The cytoskeleton in plant and fungal cell tip growth

Tip-growing cells have a particular lifestyle that is characterized by the following features: (1) the cells grow in one direction, forming a cylindrical tube; (2) tip-growing cells are able to penetrate their growth environment, thus having to withstand considerable external forces; (3) the growth velocity of tip-growing cells is among the fastest in biological systems. Tip-growing cells therefore appear to be a system well suited to investigating growth processes. The cytoskeleton plays an important role in cell growth in general, which is why tip-growing cells provide an excellent model system for studying this aspect. The cytoskeletal system comprises structural elements, such as actin filaments and microtubules, as well as proteins that link these elements, control their configuration or are responsible for transport processes using the structural elements as tracks. Common aspects as well as differences in configuration and function of the cytoskeleton in various types of tip-growing cells reveal the general principles that govern the relationship between the cytoskeleton and cell growth.     

Engineering a multi-nucleated myotube, the role of the actin cytoskeleton

Myoblasts in vitro form characteristic arrays of bipolar-shaped cells prior to fusion. We have shown that the actin cytoskeleton re-organizes in these fusing cells and that the interaction of non-muscle myosin 2A with actin at the plasma membrane helps to generate the bipolar shape of myoblasts, which is key for fusion. Here we discuss how fusion occurs, and in particular how the actin cytoskeleton is involved. Myoblast fusion is essential to form the multi-nucleated muscle fibres that make up the skeletal muscle. Skeletal muscle fibres contain many nuclei, roughly one nucleus to every 15 sarcomeres (35 microm) in adult muscle, although this varies with muscle type (Bruusgaard et al., 2006). Thus a muscle fibre 30 cm long contains about 8000 nuclei and is formed by the fusion of about 8000 cells during development. The formation of multi-nucleated myotubes has been intensively studied for many years using a number of different systems. Many different proteins have been identified using Drosophila as a model system (e.g. see reviews by Taylor, 2000, 2002) that have given an insight into what happens in mammals. However, the process of fusion of mammalian cells is less well understood, and this paper will cover some of the aspects of mammalian myoblast fusion, with a particular focus on the role of the actin cytoskeleton.     

激光共聚焦显微成像技术在植物细胞微丝骨架三维动态观察中的应用

激光扫描共聚焦显微镜(laser scanning confocal microscope,LSCM)是目前生物学领域应用最广泛、分辨率高的仪器。可用于细胞内形态结构观察及三维重建、组分空间定位、实时动态变化监测等研究,图像分析软件还能提供荧光强度、空间距离定量测定的丰富信息。本文以携带GFP融合拟南芥丝束蛋白1(AtFIM1)的肌动蛋白结合结构域2(fABD2)基因的B Y-2转基因细胞系为材料,运用LSCM技术观察到间期细胞的网络状微丝结构并重构出胞内微丝的三维网络结构;实时动态监测细胞有丝分裂过程中微丝骨架的动态变化;通过细胞内荧光强度的分布直观地看出BY-2细胞胞质分裂过程中微丝骨架的动态变化。这些结果显示出LSCM在研究植物细胞微丝骨架的三维网络动态结构及图像荧光强度分析与统计方面的优越性。     

The stabilization of labile configurations of plant cytoplasm by freeze-substitution

The system of elongated, vacuole-like structures that are seen in phase-contrast images of living plant cytoplasm, which has been called the pleomorphic canalicular system, has been shown previously to be distorted and vesiculated by conventional fixation methods. The stabilization of this labile structure in tomato hair cells by freeze-substitution is described. Structural features of the freeze-substituted cytoplasm as seen in whole mounts of resin-embedded cells resemble closely those of the living cells.     

Possible roles of extracellular matrix and cytoskeleton in leech body wall muscles

Round circomyarian fibres of leeches are peculiar helical muscles. The fibres are characterized by a lack of junctions, being separated by a thick extracellular matrix, and by scarce end-plates. Even so, the fibres grouped in units show the same degree of contraction. Biochemical, immunocytochemical and ultrastructural studies were performed in order: (a) to demonstrate the presence in the extracellular matrix of fibronectin, collagen type IV and laminin and in the cytoskeleton of desmin and α-actinin; (b) to show the possible link of extracellular matrix with the scaffold of intermediate filaments; (c) to evaluate how the extracellular matrix can play a role in the transduction of a signal during contraction–relaxation–superelongation phases.     

Ultrastructural localization of nucleic acids in plant tissues following the use of malachite green or neutral red in the fixative solution

Malachite green and neutral red, when added to glutaraldehyde for fixation of various tissues, yielded high-contrast images of cell ultrastructure. Malachite green, in acid conditions, appeared to increase contrast of heterochromatin material in the nucleus whereas neutral red gave greater clarity to the nucleolus and to cytoplasmic ribosomes. Control tissue fixed under acid conditions showed little damage but there were ‘crystalline’ areas at the periphery of the nucleolus. RNase did not digest cytoplasmic ribosomes from tissue after neutral red glutaraldehyde fixation. These results suggested that neutral red became bound to RNA in the tissues. Fixation with malachite green, at a pH below 6, did not affect the digestion of RNA by RNase but did protect chromatin against the bleaching action of the chelating agent EDTA. The addition of malachite green (pH < 6) or neutral red to glutaraldehyde are useful techniques for the investigation of the ultrastructure of nuclear material and cytoplasmic ribosomes.     

Structural organization of the cytoskeleton in SV40 human corneal epithelial cells cultured on nano- and microscale grooves

The basement membrane of human corneal epithelial cells (HCECs) has a three-dimensional nanoscale architecture, which includes pores, bumps and fibers that may influence cell-substrate adhesion and spreading in the overlying cells. We previously demonstrated that nano- and microscale groove and ridge patterns influence the morphological response and the adhesive response of HCECs to a nominal wall shear stress. Cell-substrate adhesion is mediated by adhesion receptors that bind to extracellular matrix components and anchor the cytoskeleton (CSK) of cells to extracellular elements. Here we investigate the CSK organization in SV40-transformed HCECs grown on nano- and microscale groove and ridge patterns. X-ray lithography was used to fabricate uniform groove and ridge patterns with features ranging in size from 200 nm to 2 microm grooves. Scanning electron microscopy and transmission electron microscopy were used to investigate CSK structure and the distribution of -beta1 integrin adhesion receptors. CSK elements aligned with the patterns; however, the spatial organization of these elements was influenced by feature size. Larger CSK bundles lay on top of the ridges and ran parallel to the patterns, whereas smaller CSK bundles, whose width was proportional to the groove size, spanned the grooves. -Beta1 integrins co-localized with the CSK and had a higher density at the poles of aligned spindle-shaped cells. Differences in organization seen on the different topographical feature sizes may be indicative of differences in extracellular matrix organization. This may explain, in part, previous observations regarding the dependence of cell adhesive responses on the size of topographic features in the substrate.     

电站止回阀的选型及应用

介绍了几种典型电站止回阀的主要性能、结构形式及工作原理，分析了影响止回阀密封性能及稳定工作的不利因素，提出了止回阀选型的一般要求。     

混凝土拌和站设备的选择故障及维修管理

混凝土拌合站设备的正常运行是拌合站混凝土生产的可靠保障,加强混凝土拌合站设备的质量管理势在必行。针对拌合设备管理的重要性,从混凝土拌合设备运行系统组成、设备的选择、故障及维修角度做了较为系统的阐述,明确了混凝土拌和站设备管理中的若干问题。     

新疆红枣植保机械使用现状的分析与研发

根据红枣种植的农业技术要求,通过对红枣植保机械的使用现状与存在问题的分析,指出了目前红枣植保机械存在着喷雾压力低,穿透力不强,喷雾不均匀,适应性较差的问题,提出了必须加强红枣专用植保机械的研究和开发的力度,进一步提高红枣产品的产量和品质,形成特色产业,促进新疆林果业的进一步发展。     

植物防御素的生物学活性及其应用前景

植物防御素是植物界中一类阳离子型多肽,在结构和功能上,它们与早先发现的哺乳动物和昆虫防御素类似。植物防御素的分子量在5kDa左右,其中含有8个保守的半胱氨酸残基。植物防御素的三维结构呈小球形,包括1个α-螺旋和3个反平行的β-折叠片层结构,8个半胱氨酸可以形成3对或4对二硫键,从而构成了Cys稳定的βαββ结构模序。从植物防御素的结构和体外生物学活性,可以看出植物防御素是一类十分复杂的多肽,它的作用不仅仅局限于植物免疫,而应具有广阔的应用前景。     

The role of mass spectrometry in plant systems biology

Large-scale analyses of proteins and metabolites are intimately bound to advancements in MS technologies. The aim of these non-targeted "omic" technologies is to extend our understanding beyond the analysis of only parts of the system. Here, metabolomics and proteomics emerged in parallel with the development of novel mass analyzers and hyphenated techniques such as gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) and multidimensional liquid chromatography coupled to mass spectrometry (LC-MS). The analysis of (i) proteins (ii) phosphoproteins, and (iii) metabolites is discussed in the context of plant physiology and environment and with a focus on novel method developments. Recently published studies measuring dynamic (quantitative) behavior at these levels are summarized; for these works, the completely sequenced plants Arabidopsis thaliana and Oryza sativa (rice) have been the primary models of choice. Particular emphasis is given to key physiological processes such as metabolism, development, stress, and defense. Moreover, attempts to combine spatial, tissue-specific resolution with systematic profiling are described. Finally, we summarize the initial steps to characterize the molecular plant phenotype as a corollary of environment and genotype.     

The use of model systems prepared from tobacco BY-2 cells for studies of the plant cytoskeleton

Model systems, such as plasma membrane-permeabilized cells, isolated phragmoplasts and membrane ghosts, were prepared from tobacco BY-2 cells and used for studies of the plant cytoskeleton. The use of membrane-permeabilized cells enabled us to demonstrate the occurrence in the phragmoplast of the translocation of microtubules away from the equatorial plane and led us to the isolation of a kinesin-like microtubule-translocating protein from phragmoplasts. Autoradiographic experiments using preparations of isolated phragmoplasts revealed that 1,3-β-glucan was synthesized at the equatorial plane of isolated phragmoplasts and xyloglucan was synthesized in the Golgi apparatus. Membrane ghosts from which pre-existing cortical microtubules had been removed by a cold solution of CaCl₂ bound microtubules that had polymerized in vitro , but those from which pre-existing microtubules had been removed by KCl did not. We used membrane ghosts that had been pretreated with KCl in an attempt to identify a microtubule-plasma membrane cross-linking activity in a fraction of a cell extract. We found that a fraction solubilized by KCl from isolated cortical microtubules had such activity.     

在线分析仪表在乙烯装置中的应用

随着节能降耗、提高质量、治污减排和安全生产等标准的不断提高,在线分析仪表在乙烯装置中的重要性与日俱增。该文介绍了在线分析仪表在乙烯装置中的应用现状,并以某乙烯厂为例着重分析了应用中存在的问题及其解决方案。     

Rates of exocytosis and endocytosis in Arabidopsis root hairs and pollen tubes

Exocytosis and endocytosis are pivotal in many biological processes, but remain difficult to quantify. Here we combine a new algorithm for estimating vesicle size with a detailed morphological analysis of tip-growing cells, in which exocytosis is highly localized and therefore more readily quantified. Cell preservation was rendered as life-like as possible by rapid freezing. This allowed us to produce the first estimates of exocytosis rates in the root hairs and pollen tubes of the model plant Arabidopsis. To quantify exocytosis and endocytosis rates during cell growth, we measured the diameter of vesicles located in the tips of Arabidopsis root hairs and pollen tubes and the widths of cell walls and the cell lumen in longitudinal thin transmission electron microscopic sections. In addition, we measured growth velocities of Arabidopsis root hairs and pollen tubes, using video microscopy. The number of exocytotic vesicles required for cell wall expansion, and the amount of excess membrane inserted into the plasma membrane to be internalized, were estimated from the values that were obtained. The amount of excess membrane that is inserted into the plasma membrane during cell growth was estimated as 86.7% in root hairs and 79% in pollen tubes. This membrane has to be recycled by endocytosis. From counting of the total number of vesicles that is present in thin EM sections through the pollen tube tip, we estimated the average number of vesicles that is present in the tip of pollen tubes. By calculating the total amount of membrane and cell wall material that is required for continued cell growth, assuming that all vesicles are exocytotic, we estimated that pollen tubes continue to grow for 33 s when delivery of vesicles to the tip is inhibited. We arrested vesicle delivery to the tip by application of cytochalasin D. After cytochalasin D application, pollen tubes continued to grow for 30-40 s, which is in the same range as the estimated value of 33 s and shows that in this time frame, the availability of exocytotic vesicles is not a limiting factor.     

植物及其他生物样品的ICP-AES分析

本文介绍了植物及其它生物试样中多元素的电感耦合等离子体(ICP)光谱的分析方法。介绍了植物样品的处理方法及给出了分析的精密度、准确度、检出限和变异系数。并和美国的标样及国内标准样品的比较。讨论了干扰元素的影响及其消除干扰的措施。     

The role of microtubules in cellular organization and endocytosis in the plant pathogen Ustilago maydis

Microtubules are an important part of the eukaryotic cytoskeleton, which participates in numerous essential cellular processes. In fungi interphase microtubules mediate cell polarity and participate in polar growth. However, our understanding of their detailed role in fungal growth is just at the beginning. In growing cells of the plant pathogenic fungus Ustilago maydis microtubules are organized by polar microtubule organizing centres that focus the microtubule minus ends at the small bud. Two opposing motor complexes utilize this microtubule polarity. Cytoplasmic dynein and a kinesin of the Unc104/Kif1A family of kinesins mediate rapid bi‐directional transport of early endosomes. A balance of their activity is required for cell cycle‐dependent accumulation of early endosomes at the growth site, the rear cell pole and the region of cell cleavage. Mutant phenotypes suggest that these endosomes participate in polar growth, bud site selection and cell separation. Therefore, our data suggest that endocytotic membrane recycling participates in local exocytosis, and that the microtubule cytoskeleton has a crucial role in this process.     

Olive pollen profilin (Ole e 2 allergen) co-localizes with highly active areas of the actin cytoskeleton and is released to the culture medium during in vitro pollen germination

Pollen allergens offer a dual perspective of study: some of them are considered key proteins for pollen physiology, but they are also able to trigger allergy symptoms in susceptible humans after coming in contact with their tissues. Profilin (Ole e 2 allergen) has been characterized, to some extent, as one of the major allergens from Olea europaea L. pollen, a highly allergenic species in the Mediterranean countries. In order to obtain clues regarding the biological role of this protein, we have analyzed both its cellular localization and the organization of actin throughout pollen hydration and early pollen tube germination. The localization of the cited proteins was visualized by confocal laser scanning microscopy immunofluorescence using different antibodies. Upon pollen hydration and pollen germination, a massive presence of profilin was detected close to the site of pollen tube emergence, forming a ring-like structure around the 'effective' apertural region. Profilin was also detected in the pollen exine of the germinating pollen grains and in the germination medium. After using a permeabilization-enhanced protocol for immunolocalization, profilin was also localized in the cytoplasm of the pollen tube, particularly at both the proximal and apical ends. Noticeable accumulations of actin were observed in the cytoplasm of the pollen tube; particularly, in both the apical region and the area immediately close to the aperture. Actin filaments were not observed, probably due to the need of further enhanced fixation procedures. The ultrastructural localization of profilin showed the presence of the protein in the cytoplasm of both the mature pollen grain and the pollen tube. The results shown here could be interpreted as signs of a massive dissociation of the actin-profilin complexes, mobilization of actin monomers, and therefore, an intense activity of the actin cytoskeleton. The extensive release of allergenic proteins from the pollen grain into the surrounding aqueous media, as described here for profilin, may help us to understand the mechanisms by which these allergens might come in contact with the human mucosa, therefore triggering the symptoms of allergy.     

国内中小型企业设备管理模式研究与创新

随着国内经济的快速发展,中小型企业如雨后春笋般涌现出来。国内制造业得到了蓬勃发展,企业对设备的需求日益增加、日益多样化、日益复杂化,推动了设备管理模式的创新与进步,对设备管理工作提出更高的要求。对我国中小型企业的基本实际进行研究后,总结归纳了其在设备管理的缺陷和不足,探索出一套适合中小型企业的设备管理模式。     

浅谈药品生产企业的厂址选择和厂区规划

厂址的选择与厂区的规划是药品生产企业实施GMP的第1个环节,现就药品生产企业厂址选择与厂区规划的GMP符合性进行了探讨,并提出了具体的方法与实施步骤。