动脉粥样硬化中单核细胞膜流动性研究

为研究动脉粥样硬化中单核细胞膜流动性的变化,本实验选用20只新西兰白兔建立动物粥样硬化模型,提取模型组与对照组兔外周血中单核细胞,通过荧光漂白恢复技术检测单核细胞膜流动性,并结合动脉粥样硬化动物模型病理切片揭示其与动脉粥样硬化相关性。结果显示动物动脉粥样硬化模型建立成功,模型组单核细胞膜的荧光恢复率和扩散系数均低于对照组。本研究揭示了单核细胞细胞膜的流动性与动脉粥样硬化的发生有关,为今后深入探究单核细胞与动脉粥样硬化关系提供实验基础。     

利用激光扫描共聚焦显微镜研究植物细胞发育形态学变化

通过激光扫描共聚焦显微镜,利用不同种类(波长)的激光研究植物细胞发育形态学变化。结果表明,利用紫外激光(351 nm)扫描可以清楚地观察到拟南芥叶片表皮细胞的形态及其变化,在已分化的叶片表皮上可观察到包括“铺垫”表皮细胞(epidermal pavement cells)、气孔保卫细胞(guard cell)、气孔伴胞(subsidiarycells)、表皮毛细胞(trichomes)和表皮毛的足细胞(socket cells)等多种形态不同的细胞种类;利用蓝光激光(488nm)辅助曙红浅染,可清晰地显示出拟南芥根生长区内部的各种原始细胞,包括静止区(quiescent center)细胞、皮层/内皮层原始细胞(cortex/endodermal initial cell)、表皮/根冠原始细胞(epidermal/root cap initial cell)和中柱/根冠原始细胞(columella/root cap initial cell)等。利用双光子激光(800 nm)连续扫描30 s可以诱发叶绿体产生自发荧光,并可观察到叶绿体在叶肉细胞中的运动轨迹。结果说明激光扫描共聚焦显微镜在植物细胞形态及发育研究上具有独特的功能。     

Cardiogel: A biosynthetic extracellular matrix for cardiomyocyte culture

Summary Tissue-cultured neonatal cardiomyocytes can be successfully maintained in culture on a variety of extracellular matrix components such as laminin, fibronectin, and interstitial collagens (Types I and III).In vivo, however, cardiomyocytes (as well as many other cells) exist in a highly complex extracellular matrix environment composed of, in addition to the above three components, other proteins, proteoglycans, and growth factors. We have developed a procedure for culturing cardiomyocytes on a naturally occurring complete extracellular matrix, Cardiogel. This substrate, synthesized by cardiac fibroblasts, contains laminin, fibronectin, Types I and III collagen, and proteoglycans. When compared to cardiomyocytes grown on laminin alone or fibronectin alone, Cardiogel-supported cardiomyocytes adhere more rapidly after plating, exhibit spontaneous contractility earlier, undergo cytoskeletal and myofibrillar differentiation earlier, and grow larger than their counterparts. We suggest that their superior growth characteristics reflect the synergistic effect of numerous extracellular matrix components’ signals in Cardiogel transduced by the cardiomyocyte cytoskeletal elements.     

Comparison of single cell culture derived Solanum tuberosum L. plants and a model for their application in breeding programs

Summary The techniques of microspore and protoplast regeneration starting from dihaploid Solanum tuberosum plants has been improved to such an extent that the production of more than 2000 microspore derived A₁ plant lines and of several hundred protoplast derived plantlets has become possible. Further, from the dihaploid Solanum species S. phureja the regeneration of microspores to plants, and from the species S. infundibuliforme, S. sparsipilum and S. tarijense the regeneration of protoplasts to calluses, has been achieved. The plants descending from the two single cell culture systems are compared with reference to phenotypic markers and economic qualities. Some principles characteristic for either microspore or protoplast derived plants are examined and their significance is discussed. The results are compiled into an extended analytical synthetic breeding scheme based on a stepwise reduction of the autotetraploid to the monohaploid level and a subsequent controlled combination to a new synthetic completely heterozygous tetraploid potato.     

钙结合蛋白CIB1在293T细胞中的表达及亚细胞定位研究

分析和比对钙结合蛋白CIB1在人类、大鼠、小鼠中氨基酸序列差异,并研究CIB1蛋白在293T细胞中的表达及亚细胞定位情况。通过Western Blot分析发现293T细胞本身几乎检测不到CIB1的表达。进一步采用CIB1外源性质粒转染,通过免疫荧光分析实验,在激光共聚焦显微镜下观察转染后不同时间293T细胞中CIB1的表达情况及亚细胞定位变化。实验结果表明,随着转染时间的增加,CIB1在293T细胞中的表达有逐渐增强的趋势,并且发生从细胞核向细胞质的转位现象。该实验结果对了解CIB1亚细胞定位变化及功能研究具有重要参考价值。     

A new approach for the spatially resolved qualitative analysis of the protein distribution in hydrogel beads based on confocal laser scanning microscopy

To investigate the spatial distribution of white egg albumin (WEA) in alginate beads, a new method based on confocal laser scanning microscopy (CLSM) was developed. In contrast to the existing CLSM methods, misleading conclusions are prevented with the application of the new method which does not allow the attenuation of the exciting and emitted light by the opaque hydrogel matrices to be disregarded. By the application of this method, the distribution of WEA in alginate beads was shown to be dependent on the amount of protein loading. At low quantities of protein, a higher protein concentration occurs in the shell layer of the alginate bead while at higher loadings a more or less homogeneous distribution is observed.     

细胞内F-actin的聚合与解聚对肝癌Bel-7402细胞的影响

目的：为探讨癌细胞内F—actin的解聚与聚合对癌细胞的形态、迁移、侵入的影响。方法：利用激光共聚焦显微镜对贴附培养的人肝癌：Bel-7402细胞形态及其细胞内F-actin进行观察;使用流式细胞仪对贴附的Bel一7402细胞及其脱落细胞与Cyt—B处理后Bel-7402细胞内F-actin的含量进行分析。结果：Bel-7402细胞在培养的过程中,癌细胞形态伸展,出现侵入性生长,细胞内F-actin聚合形成粗大的贯通细胞内的F-actin束,F-acfin含量增高;癌细胞在生长过程中,常出现重叠生长,细胞变圆,F_actin解聚变短,F-acfin小体增高,细胞有脱落的趋势,其脱落细胞内的F-actin含量低于贴附细胞。结论：人肝癌：Bel-7402细胞内F-actin的聚合可增加癌细胞的贴附和侵入性：细胞内F-actin的解聚,及Gactin重新聚合形成F-aefin小体可影响到癌细胞脱落及迁移。     

激光扫描共聚焦显微镜在植物学中的应用

激光扫描共聚焦显微镜(LSCM)是普通光学显微镜与激光和计算机及其相应的软件技术组合的产物,实现了连续光学切片,能在亚细胞水平观察细胞骨架的动态变化、细胞内特异蛋白、钙等离子的变化,并结合电生理等技术观察细胞生理活动与细胞形态及运动变化的相互关系。并广泛应用于生物三维结构重组及动态分析。本文综述了应用激光扫描共聚焦显微镜技术在植物细胞学、植物发育、组织化学以及基因表达、检测等领域取得的进展。     

Innocuousness and intracellular distribution of PKH67: A fluorescent probe for cell proliferation assessment

PKH dyes were initially developed by Horan et al. to provide appropriate probes for in vitro and in vivo cell tracking. It has been reported for many cell types that PKH bind irreversibly to the cell membrane without significantly affecting cell growth. Thus, these probes provide an opportunity for long-term cell monitoring and the identification of cells of interest among a heterogeneous cell population. An important feature is that upon cell division, the probe is partitioned equally between each daughter cell, making it possible to quantify tell fluorescence by flow cytometry. In this situation. the flow cytometric study of PKH67 characteristics shows that this probe does not affect the main cell-functions such as viability or proliferation. Moreover, the intracellular distribution of PKH67 is demonstrated by following its kinetics of internalization by confocal microscopy. These results present PKH67 as a probe suitable for dynamic analysis of cell proliferation as well as the study of intracellular localization and membrane recycling mechanisms.     

The use of new probes and stains for improved assessment of cell viability and extracellular polymeric substances in <Emphasis Type="Italic">Candida albicans</Emphasis> biofilms

Phenotypic and genotypic cell differentiation is considered an important feature that confers enhanced antifungal resistance in candidal biofilms. Particular emphasis has been placed in this context on the viability of biofilm subpopulations, and their heterogeneity with regard to the production of extracellular polymeric substances (EPS). We therefore assessed the utility of two different labeled lectins Erythrina cristagalli (ECA) and Canavalia ensiformis (ConA), for EPS visualization. To evaluate the viability of candidal biofilms, we further studied combination stains, SYTO9 and propidium iodide (PI). The latter combination has been successfully used to assess bacterial, but not fungal, viability although PI alone has been previously used to stain nuclei in fungal cells. Candida albicans biofilms were developed in a rotating disc biofilm reactor and observed in situ using confocal scanning laser microscopy (CSLM). Our data indicate that SYTO9 and PI are reliable vital stains that may be used to investigate C. albicans biofilms. When used together with ConA, the lectin ECA optimized EPS visualization and revealed differential production of this material in mature candidal biofilms. The foregoing probes and stains and the methodology described should help better characterize C. albicans biofilms in terms of cell their viability, and EPS production.     

The quest for four-dimensional imaging in plant cell biology: it's just a matter of time

BACKGROUND: Analysis of plant cell dynamics over time, or four-dimensional imaging (4-DI), represents a major goal of plant science. The ability to resolve structures in the third dimension within the cell or tissue during developmental events or in response to environmental or experimental stresses (i.e. 4-DI) is critical to our understanding of gene expression, post-expression modulations of macromolecules and sub-cellular system interactions. SCOPE: Microscopy-based technologies have been profoundly integral to this type of investigation, and new and refined microscopy technologies now allow for the visualization of cell dynamics with unprecedented resolution, contrast and experimental versatility. However, certain realities of light and electron microscopy, choice of specimen and specimen preparation techniques limit the scope of readily attaining 4-DI. Today, the plant microscopist must use a combinatorial strategy whereby multiple microscopy-based investigations are used. Modern fluorescence, confocal laser scanning, transmission electron and scanning electron microscopy provide effective conduits for synthesizing data detailing live cell dynamics and highly resolved snapshots of specific cell structures that will ultimately lead to 4-DI. This review provides a synopsis of such technologies available.     

A batch assay using Calcofluor fluorescence to characterize cell wall regeneration in plant protoplasts

A batch assay to study and measure the regeneration of cell walls during the early days of culture of primary protoplasts is presented. The assay involves the measurement of Calcofluor White fluorescence on a scanning fluorometer when the Calcofluor is adsorbed to the cellulosic component of the newly synthesized cell walls. The Calcofluor fluorescence, when standardized with microcrystalline cellulose, provided a measure of cell wall cellulose. The assay was used to study cell wall regeneration in Hyoscyamus muticus L. protoplasts during 8 days of culture.     

Morphogenetic plasticity of callus reinitiated from cell suspension cultures of the fern Platycerium coronarium

Cell suspension cultures were initiated from gametophyte-derived callus of the fern Platycerium coronarium. Two distinct types of callus masses, distinguished by their colouration, were obtained when the cells from suspension culture were plated on semisolid Murashige and Skoog (MS) medium containing 10 M kinetin. The two types of callus masses had distinct morphogenetic capacities despite their common origin. Morphogenesis into either gametophytes or sporophytes occurred when these callus masses were cultured on phytohormone-free MS medium depending on the type of callus used. The dark-green gametophytic callus showed a faster rate of growth and morphogenesis as compared to the pale-green sporophytic callus. Total chlorophyll content and autofluorescence and size of chloroplasts of the sporophytic callus and cell suspension cultures were lower than that of the gametophytic callus. Observations from confocal laser scanning microscopy were in agreement with the physiological parameters measured. The availability of cell cultures of the same ploidy level, but with two distinct pathways of development will be useful for comparative studies of developmental plasticity.     

Confocal microscopy as a tool to reveal the tridimensional organization of intracellular lumens and intercellular cysts in a human colon adenocarcinoma cell line

Adenocarcinoma cells often form intracellular lumens and intercellular cysts. In order to study the structural relationships between these lumens and the apical domain of normal enterocytes, we have applied electron microscopy and confocal microscopy to a cloned cell line derived from the human colon adenocarcinoma cell line LoVo which express a high number of intracellular lumens and intercellular cysts. Microvilli reminiscent of those detected in the brush border of small intestinal cells are formed in the two types of compartments. By immunofluorescence, we found that a 135 kDa membrane glycoprotein characterized by a monoclonal Ab and normally associated with the brush-border of enterocytes is expressed at the surface of the intracellular lumens and intercellular cysts present in the adenocarcinoma cells. Comparison of fluorescence and reflection contrast micrographs obtained by confocal microscopy demonstrate the presence of spherical intracellular lumens in the juxtanuclear region of single cells, and of more complex shaped intercellular cysts located within clusters of cells. The later cells form junctional complexes limiting an apical plasma membrane domain in contact with the intercellular cyst. It is suggested that the intracellular lumens may represent the abortive form of an apical plasma membrane due to the lack of components required to establish epithelial cell contacts. As opposed to conventional fluorescence microscopy, confocal microscopy allows rapid inspection of the tridimensional organization of intracellular lumens and intercellular cysts even when they are located in cell multilayers.     

Down-modulation of B cell signal transduction by ligation of mucins to CD22

Epithelial cancer cells secrete mucins carrying carbohydrate antigens such as a sialyl-Tn antigen into cancer tissues and/or the bloodstream, in which mucins may interact with CD22 (Siglec-2). Mucins isolated from colon cancer cells and bovine submaxillary mucins bound to CD22 cDNA transfectants and a human B cell line, Daudi cell, and the binding of soluble recombinant CD22 to the mucins was confirmed by means of a plate assay. The binding specificity was demonstrated by the fact that the mucins bound to the recombinant CD22 with an intact ectodomain but not to that with a mutated ectodomain. Daudi cells were stimulated with anti-IgM F(ab′)₂ in the presence or absence of mucins. Ligation of mucins to CD22 decreased the phosphorylation of CD22 and SHP-1 recruitment, and the phosphorylation of ERK-1/2 prominently. The in vivo effect of mucins on splenic B cells in the tumor-bearing state was investigated using mucin-producing (TA3-Ha) and non-producing (TA3-St) mammary adenocarcinoma-bearing mice. When fluorescence-labeled epiglycanins were administered to normal mice, a portion of them was taken up by the spleen and became associated with splenic B cells. We found that splenic B cells were reduced in TA3-Ha-bearing mice but not in TA3-St-bearing ones. These results suggest that in the tumor-bearing state a portion of the mucins in the bloodstream was taken up by the spleen and ligated to CD22 expressed on splenic B cells, which may have led to down-regulation of signal transduction.     

Fluorescent metal nanoshell and CK19 detection on single cell image

In this article, we report the synthesis strategy and optical properties of a novel type of fluorescence metal nanoshell when it was used as imaging agent for fluorescence cell imaging. The metal nanoshells were made with 40 nm silica cores and 10 nm silver shells. Unlike typical fluorescence metal nanoshells which contain the organic dyes in the cores, novel metal nanoshells were composed of Cy5-labelled monoclonal anti-CK19 antibodies (mAbs) on the external surfaces of shells. Optical measurements to the single nanoparticles showed that in comparison with the metal free labelled mAbs, the mAb-Ag complexes displayed significantly enhanced emission intensity and dramatically shortened lifetime due to near-field interactions of fluorophores with metal. These metal nanoshells were found to be able to immunoreact with target cytokeratin 19 (CK19) molecules on the surfaces of LNCAP and HeLa cells. Fluorescence cell images were recorded on a time-resolved confocal microscope. The emissions from the metal nanoprobes could be clearly isolated from the cellular autofluorescence backgrounds on the cell images as either individuals or small clusters due to their stronger emission intensities and shorter lifetimes. These emission signals could also be precisely counted on single cell images. The count number may provide an approach for quantifying the target molecules in the cells.     

The osmoregulatory and the amino acid-regulated responses of system A are mediated by different signal transduction pathways

The osmotic response of system A for neutral amino acid transport has been related to the adaptive response of this transport system to amino acid starvation. In a previous study (Ruiz-Montasell, B., M. Gómez-Angelats, F.J. Casado, A. Felipe, J.D. McGivan, and M. Pastor-Anglada. 1994. Proc. Natl. Acad. Sci. USA. 91:9569-9573), a model was proposed in which both responses were mediated by different mechanisms. The recent cloning of several isoforms of system A as well as the elucidation of a variety of signal transduction pathways involved in stress responses allow to test this model. SAT2 mRNA levels increased after amino acid deprivation but not after hyperosmotic shock. Inhibition of p38 activity or transfection with a dominant negative p38 did not alter the response to amino acid starvation but partially blocked the hypertonicity response. Inhibition of the ERK pathway resulted in full inhibition of the adaptive response of system A and no increase in SAT2 mRNA levels, without modifying the response to hyperosmolarity. Similar results were obtained after transfection with a dominant negative JNK1. The CDK2 inhibitor peptide-II decreased the osmotic response in a dose-dependent manner but did not have any effect on the adaptive response of system A. In summary, the previously proposed model of up-regulation of system A after hypertonic shock or after amino acid starvation by separate mechanisms is now confirmed and the two signal transduction pathways have been identified. The involvement of a CDK-cyclin complex in the osmotic response of system A links the activity of this transporter to the increase in cell volume previous to the entry in a new cell division cycle.     

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid–base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na⁺]_i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na⁺]_i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye–ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na⁺]_i, whereas no significant differences in branchial [Na⁺]_i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na⁺]_i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.     

The sub-membrane reticulum of the human erythrocyte: A scanning electron microscope study

A web-like reticulum underlying the human erythrocyte membrane was studied at a resolution of 5–10 nm by means of a scanning electron microscope. The network was visualized in isolated membranes (ghosts) torn open to reveal their interior space and in residues derived from ghosts extracted with Triton X-100. It formed a continuous (rather than patchy) cover over the entire cytoplasmic surface, except where lifted off or torn away. Filaments (5–40 nm in diameter), annular figures (40–60 nm in diameter), and nodes (30–100 nm in diameter) were prominent in different networks. The dimensions of the filaments and the interstices in the reticulum varied with conditions, suggesting that the network has elastic properties. This reticulum is probably related to the erythrocyte membrane proteins spectrin and actin.