福氏志贺菌硫氧还过氧化物酶的晶体生长和初步晶体学研究（英文）

过氧化物酶是一类广泛存在于生物体内的抗氧化剂,清除有氧代谢过程中产生的活性氧,对于保护机体内的生物大分子有重要的生物学功能.福氏志贺菌硫氧还过氧化物酶(SF2523)作为过氧化物酶家族的一员,通过清除福氏志贺菌体内的活性氧,在维持其活性和致病性上起重要作用.目前,SF2523的三维结构还没有得到解析,其具体的功能机制也尚不清楚.为了得到SF2523蛋白的三维结构,进而了解具体的功能机制,实验获得了均一稳定的可溶蛋白,验证具有体外活性,培养出可用于X射线衍射的蛋白质晶体.在中国科学院高能物理研究所同步辐射装置收到晶体的衍射数据供结构解析使用.SF2523晶体属于空间群P2_12_12_1,晶胞参数为a=35.80,b=50.63,c=88.52,α=β=γ=90.00°,每个晶体学不对称单位含有1个蛋白质分子,马修斯系数为2.03~3/u,溶剂含量为39.56%.     

来源于Eisenia fetida的蚯蚓纤溶酶组分A的多对同晶置换法相位求解

来源于Eisenia fetida的蚯蚓纤溶酶组分A, 既是直接的纤溶酶, 又是纤溶酶原激活物的蛋白质, 已经被结晶. 晶体属于正交晶系, 空间群为P2₁2₁2₁, 每一个不对称单位含有3个蛋白质分子. 为了解析该蛋白质的衍射相位, 使用含有1.4 mol/L Li₂SO₄, 0.1 mol/L MOPS(pH 7.2)的重原子浸泡母液制备了4种合用的重原子衍生物. 用差值Patterson法和差值Fourier法确定了衍生物晶体中重原子的位置, 并将其联合修正获得0.25 nm分辨率的初始蛋白质结构相位. 通过重原子位置关系确定了不对称单位中3个独立蛋白质分子之间的非晶体学对称关系, 并利用其对初始的电子密度进行平均, 大大提高了电子密度质量, 为进一步的结构解析奠定了基础.     

白腐菌产锰过氧化物酶条件的研究

白腐菌Phanerochaeta chrysosporium MIG. 383降解桉木时具有显著的选择性,30天内降解37．23％Klason木素,7.29％综纤维素。该菌株产胞外锰过氧化物酶,并在高碳低氧培养基中显示较高酶活。静置液体培养的优化培养条件是(L^-1)：10g葡萄糖,2mmol酒石酸铵,10mmol pH4.5醋酸钠缓冲液,1g吐温80,2gK₂PO₄,0.5g MgSO₄·7H₂O,0.1g CaCl₂·2H₂O,lmg VB₁,70ml微量元素混合液：最适产酶温度是37℃。上述条件下,该菌接种后静置培养4天,产锰过氧化物酶活达1840U／L,酶作用最适温度是37℃,最适DH是3.5。     

龋齿致病菌变形链球菌蛋白 Smu.260 的 制备、结晶及初级晶体学分析

变形链球菌 (Streptococcus mutans) 是最主要的龋齿致病菌,其基因 Smu.260 编码一个约 23 ku (200 个氨基酸 ) 的蛋白质. Smu.260的 DNA 片段被克隆到表达载体 pET28a 后在大肠杆菌 BL21(DE3) 菌株中表达得到很好的产量. 产物 Smu.260 蛋白通过 Ni²⁺亲和柱和分子筛两步法纯化,并发现纯化后的蛋白以两种形式存在,二聚体 (约46 ku) 和四聚体,前者呈亮黄色,后者无色. 采用悬滴气象扩散法得到了二聚体形式的晶体. 晶体的 X 射线衍射分辨率达到 2.3埃,晶体属正交空间群 P2₁2₁2₁,晶格参数为a=89.88埃, b=90.91埃, c=105.17埃. 晶胞不对称单元内估计含有一个二聚体,溶剂含量为 53％ .     

福氏志贺菌硫氧还过氧化物酶的表达、纯化、活性检测及晶体生长的初步研究

Sf2523蛋白属于硫氧还蛋白过氧化物酶(Prx)家族,在有氧代谢过程中消除活性氧,起到保护生命大分子的重要作用.通过构建原核表达体系,可溶性表达并纯化了Sf2523酶蛋白,并对蛋白进行了过氧化物酶活性检测,证明其依然具有天然活性,酶的核心结构并未发生变化.由分子排阻色谱结果发现,酶蛋白体内和体外聚集状态不同,离体蛋白聚集状态不稳定,趋于二体化.将纯化的单体蛋白即时进行了结晶实验,初筛长出了针状晶体.通过进一步切除标签蛋白进行优化处理,最终得到了均匀的三维单晶.     

水稻磷脂氢谷胱甘肽过氧化物酶在蛋白质水平上的组织和诱导表达特征

蛋白质是生命活动的主要承担分子,了解蛋白质在有机体中的时空分布对于正确解析蛋白质的功能十分重要．磷脂氢谷胱甘肽过氧化物酶 (PHGPx) 是目前发现的唯一能够直接还原膜上脂类过氧化物的抗氧化酶,在保护生物膜免受过氧化损伤方面有着重要作用．采用Western blot技术,分析了水稻PHGPx (OsPHGPx) 在水稻不同组织以及多种胁迫条件下的蛋白质表达特征．结果表明,OsPHGPx在成熟水稻植株内主要分布于叶组织中,以旗叶中含量最高,而在水稻幼苗中则在茎及叶组织中均检测到较强的杂交信号．OsPHGPx在幼苗中的表达受到H₂O₂和NaCl的强烈诱导,但植物激素对其表达的影响较弱．H₂O₂和NaCl的诱导效果呈现出时间及剂量的相关性,当用0.5 mmol/L H₂O₂处理12 h或用500 mmol/L NaCl处理24 h,此时OsPHGPx表达量达到最大值．对H₂O₂清除剂二甲基硫脲处理的水稻幼苗,外源H₂O₂的再处理并不能诱导OsPHGPx的表达,而NaCl的诱导效果并不受影响,说明H₂O₂可能并不介导NaCl诱导OsPHGPx的表达．这些结果为进一步研究OsPHGPx在水稻中生物学功能奠定了基础．     

鸡肝果糖-2,6-二磷酸酯酶结构域的初步晶体学研究

从鸡肝6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酯酶分离的果糖-2,6-二磷酸酯酶结构域(残基245～468)已在E.coli中获得高效表达,并经分离得到纯化,使用悬滴气相扩散法成功地培养出该果糖-2,6-二磷酸酯酶结构域单晶.该酶晶体属于四方晶系,空间群为P4₁2₁2或P4₃2₁2,晶胞参数为:a=b=10.02nm,c=13.98nm,α=β=γ=90°.晶胞内每个结晶学不对称单位含有2个果糖-2,6-二磷酸酯酶分子.利用日本 Photon Factory同步辐射光源收集了分辨率为 0.32 nm的母体衍射据.     

N-糖链加工影响里氏木霉形态发生并提高木质纤维素降解能力

[背景] 烟曲霉α-1,2-甘露糖苷酶MsdS在高尔基体中将N-糖链Man₈GlcNAc₂加工为成熟分泌糖蛋白的糖型Man₆GlcNAc₂,有研究表明MsdS与烟曲霉的形态发生、细胞壁合成及蛋白质分泌密切相关;与烟曲霉不同的是,里氏木霉的成熟分泌糖蛋白上的N-糖链结构为Man₈GlcNAc₂,细胞却能正常生长,说明丝状真菌N-糖链的加工具有物种特异性,但其生物学意义不明。[目的] 为研究N-糖链加工对里氏木霉细胞生长及蛋白质分泌的影响,本研究将烟曲霉MsdS转入里氏木霉中以改变其成熟分泌糖蛋白的糖型。[方法] 构建带有烟曲霉msdS基因的重组质粒并转入里氏木霉中,获得msdS表达菌株Tr-MsdS,分析Tr-MsdS菌株的生长表型、N-糖组、蛋白质分泌途径和纤维素酶活性的变化。[结果] 在里氏木霉msdS表达菌株Tr-MsdS中,分泌糖蛋白的主要糖型由出发株的Man₈GlcNAc₂转变为Man₆GlcNAc₂,细胞壁组分发生变化,但细胞壁完整性未受影响;与出发株相比,Tr-MsdS菌株产孢、出芽及分枝增多;另外,MsdS的表达还影响蛋白质分泌,在50℃时降解纤维素和β-葡聚糖的能力分别提高9.9%和32.2%。[结论] 研究结果表明,N-糖链的加工可影响里氏木霉蛋白质,尤其是纤维素酶的分泌,干扰N-糖链加工可能是提高里氏木酶纤维素酶产量的新策略。     

过氧化氢对培养心肌细胞损伤作用的研究

氧化应激时产生大量的自由基,造成心肌细胞的损伤.过氧化氢(H₂O₂)是有机体氧化代谢产物,同时是一种活性氧.应用不同浓度的H₂O₂,分别于不同作用时间,动态观察其对心肌细胞的损伤作用.从实验结果看到,低浓度的H₂O₂（＜0.1 mmol/L）作用2 h,使心肌细胞产生早期的生物化学的改变,如MDA产生堆积和细胞周期时相改变（G1期细胞增加,G2期细胞减少）,此时心肌酶基本无泄漏,心肌细胞的死亡率很低,HE形态学观察基本无改变;随着H₂O₂浓度的增加（1～5 mmol/L）和作用时间的延长,进一步诱导细胞损伤加剧,LDH释放和MDA积累明显升高,细胞死亡率也明显增加,已具有统计学意义.同时可观察到其病理形态学的坏死性改变;当10 mmol/L H₂O₂作用时,细胞大量死亡,形态学可见细胞极度收缩、脱落,形成大面积的细胞脱失区.因此,H₂O₂作为一种活性氧自由基,依其浓度和作用时间不同可造成不同程度的心肌细胞的损伤.辣根过氧化物酶作为一种自由基清除剂,可明显减少H₂O₂活性氧自由基对心肌细胞的损伤作用.     

八株芽孢杆菌菌株的分类及固氮活性的测定

从8个省市的不同土壤分离了200个菌株,筛选出8株（JR₁,JR₂,JR₅,ZZ₃,ZZ₁₂,ZZ₁₆,M₁,K₄）能在无氮培养基上生长良好的典型的芽抱杆菌,其中6株（JR₁,JR₂,JR₄和ZZ₃,ZZ₁₂,ZZ     

Preparation of hair for measurement of elements by inductively coupled plasma-mass spectrometry (ICP-MS)

The preparation of hair for the determination of elements is a critical component of the analysis procedure. Open-beaker, closedvessel microwave, and flowthrough microwave digestion are methods that have been used for sample preparation and are discussed. A new digestion method for use with inductively coupled plasma-mass spectrometry (ICP-MS) has been developed. The method uses 0.2 g of hair and 3 mL of concentrated nitric acid in an atmospheric pressurelow-temperature microwave digestion (APLTMD) system. This preparation method is useful in handling a large numbers of samples per day and may be adapted to hair sample weights ranging from 0.08 to 0.3 g. After digestion, samples are analyzed by ICP-MS to determine the concentration of Li, Be, B, Na, Mg, Al, P, S, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ge, As, Se, Rb, Sr, Zr, Mo, Pd, Ag, Cd, Sn, Sb, I, Cs, Ba, Pt, Au, Hg, Tl, Pb, Bi, Th, and U. Benefits of the APLTMD include reduced contamination and sample handling, and increased precision, reliability, and sample throughput.     

外泌体在白血病中的重要调控作用

目前,白血病复发是患者死亡的主要原因之一。肿瘤细胞和微环境的相互作用,以及隐匿在骨髓中的肿瘤干细胞,促进了白血病的复发和向淋巴组织的转移,因此白血病的治疗、转移和复发问题受到广泛关注。外泌体是由绝大多数细胞分泌的双层脂质膜囊泡,可以调控细胞间的交流和信息传递。在白血病细胞、基质细胞和内皮细胞之间的相互联系中都涉及到外泌体,白血病细胞来源的外泌体存在于白血病患者的血浆中,能把其携带的白血病相关抗原及微小RNA呈递给靶细胞,促进白血病肿瘤细胞的增殖,有助于肿瘤细胞实现免疫逃避,保护白血病细胞抵抗化疗药物导致的细胞毒性作用,促进血管生成及肿瘤细胞的迁移。因此,外泌体与白血病的转移、治疗及预后密切相关,可以用来检测和监测白血病的进展。本文综述了外泌体的来源、形成与分泌机制,以及外泌体在白血病发生前、发展中、预后和免疫治疗中所扮演的重要角色。     

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein^1,2. The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale^1-4.Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).     

Assessing burrowing, nest construction, and hoarding in mice

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening of possible treatments for AD currently largely focuses on cognitive testing, which frequently demands expensive equipment and lots of experimenter time. However, human episodic memory (the most severely affected aspect of memory in AD) is different to rodent memory, which seems to be largely non-episodic. Therefore the present ways of screening for new AD treatments for AD in rodents are intrinsically unlikely to succeed. A new approach to preclinical screening would be to characterise the ADL of mice. Fortuitously, several such assays have recently been developed at Oxford, and here the three most sensitive and well-characterised are presented. Burrowing was first developed in Oxford. It evolved from a need to develop a mouse hoarding paradigm. Most published rodent hoarding paradigms required a distant food source to be linked to the home cage by a connecting passage. This would involve modifying the home cage as well as making a mouse-proof connecting passage and food source. So it was considered whether it would be possible to put the food source inside the cage. It was found that if a container was placed on the floor it was emptied by the next morning., The food pellets were, however, simply deposited in a heap at the container entrance, rather than placed in a discrete place away from the container, as might be expected if the mice were truly hoarding them. Close inspection showed that the mice were performing digging ("burrowing") movements, not carrying the pellets in their mouths to a selected place as they would if truly hoarding them. Food pellets are not an essential substrate for burrowing; mice will empty tubes filled with sand, gravel, even soiled bedding from their own cage. Moreover, they will empty a full tube even if an empty one is placed next to it. Several nesting protocols exist in the literature. The present Oxford one simplifies the procedure and has a well-defined scoring system for nest quality. A hoarding paradigm was later developed in which the mice, rather than hoarding back to the real home cage, were adapted to living in the "home base" of a hoarding apparatus. This home base was connected to a tube made of wire mesh, the distal end of which contained the food source. This arrangement proved to yield good hoarding behaviour, as long as the mice were adapted to living in the "home base" during the day and only allowed to enter the hoarding tube at night.     

日本北海道虻类雌性外生殖器形态(双翅目：虻科)(英文)

【目的】利用日本北海道虻类评估和验证外生殖器在分类学上的意义。【方法】将虻类成虫标本浸渍在生理盐水中并置于双目显微镜下通过针和镊子在培养皿中进行解剖并绘图,观察第9背板、第10背板、尾叶、第8腹板、受精囊、受精囊管及生殖叉器的形态特征。【结果】在日本北海道共记录了虻科(Tabanidae) 3亚科7属38种。我们观察并描述了3亚科其中的6属24种的雌性外生殖器的主要特征。亚科之间存在明显差异;然而在一般情况下属之间很难建立一种方法来确定共同点;种之间只有在斑虻属Chrysops中有相似之处,其他属中则比较多样化。因此,亚科鉴定根据第9背板、第8腹板及受精囊足以进行区分,属及种鉴定需要结合第9背板、第10背板、尾叶、第8腹板、受精囊、受精囊管及生殖叉器各自的特征组合在一起才能区分开来。我们也制作了虻类外生殖器的检索表。【结论】和许多其他昆虫一样,外生殖器是虻科的重要分类特征,对于促进分类学和系统学的发展具有重要意义。本研究首次对分布在日本北海道的虻科雌性外生殖器进行了系统研究。     

Staining protocols for human pancreatic islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region. The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&E and IHC stains. The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.     

A phylogenetic analysis of 100+ genera and 50+ families of euasterids based on morphological and molecular data with notes on possible higher level morphological synapomorphies

 A data matrix of 143 morphological and chemical characters for 142 genera of euasterids according to the APG system was compiled and complemented with rbcL and ndhF sequences for most of the genera. The data were subjected to parsimony analysis and support was assessed by bootstrapping. Strict consensus trees from analyses of morphology alone and morphology + rbcL + ndhF are presented. The morphological data recover several groups supported by molecular data but at the level of orders and above relationships are only superficially in agreement with molecular studies. The analyses provide support for monophyly of Gentianales, Aquifoliales, Apiales, Asterales, and Dipsacales. All data indicate that Adoxaceae are closely related to Dipsacales and hence they should be included in that order. The trees were used to assess some possible morphological synapomorphies for euasterids I and II and for the orders of the APG system. Euasterids I are generally characterised by opposite leaves, entire leaf margins, hypogynous flowers, “early sympetaly” with a ring-shaped corolla primordium, fusion of stamen filaments with the corolla tube, and capsular fruits. Euasterids II often have alternate leaves, serrate-dentate leaf margins, epigynous flowers, “late sympetaly” with distinct petal primordia, free stamen filaments, and indehiscent fruits. It is unclear which of these characters represent synapomorphies and symplesiomorphies for the two groups, respectively, and there are numerous expections to be interpreted as reversals and parallelisms. Received August 28, 2000 Accepted August 7, 2001     

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells^3-5. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells⁶, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies¹. Originally published by Naal et al.¹, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease^7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function². In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε₂₈₀ = 4,200 L/M/cm)¹². This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.     

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.