重构型人caspase-8基因的表达诱导HeLa细胞凋亡

以两种大、小亚基基因次序颠倒的重构型人caspase-8基因的pIRES2-EGFP真核表达载体转染HeLa细胞,用间接免疫荧光染色、免疫细胞化学染色和电镜观察等方法,研究重构型人caspase-8基因在HeLa细胞中的表达及其促凋亡活性.结果显示,两种重构型人caspase-8基因在HeLa细胞中的表达可以有效地诱导HeLa细胞凋亡.     

Surface topography of the p3 and p6 annexin V crystal forms determined by atomic force microscopy

Annexin V is a member of a family of structurally homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca(2+)-dependent manner. The structure of the soluble form of annexin V has been solved by X-ray crystallography, while electron crystallography of two-dimensional (2D) crystals has been used to reveal the structure of its membrane-bound form. Two 2D crystal forms of annexin V have been reported to date, with either p6 or p3 symmetry. Atomic force microscopy has previously been used to investigate the growth and the topography of the p6 crystal form on supported phospholipid bilayers (Reviakine et al., 1998). The surface structure of the second crystal form, p3, is presented in this study, along with an improved topographic map of the p6 crystal form. The observed topography is correlated with the structure determined by X-ray crystallography.     

新型光敏剂分子二乙醇胺基竹红菌乙素光诱导HeLa细胞死亡中的氧化应激研究

二乙醇胺基竹红菌乙素(2-ethanolamino-2-demethoxy-17-ethanolimino-hypocrellin B,EAHB)是一种新型的可吸收600 nm以上红光的竹红菌乙素衍生物。本文研究了二乙醇胺基竹红菌乙素-光动力诱导HeLa细胞死亡的效果及其氧化应激机制。结果发现,红光诱导后,MTT法检测到二乙醇胺基竹红菌乙素-光动力作用使HeLa细胞的存活率显著降低,且存活率与光敏剂浓度和光照剂量成反比;二乙醇胺基竹红菌乙素-光动力诱导HeLa细胞内产生活性氧自由基;同时,胞内超氧化物歧化酶和还原型谷胱甘肽水平显著降低,细胞脂质过氧化标志分子丙二醛显著升高,并检测到细胞质膜损伤标志分子乳酸脱氢酶的渗出显著增加。研究结果说明新型光敏剂二乙醇胺基竹红菌乙素可有效光诱导肿瘤细胞死亡,而细胞内氧化应激反应可能是二乙醇胺基竹红菌乙素光诱导肿瘤细胞死亡的重要作用机制。     

藻蓝蛋白联合全反式维甲酸对HeLa细胞生长抑制作用的研究(英文)

目的:探讨全反式维甲酸和藻蓝蛋白单独及联合用药对HeLa细胞生长的影响,并揭示两者联合用药对细胞周期和细胞凋亡影响的分子机制。方法:MTT法检测全反式维甲酸和藻蓝蛋白单独及联合用药对HeLa细胞生长的影响,原位杂交法检测用药前后细胞内CDK-4基因mRNA的表达情况,免疫组化法检测用药前后bcl-2基因的表达情况,TUNEL法检测用药前后细胞凋亡情况。结果:全反式维甲酸和藻蓝蛋白均具有抑制HeLa细胞生长的作用,当达到相同的抑制率时,联合藻蓝蛋白使用可以显著降低全反式维甲酸的使用剂量从而达到降低毒副作用的目的。两者联合用药可以显著降低CDK-4的表达量从而对HeLa细胞的细胞周期产生影响。两者联合用药可以显著下调bcl-2的表达水平从而引发细胞凋亡。结论:通过联合藻蓝蛋白,可以显著降低全反式维甲酸的使用剂量从而降低毒副作用。全反式维甲酸和藻蓝蛋白联合用药抑制HeLa细胞生长的分子机制可能是通过抑制CDK-4和bcl-2的表达来影响细胞周期并最终导致细胞凋亡。     

Validation of Normal Human Bronchial Epithelial Cells as a Model for Influenza A Infections in Human Distal Trachea

Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic.     

天花粉蛋白对宫颈癌HeLa细胞TSLC1基因甲基化及其表达的影响

目的：检测人宫颈癌HeLa细胞中TSLC1基因甲基化的状况,研究在人宫颈癌HeLa细胞凋亡过程中TSLC1基因甲基化的变化情况,探讨肿瘤细胞凋亡与抑癌基因甲基化的相关性,并进一步证实天花粉蛋白（TCS）去甲基化作用是否存在普遍性,以促进天花粉蛋白的临床应用。方法：应用甲基化特异性PCR（MSP）法检测人宫颈癌HeLa细胞及其凋亡过程中TSLC1基因甲基化的状况;采用实时定量RT-PCR技术检测TCS处理前、后HeLa细胞TSLC1基因表达的变化。结果：肿瘤抑制基因TSLC1在人宫颈癌HeLa细胞中呈高度甲基化状态,经40μg／mL TCS处理48h后,TSLC1基因甲基化程度明显降低;RT-PCR检测结果显示,TCS处理组HeLa细胞中TSLC1 mRNA的表达量高于未处理组,提示TSLC1基因启动子区CpG岛甲基化是导致其低表达的重要机制。结论：肿瘤抑制基因TSLC1启动子甲基化在人宫颈癌癌变过程中可能是一种重要的分子调控机制;人宫颈癌HeLa细胞凋亡与抑癌基因的去甲基化之间可能存在某些密切的相关性;TCS对肿瘤抑制基因TSLC1有一定的去甲基化作用。     

Existance of a Bovine Sperm Antibody Recognizable Antigen-Epitope on the Plasma Surface of Lily Sperm Cells

By using Western blot and rabbit-anti-bovine sperm IgG as the first antibody, a 64 kD protein from sperm cells of Lilium davidii Duch. and the same molecular weight protein from generative cells of L. davidii as well as a 22 kD and a 65 kD protein from sperm cells of Zea mays could be detected. Nevertheless, proteins from filament and anther wall of L. daviclii or etiolated shoot of corn manifested negative reaction. By indirect immunofluorescence assay using the same antibody, the surface of sperm cells from L. daviclii showed positive reaction. Based on the results, the authors believed that a plant sperm cell might have the same epitope (s) as that of an animal sperm cell; the epitope (s) might locate on the surface of a sperm cell and be specific to sperm cells.     

双光子及共聚焦激光扫描显微术研究天花粉蛋白对人绒癌细胞的作用机制

天花粉蛋白(trichosanthin, TCS)是从中草药栝楼根中提取的一种核糖体失活蛋白,具有抗肿瘤和抗HIV功能.应用双光子及共聚焦激光扫描显微术结合特异性荧光探针Hoechst 33342、2′,7′-二氯荧光黄双乙酸酯 (DCFH-DA)、Indo-1和Fluo 3-AM,首次同时观察了TCS诱导人绒癌细胞（JAR细胞）凋亡过程中活性氧自由基（ROS）和细胞内钙离子浓度（［Ca²⁺］_i）的变化,实验结果表明TCS引起的［Ca²⁺］_i升高和ROS形成参与了TCS诱导的JAR细胞凋亡,并且ROS形成和［Ca²⁺］_i升高有关.共聚焦激光扫描显微术的研究结果表明,［Ca²⁺］_i升高不是导致ROS形成的主要原因,TCS诱导产生的ROS可能是通过TCS与JAR细胞膜表面受体作用介导的.     

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins. J. Cell. Biochem. 68:139–150, 1998. © 1998 Wiley-Liss, Inc.     

Raman spectroscopy as a non‐invasive technique for the quantification of melanins in feathers and hairs

The quantification of melanins is a complex task due to the chemical heterogeneity of the pigments and the difficulty of their isolation. The best accepted procedure currently consists in the chemical cleavage of melanins and the subsequent detection of degradation products by HPLC, which implies the destruction of samples. Here, we show that Raman spectroscopy is a non‐invasive technique that can be used to quantify melanins. We made parallel analyses of the characteristics of pheomelanin and eumelanin Raman spectra as measured by confocal Raman microscopy and of degradation products of pheomelanin (4‐amino‐3‐hydroxyphenylalanine, 4‐AHP) and eumelanin (pyrrole‐2,3,5‐tricarboxylic acid, PTCA) as measured by HPLC in feathers of red‐legged partridges and hairs of wild boars and humans. We found strong correlations between the spectral Raman characteristics and 4‐AHP and PTCA levels, which indicates that the Raman spectra of melanins can be used to determine their content.     

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.     

Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer

Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (<100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.     

Neuronal Cell Surface Molecules Mediate Specific Binding to Rabies Virus Glycoprotein Expressed by a Recombinant Baculovirus on the Surfaces of Lepidopteran Cells

The existence of specific rabies virus (RV) glycoprotein (G) binding sites on the surfaces of neuroblastoma cells is demonstrated. Spodoptera frugiperda (Sf21) cells expressing G of the RV strain CVS (Gcvs-Sf21 cells) bind specifically to neuroblastoma cells of different species but not to any other cell type (fibroblast, myoblast, epithelial, or glioma). Attachment to mouse neuroblastoma NG108-15 cells is abolished by previous treatment of Gcvs-Sf21 cells with anti-G antibody. Substitutions for lysine at position 330 and for arginine at position 333 in RV G greatly reduce interaction between Gcvs-Sf21 cells and NG108-15 cells. These data are consistent with in vivo results: an avirulent RV mutant bearing the same double mutation is not able to infect sensory neurons or motoneurons (P. Coulon, J.-P. Ternaux, A. Flamand, and C. Tuffereau, J. Virol. 72:273–278, 1998) after intramuscular inoculation into a mouse. Furthermore, infection of NG108-15 cells by RV but not by vesicular stomatitis virus leads to a reduction of the number of binding sites at the neuronal-cell surface. Our data strongly suggest that these specific attachment sites on neuroblastoma cells represent a neuronal receptor(s) used by RV to infect certain types of neurons in vivo.     

Chondroitin Sulfate as a Molecular Portal That Preferentially Mediates the Apoptotic Killing of Tumor Cells by Penetratin-directed Mitochondria-disrupting Peptides

The use of cell-penetrating peptides (CPPs) as drug carriers for targeted therapy is limited by the unrestricted cellular translocation of CPPs. The preferential induction of tumor cell death by penetratin (Antp)-directed peptides (PNC27 and PNC28), however, suggests that the CPP Antp may contribute to the preferential cytotoxicity of these peptides. Using PNC27 as a molecular model, we constructed three novel peptides (PT, PR9, and PD3) by replacing the leader peptide Antp with one of three distinct CPPs (TAT, R9, or DPV3), respectively. The IC₅₀ values of PNC27 in tumor cells were 2–3 times lower than in normal cells. However, all three engineered peptides demonstrated similar cytotoxic effects in tumor and normal cells. Another three chimeric peptides containing the leader peptide Antp with different mitochondria-disrupting peptides (KLA-Antp (KGA), B27-Antp (BA27), and B28-Antp (BA28)), preferentially induced apoptosis in tumor cells. The IC₅₀ values of these peptides (3–10 μm) were 3–6 times lower in tumor cells than in normal cells. In contrast, TAT-directed peptides (TAT-KLA (TK), TAT-B27 (TB27), and TAT-B28 (TB28)), were cytotoxic to both tumor and normal cells. These data demonstrate that the leader peptide Antp contributes to the preferential cytotoxicity of Antp-directed peptides. Furthermore, Antp-directed peptides bind chondroitin sulfate (CS), and the removal of endogenous CS reduces the cytotoxic effects of Antp-directed peptides in tumor cells. The overexpression of CS in tumor cells is positively correlated to the cell entry and cytotoxicity of Antp- directed peptides. These results suggest that CS overexpression in tumor cells is an important molecular portal that mediates the preferential cytotoxicity of Antp-directed peptides.     

Use of confocal scanning laser microscopy to measure the concentrations of aerial and penetrative hyphae during growth of Rhizopus oligosporus on a solid surface

In order to develop a method for use in investigations of spatial biomass distribution in solid-state fermentation systems, confocal scanning laser microscopy was used to determine the concentrations of aerial and penetrative biomass against height and depth above and below the substrate surface, during growth of Rhizopus oligosporus on potato dextrose agar. Penetrative hyphae had penetrated to a depth of 0.445 cm by 64 h and showed rhizoid morphology, in which the maximum biomass concentration, of 4.45 mg dry wt cm(-3), occurred at a depth of 0.075 cm. For aerial biomass the maximum density of 39.54 mg dry wt (-3) occurred at the substrate surface. For both aerial and penetrative biomass, there were two distinct regions in which the biomass concentration decayed exponentially with distance from the surface. For aerial biomass, the first exponential decay region was up to 0.1 cm height. The second region above the height of 0.1 cm corresponded to that in which sporangiophores dominated. This work lays the foundation for deeper studies into what controls the growth of fungal hyphae above and below the surfaces of solid substrates.     

Evidence for the presence of a receptor for the cytolethal distending toxin (CLDT) of Campylobacter jejuni on CHO and HeLa cell membranes and development of a receptor-based enzyme-linked immunosorbent assay for detection of CLDT

Abstract Using ligand blotting, it was found that partially purified cytolethal distending toxin prepared from and enterotoxigenic strain of Campylobacter jejuni , bound to two peptides of molecular masses of approximately 59 kDa and 45 kDa and to a single peptide of 59 kDa in protein blots prepared from HeLa and CHO cell membranes, respectively. In contrast, labile toxin of Escherichia coli and cholera toxin bound to a single peptide of the same molecular mass (15 kDa) on protein blots prepared from both CHO and HeLa cell crude membranes resolved by gel electrophoresis. This banding pattern was identical using SDS-solubilized membrane, with or without heat treatment, but no band was obtained when reduced (treatment with 2-mercaptoethanol) samples were used for the gel electrophoresis. The differences between receptors of cytolethal distending toxin and cholera toxin/labile toxin were exploited to develop a receptor-based enzyme-linked immunosorbent assay for detection of cytolethal distending toxin which involved the consecutive addition of either solubilized CHO or HeLa membranes, antigen and antibody. This enzyme-linked immunosorbent assay consistently detected crude cytolethal distending toxin diluted up to 16-fold. The receptor-based enzyme-linked immunosorbent assay for detection of cytolethal distending toxin developed in this study is a suitable alternative assay which can be performed easily in laboratories with minimal facilities and, more importantly, the results are available within a few hours as compared to times of up to 5 days in the conventional tissue culture detection of cytolethal distending toxin.     

Identification of the tumor metastasis suppressor Nm23-H1/Nm23-R1 as a constituent of the centrosome

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event.     

Identification of Caveolae-like Structures on the Surface of Intact Cells Using Scanning Force Microscopy

Caveolae are small, functionally important membrane invaginations found on the surface of many different cell types. Using electron microscopy, caveolae can be unequivocally identified in cell membranes by virtue of their size and the presence of caveolin/VIP22 proteins in the caveolar coat. In this study we have applied for the first time scanning force microscopy (SFM), to visualize caveolae on the surface of living and fixed cells. By scanning the membranes of Chinese hamster ovary cells (CHO), using the tapping mode of the SFM in fluid, we could visualize small membrane pits on the cell membranes of living and fixed cells. Two populations of pits with mean diameters of around 100 nm and 200 nm were present. In addition, the location of many pits visualized with the SFM was coincident with membrane spots fluorescently labeled with a green fluorescent protein-caveolin-1 fusion protein. Scanning force microscopy on cells treated with methyl--cyclodextrin, an agent that sequesters cholesterol and disrupts caveolae, abolished pits with a measured diameter of 100 nm but left pits of around 200 nm diameter intact. Thus, the smallest membrane pits measured with the SFM in CHO cells were indeed very likely to be identical to caveolae. These experiments show for the first time that SFM can be used to visualize caveolae in intact cells.     

Identification of a GM1-Binding Protein on the Surface of Murine Neuroblastoma Cells

S20Y murine neuroblastoma cells appear to express a protein component(s) able to adhere specifically to the oligosaccharide portion of GM1 (oligo-GM1). To identify proteins with which the oligo-GM1 becomes closely associated, a radiolabeled (125I), photoactivatable derivative of oligo-GM1 was prepared. This was accomplished by reductive amination of the glucosyl moiety of oligo-GM1 to 1-deoxy-1-aminoglucitol, followed by reaction of the amine with sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). Crosslinking studies using the photoactivatable probe indicated that it came in close proximity to a protein with an apparent molecular mass of approximately 71 kDa. In competition experiments, as little as a 10-fold molar excess of oligo-GM1 resulted in a selective reduction in labeling of this protein; preincubation with a 200-fold molar excess of siayllactose was necessary to observe the same change in the labeling pattern, lending additional support to the hypothesis that the approximately 71-kDa protein specifically associates with oligo-GM1. Cell surface location of the oligo-GM1 binding protein was confirmed using subcellular fractionation and morphological analyses.