甘油和蔗糖对光系统I结构与功能的影响

在光系统I(PSI)内部结合有大量的水分子，而这些水分子的生理功能还不清楚。在本工作中，我们通过外加具有渗透活性和吸水强的多羟基化合物甘油和蔗糖来改变PSI环境中水的含量，研究水的改变对PSI结构与功能的影响。主要结果如下： 1．甘油和蔗糖对PSI的电子传递产生影响，影响程度和大小与它们的浓度有关。一般地，低浓度的甘油和蔗糖可促进PSI的电子传递，而在高浓度时，这种促进作用有所减弱。但过高浓度的甘油(>60%, v/v)会抑制PSI的电子传递活性。 2．与对PSI电子传递的影响趋势相类似，在低浓度的甘油和蔗糖存在下，PSI的光化学反应活性(PSI反应中心色素P700的光氧化还原能力)大为增加，而较高浓度的甘油和蔗糖对P700的氧化还原能力有所抑制。 3．甘油和蔗糖也会改变PSI中的主体色素(bulk chlorophyll)和长波色素或红色素(red chlorophyll)之间的能量分布。它们的作用导致激发能分配失衡，使更多的激发能分配到红色素。 4．甘油和蔗糖的作用还会影响PSI的蛋白质构象。甘油使PSI蛋白质内部的色氨酸残基(Trpapolar)处于更加疏水的微环境，而蔗糖却使极性环境中的色氨酸残基(Trppolar)周围微环境的极性继续增大。它们均会使色氨酸残基邻近的具有淬灭活性的蛋白质的位置和/或方向有所变化。同时，甘油和蔗糖的作用也会导致PSI的疏水性增加。

光系统I及其亚组分的分离纯化与光破坏特性的研究

光系统I（photosystem I，PSI）是光合膜上参与光合作用原初反应过程的主要膜蛋白超分子复合体之一。高等植物的PSI是由核心复合体（14个亚基）和捕光色素蛋白复合体I（light-harvesting complex I, LHCI，含4个Lhca蛋白）组成的超分子复合体，它的主要功能是调节光诱导的从囊腔侧的质体兰素（plastocyanin，PC）向基质侧的铁氧还蛋白（ferredoxin，Fd）的电子传递。研究PSI的结构与功能对于揭示植物光合作用高效吸能、传能的分子机理具有重要意义。在本文中，我们首先建立了分离制备PSI及其亚组分的方法（Qin et al., 2007），并在此基础上对PSI在强光破坏的过程中结构与功能的变化进行了比较深入的研究。本论文的主要研究结果如下： 1．快速、高效分离纯化PSI及其亚组分方法的建立。 国际上传统的PSI分离方法（Bassi and Simpson, 1987; Croce et al., 1998; Påsllon et al.1995; Schmid et al. 2002），耗时长较长（分离PSI颗粒一般需要多于20h的蔗糖超速离心过程，而分离PSI的亚组分则需要25-60h的蔗糖超速离心过程）、得率较低，这不便于PSI方面的研究，为此我们首先改进了传统的分离纯化方法。新方法以高等植物菠菜叶片作为原材料，使用Triton X-100作为增溶剂，通过差速离心技术获得的粗制品，然后使用十二烷基麦芽糖苷(n-Dodecyl β-D-maltoside, DDM)增溶PSI粗制品，之后采用100,000×g，垂直转头（Beckman VTi 50）0.1-1 mol/L蔗糖梯度离心3h获得纯度较高的PSI颗粒。然后使用DDM和3-(N, N-Dimethylpalmitylammonio) propanesulfonate （zw 3-16）两种增溶剂处理PSI，后经100,000×g，垂直转头（Beckman VTi 50）蔗糖梯度离心4h获得纯度较高的PSI core、LHCI-680、LHCI-730复合体。采用吸收光谱、荧光光谱技术研究了各样品的基本光谱学特性，采用HPLC分析了各样品的色素组成，结果显示平均每个Lhca蛋白结合1.5-1.6黄体素，1.0紫黄质, 0.8-1.1 β-胡萝卜素，该方法制备的LHCI比传统方法制备的LHCI减少了类胡萝卜素的丢失。这一工作为以后结构与功能的研究工作奠定了良好的基础。 2．PSI复合体及其亚组分的特性研究。 PSI颗粒具有一定的适应环境酸碱变化的能力，在我们的试验条件下PSI颗粒在pH 5-10相对稳定。PSI、LHCI很难通过加入Mg2+、Ca2+、Na+阳离子聚集沉淀。经绿胶鉴定我们制备的LHCI-680、LHCI-730是二聚体形式;而把PSI绿胶后再进行第二向十二烷基硫酸钠－聚丙烯酰氨凝胶电泳（SDS-PAGE）电泳，结果发现在稍强烈的绿胶增溶条件下，LHCI-730是以二聚体的形式存在，但是LHCI-680却是以单体的形式出现。这说明LHCI形成的二聚体，尤其是LHCI-680，较容易受到增溶处理而分离成单体形式，解释了以生化分离手段得到的LHCI-680的聚集形式是单体还是二聚体这个目前国际上还有有争议的问题。 3．PSI、LHCI光破坏的基本特点。 采用白光（2500 μmol•m-2•s-1）照射PSI颗粒，通过SDS-PAGE及室温吸收光谱检测光照过程中PSI复合体的变化，结果表明：去氧处理能够大大延缓PSI的光破坏，而PSI脱辅基蛋白不会发生光破坏，这说明PSI发生的光破坏可能与Chl与O2的相互作用有关。采用白光（1000 μmol•m-2•s-1、300 μmol•m-2•s-1）处理LHCI-680、LHCI-730，发现LHCI-680被破坏的速度明显快于LHCI-730被破坏的速度，这是首次在体外分离的水平上揭示了不同LHCI光破坏方面的差异。LHCI-680与LHCI-730在光破坏方面的差异可能与两种天线蛋白结合的类胡萝卜素的种类和数量不同有关，还可能与二者结合的长波长Chl的情况有关，但是具体的原因还有待于进一步的研究。 4．结合不同的捕光色素蛋白复合体（light-harvesting complex，LHC）对PSI光破坏的影响。 为了研究结合不同的捕光天线对PSI光破坏的影响，我们制备了PSI-LHCII、PSI、PSI core三种复合体。使用白光（2500 μmol•m-2•s-1）照射这三种复合体，并通过测定各复合体在光破坏过程中蛋白亚基、吸收光谱、PSI活性及P700含量的变化，对比三者光破坏的速度，结果发现PSI-LHCII在这三种复合体中光破坏速度最快，而PSI和PSI core两种复合体光破坏速度基本一致。我们推测在光照过程中部分光系统II捕光Chl a/b蛋白复合体II（light-harvesting complex II，LHCII）能够向PSI core传递能量，另外PSI-LHCII绿胶分析的结果表明发生了LHCII三聚体向单体的转变，这种强光下发生的LHCII聚合形式的转化可能是高光强下调节光能捕获的一种机制，由于植物体内具有较完整的保护系统，体内PSI-LHCII的光破坏可能与体外情况不同;另外LHCI与PSI core的解离可能发生在强光照射的早期，具有保护PSI core减少光破坏的积极作用。该部分的研究首次观察了结合不同的捕光天线对PSI光破坏的影响。

磷脂酰甘油对光系统I的调控作用

磷脂酰甘油(PG)是光系统I(PSI)中唯一的磷脂，也是PSI重要的组成部分。在本工作中，我们通过改变PSI中PG的含量(体外重组至PG脂质体或专一性磷脂酶降解)，研究了PG对PSI的调控作用。主要结果如下： 1. 外加PG导致PSI色素的结合状态和激子相互作用发生改变。吸收光谱中，Chl a特征峰蓝移且吸收降低。低温荧光光谱中，680nm处的峰逐渐明显，F730-735 /F680的比值下降，LHCI-730激发峰蓝移。可视CD光谱中Chl a、Chl b蓝移，它们的相互作用增强;类胡萝卜素分子发生红移。 2. PSI的重组引起了PSI蛋白质结构的改变，即蛋白的α-螺旋结构增加而无序结构含量减少。同时，PSI蛋白质内部的色氨酸残基处于更极性的环境。 3. PG对PSI的电子传递的影响具有浓度效应。低浓度时可以促进PSI的电子传递活性，而在相对较高浓度时抑制PSI的电子传递。 4. PLA2的处理导致PSI中PG的缺失，抑制了PSI反应中心P700的暗还原反应，即延长了其还原所用的时间。P700的暗还原反应存在快相和慢相两相反应。PG的缺失降低了这两相反应的反应速率，抑制了电子从质体蓝素(PC)到P700+的传递。

Identification and cloning of a trypsin inhibitor from skin secretions of Chinese red-belly toad Bombina maxima

A novel trypsin inhibitor was identified and purified from skin secretions of Chinese red-belly toad Bombina maxima. The partial N-terminal 29 amino acid residues of the peptide, named BMTI, were determined by automated Edman degradation. This allowed the cloning of a full-length cDNA encoding BMTI from a cDNA library prepared from the toad skin. The deduced complete amino acid sequence of BMTI indicates that mature BMTI is composed of 60 amino acids. A FASTA search in the databanks revealed that BMTI exhibits 81.7% sequence identity with BSTI, a trypsin/thrombin inhibitor from European toad Bombina bombina skin secretions. Sequence differences between BMTI and BSTI were due to 11 substitutions at positions 2, 9, 25, 27, 36-37, 39, 41-42, 50 and 56. BMTI potently inhibited trypsin with a K-i value of 0.06 muM, similar to that of BSTI. However, unlike BSTI, which also inhibited thrombin with a K-i value of 1 muM, no inhibitory effect of BMTI on thrombin was observed under the assay conditions. (C) 2002 Elsevier Science Inc. All rights reserved.

Isolation and preliminary characterization of a 22-kDa protein with trypsin inhibitory activity from toad Bufo andrewsi skin

A novel trypsin inhibitor termed BATI was purified to homogeneity from the skin extracts of toad Bufo andrewsi by successive ion-exchange, gel-filtration and reverse-phase chromatography. BATI is basic single chain glycoprotein, with apparent molecular weight of 22 kDa in SDS-PAGE. BATI is a thermal stable competitive inhibitor and effectively inhibits trypsin's catalytic activity on peptide substrate with the inhibitor constant (K-i) value of 14 nM and shows no inhibitory effect on chymotrypsin, thrombin and elastase. The N-terminal sequence of BATI is EKDSITD, which shows no similarity with other known trypsin inhibitors. (c) 2005 Elsevier Ltd. All rights reserved.

Two serine protease inhibitors from the skin secretions of the toad, Bombina microdeladigitora

Two serine protease inhibitors (named BMSI 1 and BMSI 2, respectively) were identified from the skin secretions of the toad, Bombina microdeladigitora. The cDNAs encoding BMSIs were cloned from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences of BMSIs indicate that mature BMSI1 and BMSI2 are composed of 60 amino acids including 10 half-cystines to form 5 disulfide bridges. A FASTA search in the databanks revealed that BMSIs exhibit sequence similarity with other serine protease inhibitors from amphibians of the genus Bombina. BMSI1 potently inhibited trypsin and thrombin with a K(i) value of 0.02 mu M and 0.15 mu M, respectively. Sequence analysis revealed that all serine protease inhibitors from five amphibians of the genus Bombina share highly conserved primary structures. (c) 2007 Elsevier Inc. All rights reserved.

Comparative study of three short-chain neurotoxins from the venom of Naja kaouthia (Yunnan, China)

Three short-chain neurotoxins named NT-I, NT-II, and NT-III were purified from the venom of Naja kaouthia, a snake distributed throughout the south of Yunnan province, China, by a series of chromatographic steps, including an FPLC Resource S column. Their molecular weights, determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS, were 6952.19 Da, 6854.92 Da, and 6828.80 Da, respectively. NT-I consisted of 62 amino acid residues, and the other two consisted of 61 amino acid residues, including 8 cysteines. After hydrolysis by endoproteinase Glu-C, their primary sequences were determined. A test of their activities demonstrated that they effectively inhibited muscle contractions induced by electric stimulation. Furthermore, the extent of inhibition caused by NT-II and NT-III was less than that of NT-I. The IC(50)s were 0.04 mug/ml, 0.20 mug/ml, and 0.23 mug/ml for NT-I, NT-II, and NT-III, respectively. Compared with NT-II and NT-III, the higher activity of NT-I may be a result of the amino acid residue substitution Ile36 to Arg36.

A revision of the white-toothed shrews (Crocidura) of Southern China

Many systematic relationships among Chinese white-toothed shrews of genus Crocidura are presently unresolved. In this paper, a taxonomic revision of Crocidura from Southern China is presented. We studied 338 specimens from Burma, China, Korea, Pakistan, Turkey, Middle and Central Asia, and Russia (Appendix I), 285 of which had complete skulls that were analyzed with principal component and discriminant analyses. Results indicated that 6 species of Crocidura can be recognized in South China. C. fuliginosa occurs in Southwestern and Eastern China, C. attenuata is broadly distributed throughout Southern China, and C. horsfieldii is restricted to the southern part of China. C vorax and C. rapax, usually placed as synonyms of the European C russula, are recognized as 2 valid species whose ranges overlap in Southwestern China. C. shantungensis of Eastern Asia extends to the northern part of Southern China and is distinct from C. suaveolens and C. gmelini of Middle and Central Asia, respectively.

Annual Report of the East African Agricultural and Fisheries Research Council 1958

The annual report present activities carried out by the different organizations that make up the East African Agricultural & Fisheries Research Council which covers reports from the following Organisations: I. Report of the East African Agriculture and Forestry Research Organization 2. Report of the East African Fishery Research Organization 3. Report of the East African Marine Fisheries Research Organization 4. Report of the East African Trypanosomiasis Research Organization 5. Report of the East African Veterinary Research Organization The activities reported are for the period 1958

Annual Report of the East African Agricultural and Fisheries Research Council 1956-57

The annual report present activities carried out by the different organizations that make up the East African Agricultural & Fisheries Research Council which covers reports from the following Organisations: I. Report of the East African Agriculture and Forestry Research Organization 2. Report of the East African Fishery Research Organization 3. Report of the East African Marine Fisheries Research Organization 4. Report of the East African Trypanosomiasis Research Organization and 5. Report of the East African Veterinary Research Organization The activities reported are for the period 1956-57

Measurements of triggering and transient growth in a model lean-premixed gas turbine combustor

Instability triggering and transient growth of thermoacoustic oscillations were experimentally investigated in combination with linear/nonlinear flame transfer function (FTF) methodology in a model lean-premixed gas turbine combustor operated with CH 4 and air at atmospheric pressure. A fully premixed flame with 10kW thermal power and an equivalence ratio of 0.60 was chosen for detailed characterization of the nonlinear transient behaviors. Flame transfer functions were experimentally determined by simultaneous measurements of inlet velocity fluctuations and heat release rate oscillations using a constant temperature anemometer and OH */CH * chemiluminescence emissions, respectively. The phase-resolved variation of the local flame structure at a limit cycle was measured by planar laser-induced fluorescence of OH. Simultaneous measurements of inlet velocity, OH */CH * emission, and acoustic pressure were performed to investigate the temporal evolution of the system from a stable to a limit cycle operation. This measurement allows us to describe an unsteady instability triggering event in terms of several distinct stages: (i) initiation of a small perturbation, (ii) exponential amplification, (iii) saturation, (iv) nonlinear evolution of the perturbations towards a new unstable periodic state, (v) quasi-steady low-amplitude periodic oscillation, and (vi) fully-developed high-amplitude limit cycle oscillation. Phase-plane portraits of instantaneous inlet velocity and heat release rate clearly show the presence of two different attractors. Depending on its initial position in phase space at infinitesimally small amplitude, the system evolves towards either a high-amplitude oscillatory state or a low-amplitude oscillatory state. This transient phenomenon was analyzed using frequency- and amplitude-dependent damping mechanisms, and compared to subcritical and supercritical bifurcation theories. The results presented in this paper experimentally demonstrate the hypothesis proposed by Preetham et al. based on analytical and computational solutions of the nonlinear G-equation [J. Propul. Power 24 (2008) 1390-1402]. Good quantitative agreement was obtained between measurements and predictions in terms of the conditions for the onset of triggering and the amplitude of triggered combustion instabilities. © 2011 The Combustion Institute.

The effect of remodelling and contractility of the actin cytoskeleton on the shear resistance of single cells: a computational and experimental investigation.

The biomechanisms that govern the response of chondrocytes to mechanical stimuli are poorly understood. In this study, a series of in vitro tests are performed, in which single chondrocytes are subjected to shear deformation by a horizontally moving probe. Dramatically different probe force-indentation curves are obtained for untreated cells and for cells in which the actin cytoskeleton has been disrupted. Untreated cells exhibit a rapid increase in force upon probe contact followed by yielding behaviour. Cells in which the contractile actin cytoskeleton was removed exhibit a linear force-indentation response. In order to investigate the mechanisms underlying this behaviour, a three-dimensional active modelling framework incorporating stress fibre (SF) remodelling and contractility is used to simulate the in vitro tests. Simulations reveal that the characteristic force-indentation curve observed for untreated chondrocytes occurs as a result of two factors: (i) yielding of SFs due to stretching of the cytoplasm near the probe and (ii) dissociation of SFs due to reduced cytoplasm tension at the front of the cell. In contrast, a passive hyperelastic model predicts a linear force-indentation curve similar to that observed for cells in which the actin cytoskeleton has been disrupted. This combined modelling-experimental study offers a novel insight into the role of the active contractility and remodelling of the actin cytoskeleton in the response of chondrocytes to mechanical loading.

Comparison of Civil Infrastructure Optical-based Spatial Data Acquisition Techniques

Infrastructure spatial data, such as the orientation and the location of in place structures and these structures' boundaries and areas, play a very important role for many civil infrastructure development and rehabilitation applications, such as defect detection, site planning, on-site safety assistance and others. In order to acquire these data, a number of modern optical-based spatial data acquisition techniques can be used. These techniques are based on stereo vision, optics, time of flight, etc., and have distinct characteristics, benefits and limitations. The main purpose of this paper is to compare these infrastructure optical-based spatial data acquisition techniques based on civil infrastructure application requirements. In order to achieve this goal, the benefits and limitations of these techniques were identified. Subsequently, these techniques were compared according to applications' requirements, such as spatial accuracy, the automation of acquisition, the portability of devices and others. With the help of this comparison, unique characteristics of these techniques were identified so that practitioners will be able to select an appropriate technique for their own applications.