Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins. (C) 2010 Elsevier B.V. All rights reserved.

中国明对虾免疫系统中抗氧化相关基因的克隆与表达分析

对虾病害在世界范围内的广泛传播，给水产养殖和沿海农村经济造成了重大损失。深入开展对虾免疫机制研究并在此基础上寻找对虾疾病防治的有效方法已成为当务之急。研究表明，当对虾等甲壳动物受到外界病原刺激时，其体内的吞噬细胞在吞噬活动中会激活磷酸己糖支路的代谢，引起呼吸爆发，产生多种活性氧分子。另外，受到病原侵染的对虾还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发大量活性氧的产生。这些活性氧分子可以杀灭入侵的病原微生物，但同时由于活性氧分子反应的非特异性，它们也会对宿主的细胞、组织和器官造成严重伤害，进而导致对虾生理机能的损伤和免疫系统的破坏。所以，消除对虾体内因过度免疫反应产生的过量氧自由基将能够增强其抵御病原侵染的能力，提高免疫力。本论文从中国明对虾体内克隆了线粒体型超氧化物歧化酶（mMnSOD）、胞质型超氧化物歧化酶（cMnSOD）、过氧化氢酶（Catalase）和过氧化物还原酶（Peroxiredoxin）等四种与免疫系统相关的抗氧化酶基因，分析了它们的分子结构特征，组织分布及应答不同病原刺激的表达变化模式，并对其中的mMnSOD基因和Peroxiredoxin基因进行了体外重组表达、分离纯化和酶活性分析。 采用RACE技术从中国明对虾血细胞中克隆了两个超氧化物歧化酶（SOD）基因，通过序列比对分析发现，其中一个为mMnSOD基因，另一个为cMnSOD基因。mMnSOD基因的cDNA全长为1185个碱基，其中开放阅读框为660个碱基，编码220个氨基酸，其中推测的信号肽为20个氨基酸。多序列比对结果显示中国明对虾mMnSOD基因的推导氨基酸序列与罗氏沼虾、蓝蟹的推导氨基酸序列同源性分别为88%和82%。Northern blot结果表明，该基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。半定量RT-PCR结果显示，对虾感染病毒3 h时，该基因在血细胞和肝胰脏中的转录水平显著升高。此外，通过构建原核表达载体，本研究对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活分析。cMnSOD基因的cDNA全长为1284个碱基，其中开放阅读框为861个碱基，编码287个氨基酸。多序列比对结果显示中国明对虾cMnSOD基因的推导氨基酸序列与斑节对虾和凡纳滨对虾的同源性高达98%和94%。组织半定量结果显示，cMnSOD基因在对虾被检测的各个组织中均有表达。 另外，半定量RT-PCR结果表明，对虾感染病毒23h时，该基因在肝胰脏中的转录上升到正常水平的3.5倍；而感染后59 h时，该基因在血细胞中的转录上升到正常水平的2.5倍。 利用根据其他生物过氧化氢酶保守氨基酸序列设计的简并引物，结合RACE技术，从中国明对虾肝胰脏中克隆到了过氧化氢酶基因的部分片段，片段长1725个碱基。多序列比对结果发现目前所得中国明对虾Catalase基因部分片段的推导氨基酸序列与罗氏沼虾和皱纹盘鲍Catalase氨基酸序列的同源性分别达到95%和73%。通过实时荧光定量PCR技术对中国明对虾Catalase基因在各个组织中的分布情况及病毒感染后该基因在血细胞和肝胰脏中的转录变化进行了研究。结果发现，该基因在肝胰脏、鳃、肠和血细胞中表达水平较高，在卵巢、淋巴器官和肌肉中的表达水平相对较弱；感染病毒23 h和37 h时，对虾血细胞和肝胰脏中该基因mRNA的表达量分别出现显著性上升。 依据中国明对虾头胸部cDNA文库提供的部分片段信息，结合SMART-RACE技术，从中国明对虾肝胰脏中克隆到了过氧化物还原酶基因（Peroxiredoxin）， 该基因的cDNA全长为942个碱基，其中开放阅读框为594个碱基，编码198个氨基酸。中国明对虾Peroxiredoxin基因的推断氨基酸序列与伊蚊、文昌鱼和果蝇等Peroxiredoxin基因的推断氨基酸序列同源性分别为77%、76%和73%。其蛋白理论分子量为22041.17 Da，pI为5.17。Northern blot结果表明，Peroxiredoxin基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。实时荧光定量PCR结果显示，弧菌感染后，该基因在对虾血细胞和肝胰脏中的转录水平都有明显变化并且表达模式不同。另外，对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活性分析。酶活性分析表明，复性后的重组蛋白能在DTT存在的条件下还原H2O2。

Effects of increased pCO(2) and temperature on the North Atlantic spring bloom. I. The phytoplankton community and biogeochemical response

The North Atlantic spring bloom is one of the largest annual biological events in the ocean, and is characterized by dominance transitions from siliceous (diatoms) to calcareous (coccolithophores) algal groups. To study the effects of future global change on these phytoplankton and the biogeochemical cycles they mediate, a shipboard continuous culture experiment (Ecostat) was conducted in June 2005 during this transition period. Four treatments were examined: (1) 12 degrees C and 390 ppm CO2 (ambient control), (2) 12 degrees C and 690 ppm CO2 (high pCO(2)) (3) 16 degrees C and 390 ppm CO2 (high temperature), and (4) 16 degrees C and 690 ppm CO2 ('greenhouse'). Nutrient availability in all treatments was designed to reproduce the low silicate conditions typical of this late stage of the bloom. Both elevated pCO(2) and temperature resulted in changes in phytoplankton community structure. Increased temperature promoted whole community photosynthesis and particulate organic carbon (POC) production rates per unit chlorophyll a. Despite much higher coccolithophore abundance in the greenhouse treatment, particulate inorganic carbon production (calcification) was significantly decreased by the combination of increased pCO(2) and temperature. Our experiments suggest that future trends during the bloom could include greatly reduced export of calcium carbonate relative to POC, thus providing a potential negative feedback to atmospheric CO2 concentration. Other trends with potential climate feedback effects include decreased community biogenic silica to POC ratios at higher temperature. These shipboard experiments suggest the need to examine whether future pCO2 and temperature increases on longer decadal timescales will similarly alter the biological and biogeochemical dynamics of the North Atlantic spring bloom.

水葫芦苗(Halerpestes cymbalaris)的生长特征研究

以调查统计的方法在中国科学院海北定位站研究了高寒湿地植物水葫芦苗无性系的生长特征、形态特征以及能量分配规律.结果表明:匍匐茎只有1条的水葫芦苗最多,占35.29%,匍匐茎有4条的水葫芦苗只占 8.82%.同一水葫芦苗无性系中,随着匍匐茎数目的增多,分株数、间隔子数、茎总长和匍匐茎比节间重变小.分株一般在第一级最高,末级较低;第1条匍匐茎的间隔子较长.随水葫芦苗匍匐茎数目的增多,用于无性繁殖的分株干重比例逐渐增加,用于有性繁殖的花的干重比例下降.水葫芦苗无性系这种生长特征、形态特征以及能量分配规律是其生物-生态学特性和所处高寒湿地 生境共同决定的.

智能机器人分层系统中的人机交互

在非结构环境里工作的智能机器人,人智能的介入有时是必不可少的.本文研究了这类机器人系统人机接口某些问题,提出了人智能输入的分层输入原则,实时性原则和协调性原则,讨论了人机接口的结构和实现问题,给出了人机接口命令的一种格式,并提出了人机操作的实际例子.

渤海湾地区末次冰期古环境

The loess-paleosol sequence in China is one of the best archives for studying paleoenvironmental and paleoclimatic processes. The loess deposits in the coast of the Bohai Sea are suitable for the study of aridification in the northern China during glacial periods (fig.2-1). In this paper, stratigraphy was correlated by using magnetic susceptibility, grain size, and thermoluminence (TL) and accelerater mass spectrometer (AMS) ~(14)C ages. Based on the loess records, an interpretation has been made for paleoenvironmental changes on the coast of the Bohai Sea during the last glacials. The results of magnetic susceptibility, grain size, biostratigraphy, TL and ~(14)C dating, suggest that the loess-paleosol sequence in the coast of the Bohai Sea is discontinuous. The loess deposits correlated with the marine δ~(18)O stage 2 are usually absent in some profiles. Also, the thickness of the loess deposits in the same period varied significantly in different sections. In the coast of the Bohai Sea, the higher magnetic susceptibility corresponds to the finer grain size, consistent with the results of the Loess Plateau. It is indicated that the changes in magnetic susceptibility and grain size may record the paleoclimatic fluctuations of the last glacial. Although the loess deposits during the last glacial have been slightly altered by slope runoff, they have still remained main characters as the representative loess deposits of the Loess Plateau. During the last glacial, the less accumulation rate in the coast of the Bohai Sea is similar to that of the desert-loess transition zone in the northwestern Loess Plateau, and the all section contain high concentrations of sand (>60μm), indicate that the aridification in the Bohai Sea occurred during the glacial. But the changes in sand content of loess deposits along a north-south transect of the Bohai Sea and the changes of magnetic susceptibility implicate that desertification might not occur in the shelf of the Bohai Sea during the last glacial. The frequent fluctuations of summer monsoon during the marine δ~(18)O stage 4 are demonstrated by magnetic susceptibility, frequency-dependent susceptibility and the abundance of foraminifera. 46 genera, 71 species of foraminifera were identified from 138 loess samples. Almost all of the foraminifera are present in the last glacial loess, but the distribution patterns of foraminifera show significant temporal changes. The results of magnetic susceptibility, grain size and XRD indicate that not only sea-level changes had influences on foraminifera abundance, but also variations in sediment flux by rivers when sea-level drops might control the abundance of foraminifera. In addition, the diversity and exquitability of the foraminifera suggest that the frequent fluctuations of foraminifera fossil abundance during the marine δ~(18)O stages could be partly attributed to leaching. In summary, the changes in foraminifera of abundance related to the paleoclimatic variations, and the aridification extends to the coast of the eastern China during the last glacial.

喀斯特石漠化的研究现状与存在的问题

喀斯特石漠化是土地荒漠化的主要类型之一。 喀斯特生态系统的脆弱性决定了喀斯特生态系统的易损性和退化喀斯特生态系统的难恢复性，但它与发生石漠化并没必然的联系，喀斯特石漠化是岩溶生态系统退化的结果，而不是喀斯特生态系统脆弱性的必然表现。故所指的喀斯特石漠化，特指人为加速的石漠化，属于人为荒漠化的一种类型，但强调喀斯特石漠化的人为成因性，并不是否定自然背景对喀斯特生态系统的制约作用。石漠化土地的分类既要能反映出石漠化发生的严重程度，也要能体现石漠化土地的成因和岩性地貌等地质背景对生态学过程的影响，因此，作者提出以“土地利用类型+植被+岩性+地貌+石漠化程度”对人为加速石漠化过程中石漠化土地进行类型划分，以正确评价石漠化土地的生态环境价值。

Sensoriamento remoto e geoprocessamento aplicados à reconstituição histórica de parte da "Estrada dos Goiáses".

O trabalho elabora considerações, sobre o debate do bicentenário da morte de Morgado de Mateus (1798-1998), no âmbito das celebrações dos quinhentos anos do descobrimento. Foi realizado uma reconstituição do trecho campineiro do antigo percurso da estrada dos Goiáses, trajeto dos bandeirantes, concentrando-se no trecho que vai de Jundiaí a Moji Mirim, com ênfase à região de Campinas.

Analysis of the mechanism and regulation of lactose transport and metabolism in Clostridium acetobutylicum ATCC 824.

Although the acetone-butanol-ethanol (ABE) fermentation of Clostridium acetobutylicum is currently uneconomic, the ability of the bacterium to metabolise a wide range of carbohydrates offers the potential for revival based on the use of cheap, low grade substrates. We have investigated the uptake and metabolism of lactose, the major sugar in industrial whey waste, by C. acetobutylicum ATCC 824. Lactose is taken up via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) comprising both soluble and membrane-associated components, and the resulting phosphorylated derivative is hydrolysed by a phospho--galactosidase. These activities are induced during growth on lactose, but are absent in glucose-grown cells. Analysis of the C. acetobutylicum genome sequence identified a gene system, lacRFEG, encoding a transcriptional regulator of the DeoR family, IIA and IICB components of a lactose PTS, and phospho--galactosidase. During growth in medium containing both glucose and lactose, C. acetobutylicum exhibited a classical diauxic growth, and the lac operon was not expressed until glucose was exhausted from the medium. The presence upstream of lacR of a potential catabolite responsive element (cre) encompassing the transcriptional start site is indicative of the mechanism of carbon catabolite repression characteristic of low-GC Gram-positive bacteria. A pathway for the uptake and metabolism of lactose by this industrially important organism is proposed.

Improving accessibility in discussion forums

No data (2013)

Memorias políticas y militares, para servir de continuacion a los Comentarios del Marques de San Felipe : desde el año de MDCCXXV, en que concluyó este autor su obra, hasta el presente, con los Tratados de Paz Y Alianzas de España correspondientes

Signaturas: [ ]1, A-Z4, 2A-2R4, 2S1.

SS.PP. Toletanorum quotquot extant Opera : tomus primus [-tertius]

Nunc primum simul edita, ad codices mss. recognita, nonnullis notis illustrata atque in duos tomos distributa.

Memoir of Mrs. Ann H. Judson, wife of the Rev. Adoniraum Judson, missionary to Burmah, including a history of the American Baptist Mission in the Burman Empire.

http://www.archive.org/details/memoirofmrsannhj00judsuoft

An account of the life of Mr. David Brainerd : missionary from the Society for Propagating Christian Knowledge, & pastor of a church of Christian Indians in New-Jersey / published by Jonathan Edwards ; with Mr. Brainerd's public journal ; to this edition is added, Mr. Beatty's mission to the westward of the Allegheny Mountains.

http://www.archive.org/details/accountoflifeofm00brairich

A proposal for the better supplying of churches in our foreign plantations, and for converting the savage Americans to Christianity, by a college to be erected in the Summer Islands, otherwise called the Isles of Bermuda.

http://www.archive.org/details/proposalforbette00berkrich