was assessed by analysis of complete genomic sequences of both species. The average nucleotide identity between the genomic sequences is 70-75% within ORFs. The P. furiosus genome (1.908 mbp) is 170 kbp larger than the P. horikoshii genome (1.738 mbp) and the latter displays significant deletions in coding regions, including the trp, his, aro, leu-ile-val, arg, pro, cys, thr, and mal operons. P. horikoshii is auxotrophic for tryptophan and histidine and is unable to utilize maltose, unlike P. furiosus. In addition, the genomes differ considerably in gene order, displaying displacements and inversions. Six allelic intein sites are common to both Pyrococcus genomes, and two intein insertions occur in each species and not the other. The bacteria-like methylated chemotaxis proteins form a functional group in P. horikoshii, but are absent in P. furiosus. Two paralogous families of ferredoxin oxidoreductases provide evidence of gene duplication preceding the divergence of the Pyrococcus species.  相似文献   
4.
Unlike typical eukaryotes, the Dinoflagellate Crypthecodinium cohnii does not contain histones but six major basic, low molecular weight nuclear proteins which represent only 10% of the DNA mass and differ from histones in their electrophoretic and DNA-binding properties. These proteins are resolved in two-dimensional electrophoresis (AUT-PAGE x SDS-PAGE). Three proteins with an apparent molecular mass of 16, 16.5 and 17 kDa (p16, p16.5 and p17) are present in addition to the major 14 kDa basic nuclear component (HCc). HCc itself is resolved in three proteins (alpha, beta and gamma). When the proteins are not reduced with 2-mercaptoethanol before 2D-PAGE, the migration of HCc alpha, beta and gamma is modified in a way which suggests the formation of both inter- and intramolecular disulfide bridges and thus, the presence of at least two cysteines. The amino-acid analysis of HCc proteins resolved in 2D gels confirms that they are lysine-rich. HCc alpha, beta and gamma as well as p16, p16.5 and p17 are removed from isolated chromatin with 0.6 M NaCl, indicating that their affinity for DNA in vivo is lower than that of core histones. Furthermore, in vitro, they bind more tightly to single-stranded than to double-stranded DNA.  相似文献   
5.
6.

Background

Elevated cardiac troponin I (cTnI) is frequently observed in patients with severe sepsis and septic shock. However, the mechanisms underlying cTnI release in these patients are still unknown. To date no data regarding coagulation disturbances as a possible mechanism for cTnI release during sepsis are available.

Methodology/Principal Findings

Consecutive patients with systemic inflammatory response syndrome (SIRS), sepsis or septic shock without evidence of an acute coronary syndrome were analyzed. Coagulation parameters (clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF), α-angle) were assessed in native whole blood samples, and using specific activators to evaluate the extrinsic and intrinsic as well as the fibrin component of the coagulation pathway with the use of rotational thrombelastometry (ROTEM).Thirty-eight patients were included and 22 (58%) were cTnI-positive. Baseline characteristics between TnI-positive and -negative patients were similar. The CT, CFT, MCF and the α-angle were similar between the groups with trends towards shorter CT in the extrinsic and fibrin activation.

Conclusions/Significance

We found no differences in coagulation parameters analyzed with rotational thrombelastometry between cTnI-positive and -negative patients with SIRS, severe sepsis, and septic shock. These findings suggest that pathophysiological mechanisms other than thrombus-associated myocardial damage might play a major role, including reversible myocardial membrane leakage and/or cytokine mediated apoptosis in these patients.  相似文献   
7.
Exclusion has been applied as a main measure for re-vegetation all over the world.This paper,by comparing the results of year-round exclusion,seasonal exclusion,and non-exclusion,quantified the vegetation variations under three different exclusion measures and their correlation to soil factors.The analysis results for community species component and plant diversity using multi-response permutation procedures (MRPPs)showed that exclusion did change the species component and increase plant diversity remarkably,while the period of exclusion had no significant influence on these two community features.The indicator species analysis and calculation of similarity indices indicated that community for year-round exclusion were becoming xerophytization and unpalatability,and showed highly spatial heterogeneity of plant species distribution,whereas community for seasonal exclusion was under stable non-equilibrium condition.Detrended correspondence analysis (DCA)and detrend canonical correspondence analysis (DCCA)results of relationship between plant species and soil variables demonstrated that soil moisture was a controlling factor for plant species component,microbiotic soil crust cover,soil organic matter,and soil bulk density had significant effects on soil moisture,among which microbiotic soil crust was a leading factor owing to its limitation to rainfall infiltration on the one hand,and its constraints to entrance of herbaceous seeds into soil or to germination of soil seeds on the other hand.As a result of long-term removal of animal grazing,crust kept intact in year-round exclusion community,which was a main reason of community xerophytization.It was also obvious from ordination results that some important environmental factors,such as tempo-spatial change of rainfall and corresponding tempo-spatial change of soil moisture,were neglected during direct gradient analysis.In addition,biodiversity was close related to soil nutrients as well as to soil moisture condition (soil water content and crust cover),and it had positive relation to available N,and negative relation to available P.Higher soil N had advantage to non-leguminous plants growth on nutrition-poor sand land definitely.The impact of P to community component was unclear and should be studied from plant physiology.Further researches on nonequilibrium theory in semi-arid rangeland will provide a scientific and flexible animal development paradigm for being implementing livestock fen-raising and grazing-forbidden policies in China.  相似文献   
8.
目的: 心力衰竭患者呼吸调控异常的机制众说纷纭,特别是动脉血气周期性波浪式变化信号的改变及其与心功能的关系尚缺乏直接的试验证据。本文依据心力衰竭患者动脉血气周期性波浪式变化信号的降低幅度,探讨心力衰竭导致呼吸调控异常的机制。方法: 选择5名心力衰竭患者,连续桡动脉逐搏取血,测定PaO2,PaCO2,pHa和SaO2。选取2个典型呼吸周期,用于分析动脉血气的波浪式变化。比较患者相邻最高和最低值,以验证是否存在周期性波浪式信号变化。此外,将心力衰竭患者与心功能正常患者动脉血气周期性波浪式信号的变化幅度进行统计学t检验分析,比较有无差异。结果: 心力衰竭患者包括2例外科手术和3例ICU住院监护患者,4男1女,年龄(69±7)岁,身高(169±10)cm,体质量(75±19)kg,左心射血分数(LVEF)=(38±3)%。动脉血液充满肝素化细长塑化管需要17±2次心跳,即取血需要17±2次心跳,覆盖超过2个呼吸周期。心力衰竭患者PaO2,PaCO2,[H+]a和SaO2均呈现明显的波浪式变化(P<0.05),幅度分别是(7.94±2.02)mmHg,(1.18±0.56)mmHg,(0.54±0.17)nmol/L和(0.21±0.07)%,分别是各自均值的(6.1±1.5)%,(3.2±1.5)%,(1.5±0.5)%和(0.2±0.1)%。与心功能正常患者比较,动脉血气波浪式变化幅度呈现明显降低趋势,但仅PaO2和[H+]a有明显统计学差异(P<0.05)。结论: 采用连续逐搏动脉取血血气分析法证实,心力衰竭患者自主呼吸时动脉血气也有周期性波浪式变化信号,但其变化幅度较心功能正常患者明显降低。  相似文献   
9.
目的: 在完成吸入室内空气状态下症状限制性最大极限心肺运动试验(CPET)和动脉血气指标动态变化规律的基础上,进一步探讨体液酸碱度和CO2含量对呼吸调控的影响。方法: 选正常志愿者5名,给予5%NaHCO3(总量0.3 g/kg)分次口服,每5 min口服75 ml(3.75g )。总量服完1 h后,重复CPET。于静息、热身、运动及恢复期,连续测定肺通气指标及每分钟动脉取样的血气指标变化,并与本人在非碱化血液条件下对照数据进行配对t检验比较。结果: 碱化血液之后,CPET期间随着运动功率逐步递增,气体交换和血气指标的反应模式与非碱化血液对照相似(P>0.05);即与静息状态比较,每分通气量、潮气量、呼吸频率、VO2、VCO2均呈现近于线性渐进性递增(P<0.05~0.001)。与碱化血液前吸入室内空气的对照比较:在碱化血液条件下,所有时间点血红蛋白浓度,PaCO2与pH均显著提高(P<0.05);除无氧阈PaCO2减低外,只有热身状态呈增高态势,统计学有显著差异(P<0.05);而PaO2无差异(P>0.05),各状态均较对照状态减低,除恢复期外均有统计学差异(P<0.05)。与非碱化血液对照比较,除静息每分通气量低于对照(P<0.05)外,所有通气指标均无统计学差异(P>0.05)。结论: 碱化血液条件下, 尽管有更高的CaCO2, PaCO2 和 pHa平均水平及更低的Hba和[H+]a平均水平,机体对CPET的呼吸反应模式基本相似。  相似文献   
10.
50年来山东塔山植被与物种多样性的变化   总被引:3,自引:0,他引:3       下载免费PDF全文
高远  陈玉峰  董恒  郝加琛  慈海鑫 《生态学报》2011,31(20):5984-5991
为分析塔山植被与物种多样性50a自然演替和动态变化,2009-2010年,采用系统勘踏法和典型取样法进行了调查。当前塔山主要植被类型为\"黑松林-赤松林-栓皮栎林\"。50a间,该区针叶林从黑松(Pinus thunbergii)林演替为黑松林、赤松(P. densiflora)林和油松林(P. tabulaeformis)为主的混合针叶林,但针叶林的整体优势度下降,以栓皮栎(Quercus variabilis)和麻栎(Q. acutissima)为建群种的阔叶林面积明显增大,由针叶林向阔叶林的演替趋势明朗。物种丰富度为草本层>灌木层>乔木层,Shannon-wiener指数和Simpson指数为灌木层>草本层>乔木层,物种多样性较低,处于森林演替初期。  相似文献   
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