The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.     

Affine extensions of the icosahedral group with applications to the three-dimensional organisation of simple viruses

Since the seminal work of Caspar and Klug on the structure of the protein containers that encapsulate and hence protect the viral genome, it has been recognised that icosahedral symmetry is crucial for the structural organisation of viruses. In particular, icosahedral symmetry has been invoked in order to predict the surface structures of viral capsids in terms of tessellations or tilings that schematically encode the locations of the protein subunits in the capsids. Whilst this approach is capable of predicting the relative locations of the proteins in the capsids, information on their tertiary structures and the organisation of the viral genome within the capsid are inaccessible. We develop here a mathematical framework based on affine extensions of the icosahedral group that allows us to describe those aspects of the three-dimensional structure of simple viruses. This approach complements Caspar-Klug theory and provides details on virus structure that have not been accessible with previous methods, implying that icosahedral symmetry is more important for virus architecture than previously appreciated.      

3-D analysis of the Plasmodium falciparum Maurer's clefts using different electron tomographic approaches

The human malaria parasite Plasmodium falciparum exports a large number of proteins into its host erythrocyte to install functions necessary for parasite survival. Important structural components of the export machinery are membrane profiles of parasite origin, termed Maurer's clefts. These profiles span much of the distance between the parasite and the host cell periphery and are believed to deliver P. falciparum-encoded proteins to the erythrocyte plasma membrane. Although discovered more than a century ago, Maurer's clefts remain a mysterious organelle with little information available regarding their origin, their morphology or their precise role in protein trafficking. Here, we evaluated different techniques to prepare samples for electron tomography, including whole cell cryo-preparations, vitreous sections, freeze-substitution and chemical fixation. Our data show that the different approaches tested all have their merits, revealing different aspects of the complex structure of the Maurer's clefts.     

Biochemical and structural characterization of the capsid-bound tegument proteins of human cytomegalovirus

Human cytomegalovirus (HCMV) is the most genetically and structurally complex human herpesvirus and is composed of an envelope, a tegument, and a dsDNA-containing capsid. HCMV tegument plays essential roles in HCMV infection and assembly. Using cryo electron tomography (cryoET), here we show that HCMV tegument compartment can be divided into two sub-compartments: an inner and an outer tegument. The inner tegument consists of densely-packed proteins surrounding the capsid. The outer tegument contains those components that are loosely packed in the space between the inner tegument and the pleomorphic glycoprotein-containing envelope. To systematically characterize the inner tegument proteins interacting with the capsid, we used chemical treatment to strip off the entire envelope and most tegument proteins to obtain a tegumented capsid with inner tegument proteins. SDS-polyacrylamide gel electrophoresis analyses show that only two tegument proteins, UL32-encoded pp150 and UL48-encoded high molecular weight protein (HMWP), remains unchanged in their abundance in the tegumented capsids as compared to their abundance in the intact particles. Three-dimensional reconstructions by single particle cryo electron microscopy (cryoEM) reveal that the net-like layer of icosahedrally-ordered tegument densities are also the same in the tegumented capsid and in the intact particles. CryoET reconstruction of the tegumented capsid labeled with an anti-pp150 antibody is consistent with the biochemical and cryoEM data in localizing pp150 within the ordered tegument. Taken together, these results suggest that pp150, a betaherpesvirus-specific tegument protein, is a constituent of the net-like layer of icosahedrally-ordered capsid-bound tegument densities, a structure lacking similarities in alpha and gammaherpesviruses.     

Post-imaging fiducial markers aid in the orientation determination of complexes with mixed or unknown symmetry

During the entry process many icosahedral viruses must adopt a lower-order symmetry or incur a symmetry mismatch to release their genome through a single site. A membrane model system in which poliovirus was bound to receptor-decorated liposomes was used to pioneer techniques that studied the break in the symmetry of the initial attachment complex by cryo-electron microscopy. Novel methods involving a fiducial marker for the membrane contact point were developed to objectively determine the symmetry of this complex and provide a starting model to initiate a bootstrap orientation refinement. Here we analyze how errors in the subjective assignment of this position affect the determination of symmetry, and the accuracy of calculating Euler angles for each raw image. In this study we have optimized the method and applied it to study the membrane-attachment complex of Semliki Forest virus (SFV), a model system for enveloped virus fusion. The resulting reconstruction of the SFV-membrane complex with a fiducial provides the first experimental evidence that this pre-fusion cell entry intermediate approaches the membrane along the viral 5-fold axis. The analysis reported here, and its subsequent application to enveloped virus fusion, indicate that this is a robust tool for solving the structures of mixed-symmetry complexes.     

New insights into the structural organization of eukaryotic and prokaryotic cytoskeletons using cryo-electron tomography

Cryo-electron tomography (cryo-ET) is an emerging imaging technology that combines the potential of three-dimensional (3-D) imaging at molecular resolution (<5 nm) with a close-to-life preservation of the specimen. In conjunction with pattern recognition techniques, it enables us to map the molecular landscape inside cells. The application of cryo-ET to intact cells provides novel insights into the structure and the spatial organization of the cytoskeleton in prokaryotic and eukaryotic cells.     

钙酸体的结构特征和功能

钙酸体是一类存在于原生寄生虫中的特殊的细胞器。它的结构及元素成分特殊,在寄生虫的生长、生存及毒性方面具有重要作用。本文主要介绍了钙酸体的发现和命名、结构特征及其在寄生虫中的功能。     

Reactions of N-cyanomethyl groups attached to a tetraaza macrocyclic copper(II) complex leading to the formation of various hetero-functionalized macrocyclic complexes

New hetero-functionalized macrocyclic complexes [CuL²](ClO₄)₂ (I) and [CuL³](ClO₄)₂ (II) bearing one N-CH₂CONH₂ or one N-CH₂C(NH)NH(CH₂)₂CH₃ pendant arm as well as one N-CH₂CN group have been prepared by the selective reaction of water or n-propylamine with one of the two N-CH₂CN groups in [CuL¹](ClO₄)₂ (L¹ = 2,13-bis(cyanomethyl)-5,16-dimethyl-2,6,13,17-tetraazatricyclo[16.4.0.^1.180^7.12]docosane). The complex [CuL⁴](ClO₄)₂ (III) bearing both N-CH₂CONH₂ and N-CH₂C(NH)NH(CH₂)₂CH₃ pendant arms can be prepared by either the reaction of I with n-propylamine or the hydrolysis of II. The N-CH₂CONH₂ and/or N-CH₂C(NH)NH(CH₂)₂CH₃ groups of I, II, and III are coordinated to the metal ion. The crystal structure of II shows that the complex has distorted square-pyramidal coordination polyhedron with a considerably strong apical Cu-N (N-CH₂C(NH)NH(CH₂)₂CH₃) bond (2.101(6) Å). The addition of HClO₄ (?0.01 M) to an acetonitrile (or DMSO) solution of II or III produces [Cu(HL³)](ClO₄)₃ (IIa) or [Cu(HL⁴)](ClO₄)₃ (IIIa), showing that the N-CH₂C(NH)NH(CH₂)₂CH₃ pendant arm of each complex is readily protonated in the non-aqueous solvent; the resulting N-CH₂C()NH(CH₂)₂CH₃ group of IIa or IIIa is not involved in coordination. However, the N-CH₂C(NH)NH(CH₂)₂CH₃ group of II is not protonated even in ?1.0 M HClO₄ aqueous solution. In the case of III, most of the complex exists as the protonated form [Cu(HL⁴)]³⁺ in ?0.1 M HClO₄ aqueous solutions.     

Novel approach using substrate-mediated radiolabelling of RNA to link metabolic function with the structure of microbial communities

A novel concept was developed applying radioisotope-labelled substrate incorporation into the biomass. The resulting radiolabelled RNA was used both as an indicator of activity and as a template for gaining structural and functional information about a substrate-utilizing microbial community. Sequences of PCR products are separated via cloning or using molecular fingerprinting techniques. Nucleic acids from predominant clones or the whole molecular fingerprinting pattern are transferred to a membrane and hybridized with the radiolabelled sample RNA. Scanning of the hybridized blots for radioactivity indicates the members involved in the utilization of the substrate. This novel 'random walk' approach using radioisotope probing was evaluated in a model community experiment.     

P—selectin表位结构与功能研究进展

P－selectin是细胞粘附分子选择素家族成员,作为血小板／内皮细胞活化标志和粘附分子,已证明其在介导活化血小板,内皮细胞与白细胞相互粘附作用,参与免疫扣伤,炎症反应,血栓形成及肿瘤转移等多种生理,病理过程中发挥重要作用,近年研究表明,P－selectin分子中不同结构域及其功能表位在其识别,粘附及信号传导中各具重要作用,进一步揭示这种分子构效关系,将有助于阐明P－selectin的生理,病理意义。     

Structure and function of the protein kinase C gene family

Protein kinase C is a serine/threonine protein kinase which is activated in the cell in response to production of diacylglycerol. Gene cloning has revealed the presence of a highly related family of enzymes, which can be sub-divided into groups on the basis of sequence conservation. Differences are seen in both isoform distribution and associated biochemical activity, for example in substrate specificity and activator requirements. Comparison of the protein sequences andin vitro activities of the protein kinase C isoforms has identified regions important for particular aspects of kinase function. Some of these regions are also found associated with other proteins, allowing confirmation of the assigned activity. Site-directed mutagenesis has confirmed the presence of an autoinhibitory sequence involved in protein kinase C regulation and generated constitutively activated proteins which can be used to study differential isoform function. These same sequences have been shown to play a role in substrate selection, perhaps by competition for binding to the active site. Protein kinase C is known to be a phosphoprotein and the identification of regulatory sites phosphorylated by a ‘PKC-kinase’ suggest a possible alternative route for regulation of protein kinase C activity.     

Interspecific variation in the tetradactyl manus of modern tapirs (Perissodactyla: Tapirus) exposed using geometric morphometrics

The distal forelimb (autopodium) of quadrupedal mammals is a key morphological unit involved in locomotion, body support, and interaction with the substrate. The manus of the tapir (Perissodactyla: Tapirus ) is unique within modern perissodactyls, as it retains the plesiomorphic tetradactyl (four‐toed) condition also exhibited by basal equids and rhinoceroses. Tapirs are known to exhibit anatomical mesaxonic symmetry in the manus, although interspecific differences and biomechanical mesaxony have yet to be rigorously tested. Here, we investigate variation in the manus morphology of four modern tapir species (Tapirus indicus , Tapirus bairdii , Tapirus pinchaque , and Tapirus terrestris ) using a geometric morphometric approach. Autopodial bones were laser scanned to capture surface shape and morphology was quantified using 3D‐landmark analysis. Landmarks were aligned using Generalised Procrustes Analysis, with discriminant function and partial least square analyses performed on aligned coordinate data to identify features that significantly separate tapir species. Overall, our results support the previously held hypothesis that T. indicus is morphologically separate from neotropical tapirs; however, previous conclusions regarding function from morphological differences are shown to require reassessment. We find evidence indicating that T. bairdii exhibits reduced reliance on the lateral fifth digit compared to other tapirs. Morphometric assessment of the metacarpophalangeal joint and the morphology of the distal facets of the lunate lend evidence toward high loading on the lateral digits of both the large T. indicus (large body mass) and the small, long limbed T. pinchaque (ground impact). Our results support other recent studies on T. pinchaque , suggesting subtle but important adaptations to a compliant but inclined habitat. In conclusion, we demonstrate further evidence that the modern tapir forelimb is a variable locomotor unit with a range of interspecific features tailored to habitual and biomechanical needs of each species.     

蛇毒丝氨酸蛋白酶结构与功能关系

蛇毒丝氨酸蛋白酶(snake venome serine proteinases,SVSP)是分布于多种蛇毒中的一类蛋白质水解酶,它们与凝血及纤溶系统关系密切,具有一定的临床应用价值。本文主要介绍了几种空间结构已知的SVSP在结构上的特征,以及由此而产生的底物特异性及生化特性的差异,显示了天然SVSP中存在着与功能相关的结构变异多样性,这为进一步开发及医学利用SVSP提供了有益的信息。     

SmpB蛋白结构与功能研究进展

SmpB是一类普遍存在于细菌中的小RNA结合蛋白。研究表明SmpB除了在反式翻译中起着辅助tmRNA分子拯救滞留核糖体的作用,其也可以作为RNA分子伴侣调节体内RpoS的表达,以及具有直接调控RNase R及双组份系统的功能。SmpB参与的调控作用对于细菌蛋白质合成质量控制、致病菌中毒力系统调控、维持机体正常生长及发育等过程具有关键作用。本综述主要从SmpB蛋白结构及其对RNA、蛋白质调控功能等方面进行论述,以期对发掘细菌性疾病治疗靶点,研发新型抗生素,提供新的方向和思路。     

真菌漆酶的结构与功能

漆酶是一种含铜的多酚氧化酶,能催化氧化酚类和芳香类化合物,同时伴随4个电子的转移,并将分子氧还原成水。漆酶结构的解析是阐明其催化作用机理、了解蛋白质结构与功能关系的基础。综述近年来对真菌漆酶蛋白结构及其功能研究的进展。     

Ecological assessment of an intermittent Mediterranean river using community structure and function: evaluating the role of different organism groups

1. Reliable lotic ecological monitoring requires knowledge of river typology, environmental factors, the effect of stressors known here as ‘pressures’ and appropriate indicators of anthropogenically induced change. We sampled benthic macroinvertebrate, fish, bird and macrophyte communities along an intermittent Mediterranean river and analysed community structure (relative abundance) and function (metrics) relative to environmental and pressure gradients in order to identify suitable indicator group(s) for future monitoring and mitigation programmes. 2. Principal components analysis revealed that scale‐dependent longitudinal differences in valley form separated narrower higher lying sites and tributaries with good quality habitats from more open degraded sites lower down the river continuum on a small floodplain and large scale pressures describing changes in land use related to agriculture with associated physical bankside and channel impacts. 3. Forward selection of variables in redundancy analysis (RDA) showed that reach scale environmental variables were selected more frequently than pressure variables for each organism group. Altitude and pH were highly redundant within and between groups, indicating essentially longitudinal structural and functional distribution patterns. Redundancy was far lower between selected pressure variables, but single or no pressure variables were retained for some organism groups indicating poor association of functional data, in particular, with the identified pressures. All RDA results indicated a longitudinal pH gradient, highlighting the combined effect of multiple environmental and pressure based mechanisms on organism groups. 4. Large, mobile organisms such as fish and birds provided a reliable link between organism structure and function, environmental factors and physical disturbance of the channel, bankside and wider river corridor. Benthic macroinvertebrate and macrophyte structural data revealed distribution patterns in relation to water velocity, a key parameter for developing appropriate compensation measures. 5. Results clearly show the importance of assessing patterns of both functional and structural change across multiple organism groups in order to identify typologically appropriate links with complex environmental and pressure gradients and develop and implement appropriate monitoring systems.     

Curve fitting of germination data using the Richards function

Abstract. The fitting of the generalized Richards function to germination data by using two nested iterative and least squares regression procedures to estimate the four parameters (all of which can be associated with features of biological growth) is demonstrated. The program also involves a procedure of parallel curve analysis which makes comparisons between two curves by examining the whole process represented by the curve and not just a point or portion thereof. Excellent agreement between observed and expected values was obtained by analyzing data which defined patterns of germination exhibiting a range of rates and final percentages. The program also calculates a number of derived quantities including maximum daily rate of germination and time to 50% of final germination.     

Evaluation of the chemical reactivity in lignin precursors using the Fukui function

The hydroxycinnamyl alcohols: p-coumarol, coniferol and sinapol are considered the basic units and precursors of lignins models. In this work, the specific reactivity of these molecules was studied. We investigate their intrinsic chemical reactivity in terms of the Fukui function, applying the principle of hard and soft acids and bases (HSAB) in the framework of the density functional theory (DFT). Comparisons of their nucleophilic, electrophilic and free radical reactivity show their most probably sites to form linkages among them. It is found that the most reactive sites, for reactions involving free radicals, are the carbons at the β-position in the p-coumarol and sinapol molecules, whilst the regions around the carbon-oxygen bond of the phenoxyl group are the most reactive in coniferol. Figure Isocontour plots for the free radical form of the Fukui function f ⁰ (r), showing the reactive sites toward electron-rich/poor reactants     

线虫区系分析指示土壤食物网结构和功能研究进展

土壤食物网结构复杂,功能众多,直接测定土壤食物网各功能群生物量并结合数学模型来推断土壤食物网结构和功能,工作量大且分析过程繁琐。线虫生态学的发展为土壤食物网的研究开辟了一条新的思路,即利用线虫区系分析来定性推断食物网的结构和功能。线虫作为土壤中数量最丰富的后生动物,占据着土壤食物网的中心位置,其物种多样性、食性多样性、生活史策略多样性、功能团多样性奠定了其作为土壤食物网结构和功能指示生物的生态学基础。线虫区系分析根据发展历史可以分为个体分类、生活史策略分类、功能团分类和代谢足迹分类四个时期,其中后两个时期主要用于推断土壤食物网结构和功能。基于功能团的线虫区系分析将线虫的食性和生活史策略结合起来,发展出一系列指数来判断土壤食物网的连通性、食物网链长度、外界养分投入情况、分解途径及对外界干扰的响应等。基于代谢足迹的线虫区系分析在功能团分析基础上,加入线虫能流分析,从而定性反映了土壤食物网功能的大小。两者在指示土壤食物网自下而上调节及对植物线虫控制等方面起着重要的作用。