Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals.     

应用Steedman's wax切片法观察植物细胞微管骨架

微管骨架在植物发育过程中起重要作用.由于植物细胞的特殊性,与动物细胞相比植物微管骨架的研究遇到更多的困难.简略地介绍了曾被国内外学者应用的植物微管骨架的各种研究方法及其局限性.Steedman's wax是一种多脂蜡.它熔点低(35～37℃),具有与石蜡相同的切片性质,能够切成不同厚度的连续切片,适合深埋于器官内部的组织或细胞的免疫细胞化学研究.介绍了应用Steedman's wax切片法观察植物细胞微管骨架的一般程序和方法以及经过作者检验且切实可行的一些技术改进.     

Prereplicative increase of nuclear matrix-bound DNA polymerase-α and primase activities in HeLa S3 cells following dilution of long-term cultures

We investigated the association of DNA polymerase and DNA primase activity with the nuclear matrix in HeLa S3 cells diluted with fresh medium after having been cultured without any medium change for 7 days. Flow cytometric analysis demonstrated that just before dilution about 85% of the cells were in the G₁ phase of the cycle, whereas 8% were in the S phase. After dilution with fresh medium, 18–22 h were required for the cell population to attain a stable distribution with respect to the cell cycle. At that time, about 38% of the cells were in the S phase. DNA polymerase and DNA primase activity associated with the nuclear matrix prepared from cells just before dilution represented about 10% of nuclear activity. As judged by [³H]-thymidine incorporation and flow cytometric analysis, an increase in the number of S-phase cells was evident at least 6 h after dilution. However, as early as 2 h after dilution into fresh medium, a striking prereplicative increase of the two activitites was seen in the nuclear matrix fraction but not in cytosol or isolated nuclei. Both DNA polymerase and primase activities bound to the matrix were about 60% of nuclear activity. Overall, the nuclear matrix was the cell fraction where the highest induction (about 10-fold) of both enzymatic activities was seen at 30 h after dilution, whereas in cytosol and isolated nuclei the increase was about two- and fourfold, respectively. Typical immunofluorescent patterns given by an antibody to 5-bromodeoxyuridine were seen after dilution. These findings, which are at variance with our own previous results obtained with cell cultures synchronized by either a double thymidine block or aphidicolin exposure, strengthen the contention that DNA replication is associated with an underlying nuclear structure and demonstrate the artifacts that may be generated by procedures commonly used to synchronize cell cultures. J. Cell. Biochem. 71:11–20, 1998. © 1998 Wiley-Liss, Inc.     

应用激光共聚焦扫描显微镜技术检测缺氧人肺微血管内皮细胞内钙离子浓度动态变化

目的：探讨应用激光共聚焦扫描显微镜（LSCM）技术检测缺氧状态的人肺微血管内皮细胞（HPMVEC）内钙离子（Ca2＋）浓度动态变化的价值。方法：HPMVEC常规培养,按观察时间点不同分为5个缺氧培养组（1h hyp组、2h hyp组、4h hyp组、6h hyp组和8h hyp组）以及1个对照组（0h con组）共6个组,每组设8个复孔,应用LSCM技术测定缺氧后HPMVEC内Ca2＋浓度水平及随时间推移的变化。结果：LSCM技术显示HPMVEC内Ca2＋的荧光强度1h hyp组与0h con组比较、2h hyp组与1h hyp组比较、4h hyp组与2h hyp组比较、6h hyp组与4h hyp组比较、8h hyp组与6h hyp组比较有显著差异（P〈0.05）。线性回归分析结果显示Ca2＋荧光强度与缺氧时间成正相关（r=0.969,P〈0.01）。结论：HPMVEC内Ca2＋浓度随缺氧时间增长而增高;LSCM在动态检测缺氧状态下HPMVEC内的Ca2＋浓度变化中具有明显优势。     

Dichloromethane as an economic alternative to chloroform in the extraction of DNA from plant tissues

Processing of large numbers smaples of plant tissue samples for molecular mapping and gene tagging requires methods that are quick, simple, and cheap, and that eventually can be automated. Organic solvents used for DNA extraction can represent a significant proportion of the overall cost. In this study we examined dichloromethane as a replacement for chloroform to be used in combination with phenol.     

利用核磁共振技术检测植物细胞凋亡

在细胞凋亡的研究中,通常以检测细胞核和细胞器的形态改变或生物化学特性变化为指标进行分析。已有的实验表明: 动物细胞在凋亡过程中, 细胞膜的脂质双分子层发生了一系列生物物理和生物化学改变,如膜电位的改变、磷脂酰丝氨酸由细胞膜内侧向外翻转、细胞膜微粘度的改变等,这些变化会导致细胞中亚甲基信号强度的增加。我们利用质子核磁共振光谱分析(1H-NMR)方法, 首次发现用物理和化学方法诱导的烟草(Nicotiana tabacumL. cv. BY-2)和胡萝卜(Daucus carota L.)细胞在凋亡过程中伴随有亚甲基信号强度的明显增加。在用烟酰胺处理的烟草细胞中, 亚甲基信号强度的增加与DNA Ladder 几乎同时出现,随诱导时间的延长, 亚甲基信号强度也逐渐增大,在24 h亚甲基信号强度增加约2倍。而这种特征在坏死的细胞中并不存在。说明亚甲基信号强度的增加是动、植物细胞凋亡过程中所具有的共同特征,1H-NMR技术提供了一种精确可靠的分析植物细胞凋亡的手段,同时由于它所具有的非侵害性的特点,可能在揭示细胞凋亡机制的研究中具有一定的意义。     

Plant polyphenols mobilize nuclear copper in human peripheral lymphocytes leading to oxidatively generated DNA breakage: Implications for an anticancer mechanism

It was earlier proposed that an important anti-cancer mechanism of plant polyphenols may involve mobilization of endogenous copper ions, possibly chromatin-bound copper and the consequent pro-oxidant action. This paper shows that plant polyphenols are able to mobilize nuclear copper in human lymphocytes, leading to degradation of cellular DNA. A cellular system of lymphocytes isolated from human peripheral blood and comet assay was used for this purpose. Incubation of lymphocytes with neocuproine (a cell membrane permeable copper chelator) inhibited DNA degradation in intact lymphocytes. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. This study has further shown that polyphenols are able to degrade DNA in cell nuclei and that such DNA degradation is inhibited by neocuproine as well as bathocuproine (both of which are able to permeate the nuclear pore complex), suggesting that nuclear copper is mobilized in this reaction. Pre-incubation of lymphocyte nuclei with polyphenols indicates that it is capable of traversing the nuclear membrane. This study has also shown that polyphenols generate oxidative stress in lymphocyte nuclei which is inhibited by scavengers of reactive oxygen species (ROS) and neocuproine. These results indicate that the generation of ROS occurs through mobilization of nuclear copper resulting in oxidatively generated DNA breakage.     

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.     

The Involvement of Protein Synthesis Elongation Factor 1α in the Organization of Microtubules on the Perinuclear Region during the Cell Cycle Transition from M Phase to G1 Phase in Tobacco BY-2 Cells

Association of the organization of microtubules (MTs) in the perinuclear region with a 49-kDa protein, that is immunologically cross-reactive to a 51-kDa protein isolated from sea urchin centrosomes and has been shown to play some roles in the organization of MTs in animal cells (Toriyama et al.: Cell Motil. Cytoskeleton 9, 117–128, 1988), was examined during the cell cycle transition from M phase to G₁ phase using the highly synchronized tobacco BY-2 cells under confocal laser scanning microscopy (CLSM). After double staining with an antibody against the 51-kDa protein and with an antibody against tubulin, it was revealed that the 49-kDa protein was closely associated with the organization of MTs on the perinuclear regions during this stage under the CLSM. Notably, microfilaments (MFs) were not associated with the organization of MTs in the perinuclear region. This observation suggests that the 49-kDa protein plays a specific role in the organization of MTs on the perinuclear regions during the cell cycle transition from M phase to G₁ phase. To understand the molecular characteristics of the 49-kDa protein further, the search for cDNA encoding the 49-kDa protein was conducted in a cDNA expression library prepared from rapidly growing tobacco BY-2 cells using monoclonal antibodies against the 51-kDa protein. Determination of the base sequence of the isolated clone revealed that it encodes protein synthesis elongation factor (EF)-1α. Thus the significance of the involvement of the 49-kDa protein as EF-1α in the organization of MTs on the perinuclear regions is discussed in relation to other cellular functions.     

Genetic Control of Replication through N1-methyladenine in Human Cells

N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesion is mediated via three different pathways in which Pols ι and θ function in one pathway and Pols η and ζ, respectively, function in the other two pathways. Our biochemical studies indicate that in the Polι/Polθ pathway, Polι would carry out nucleotide insertion opposite 1-MeA from which Polθ would extend synthesis. In the Polη pathway, this Pol alone would function at both the nucleotide insertion and extension steps of TLS, and in the third pathway, Polζ would extend from the nucleotide inserted opposite 1-MeA by an as yet unidentified Pol. Whereas by pushing 1-MeA into the syn conformation and by forming Hoogsteen base pair with the T residue, Polι would carry out TLS opposite 1-MeA, the ability of Polη to replicate through 1-MeA suggests that despite its need for Watson-Crick hydrogen bonding, Polη can stabilize the adduct in its active site. Remarkably, even though Pols η and ι are quite error-prone at inserting nucleotides opposite 1-MeA, TLS opposite this lesion in human cells occurs in a highly error-free fashion. This suggests that the in vivo fidelity of TLS Pols is regulated by factors such as post-translational modifications, protein-protein interactions, and possibly others.     

A New Organic Dye-Based Staining for The Detection of Plant DNA in Agarose Gels

Ethidium bromide (EtBr) is used to stain DNA in agarose gel electrophoresis, but this dye is mutagenic and carcinogenic. We investigated N-719, which is a visible, reliable and organic Ruthenium-based dye, and five fluorescent alternatives for staining plant DNA. For prestaining and poststaining, N-719, GelRed, and SYBR Safe stained both DNA and PCR product bands as clearly as EtBr. SYBR Green I, methylene blue, and crystal violet were effective for poststaining only. The organic dye N-719 stained DNA bands as sensitively and as clearly as EtBr. Consequently, organic dyes can be used as alternatives to EtBr in plant biotechnology studies.     

The osmium ammine-SO2 staining method for studying the in situ configuration of viral genomes in ultrathin sections of DNA virus infected cells

Summary— The Feulgen-like osmium ammine-SO² method developed by Cogliati and Gautier (CR Acad Sci Ser D 1973, 276, 3041–3044) stains the DNA at the ultrastructural level. Compared to several other techniques for detecting DNA, this method remains the only one revealing the configuration of the DNA molecules within the cell whatever their compactness. In the present article we summarize the results we obtained with the osmium ammine method in the study of the fate of viral genomes along the infectious cycles in several DNA virus infected cells including adenovirus, herpes simplex virus, simian virus 40 and poxvirus. The results are discussed in relation to the replicative and transcribing activities of viral DNA.     

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。     

The evolution of long interspersed repeated DNA (L1, LINE 1) as revealed by the analysis of an ancient rodent L1 DNA family

Summary All modern mammals contain a distinctive, highly repeated (⩾50,000 members) family of long interspersed repeated DNA called the L1 (LINE 1) family. While the modern L1 families were derived from a common ancestor that predated the mammalian radiation ∼80 million years ago, most of the members of these families were generated within the last 5 million years. However, recently we demonstrated that modern murine (Old World rats and mice) genomes share an older long interspersed repeated DNA family that we called Lx. Here we report our analysis of the DNA sequence of Lx family members and the relationship of this family to the modern L1 families in mouse and rat. The extent of DNA sequence divergence between Lx members indicates that the Lx amplification occurred about 12 million years ago, around the time of the murine radiation. Parsimony analysis revealed that Lx elements were ancestral to both the modern rat and mouse L1 families. However, we found that few if any of the evolutionary intermediates between the Lx and the modern L1 families were extensively amplified. Because the modern L1 families have evolved under selective pressure, the evolutionary intermediates must have been capable of replication. Therefore, replicationcompetent L1 elements can reside in genomes without undergoing extensive amplification. We discuss the bearing of our findings on the evolution of L1 DNA elements and the mammalian genome.     

免疫荧光检测HIV—1抗体方法的建立和应用

免疫荧光法检测HIV抗体是确认HIV感染的方法之一［l］,与蛋白印迹法相比,它的特异性更高,易于鉴别非特异反应,更为经济、快速、容易操作。为了拥有更多的HIV抗体检测手段,我们用感染和未感染HIV－l的HeLaCD“细胞制备抗原片,建立了免疫荧光检测HIV抗体的方法,并进行了初步应用。材料和方法1细胞和病毒HeLaCD4”细胞,引自美国BruceChesebro博士的实验室,用含10％热灭活小牛血清的RPMI1640培养基培养和传代。HIV－l株引自美国。2血清艾滋诊断试剂国家参比品,批号9602Z16份经蛋白印迹确认的HIV抗体阳性血清;10份健康…     

Confocal Raman microspectroscopy as a tool for studying the chemical heterogeneities of biofilms in situ

AIMS: To investigate the use of confocal Raman microspectroscopy (CRM) for the analysis of the structure, composition and development of fully hydrated biofilms. METHODS AND RESULTS: Pseudomonas aeruginosa PAO1 biofilms were cultured in a flow cell in minimal nutrient medium (artificial sea water) and their development was followed for up to 3 weeks. The spectroscopic signature of the biofilm cells and extracellular polymeric substances (EPS) were differentiated and their distribution in biofilm colonies and within water channels was mapped in-plane and -depth. The colonies were initially amorphous, mainly composed of cells with no detectable amount of EPS. They developed rapidly to give round colonies composed of a cellular core enclosed in a sheath of EPS. The EPS continued to increase and spread throughout the biofilm to become the dominating feature of aged colonies. Colonies with a liquid core morphology - characteristic of the seeding dispersal process - were also observed. CONCLUSIONS: This study demonstrated that CRM can be used to monitor the distribution of biofilm components in fully hydrated undisturbed biofilms over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Confocal Raman microspectroscopy facilitates the analysis of hydrated, live bacterial biofilms as a function of space and time, thus making it a suitable technique for investigating the effects of various additives and environmental factors on biofilm growth.     

Karyotyping of Brassica oleracea L. based on rDNA and Cot-1 DNA fluorescence in situ hybridization

To explore an effective and reliable karyotyping method in Brassica crop plants,Cot-1 DNA was isolated from Brassica oleracea genome,labeled as probe with Biotin-Nick Translation Mix kit,in situ hybridized to mitotic spreads,and where specific fluorescent bands showed on each chromosome pair.25S and 5S rDNA were labeled as probes with DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit,respectively,in situ hybridized to mitotic preparations,where 25S rDNA could be detected on two chromosome pairs and 5S rDNA on only one.Cot-1 DNA contains rDNA and chromosome sites identity between Cot-1 DNA and 25S rDNA was determined by dual-colour fluorescence in situ hybridization.All these showed that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome landmarks is superior to all but one.A more exact karyotype ofB.oleracea has been analyzed based on a combination of rDNA sites,Cot-1 DNA fluorescent bands,chromosome lengths and arm ratios.     

Validation of the boronate sensor ContPY1 as a specific probe for fluorescent detection of hydrogen peroxide in plants

Studying the implication of hydrogen peroxide in biological processes in plants remains a challenge due to the current shortcomings of H₂O₂-responsive probes. The use of ContPY1, a new fluorescent probe, which is highly selective and sensitive for H₂O₂, was investigated. To validate the use of ContPY1 on plants, we have generated protocols employing cells suspensions and leaves, and measured specifically H₂O₂ production by plants using spectrofluorometry and microscopy.     

白菜rDNA及C0t-1DNA的荧光原位杂交及其核型分析

以早熟白菜苔为实验材料,从其基因组DNA中分离出C0t-1DNA并用生物素标记作探针,25SrDNA用地高辛标记作探针,对有丝分裂中期相染色体进行双色荧光原位杂交。每对染色体上均显示出了特定的C0t-1DNA荧光原位杂交带型,5对染色体上显示出了25SrDNA荧光原位杂交带型。双色荧光原位杂交证实了C0t-1DNA与25SrDNA二者具有一致的染色体位置特征,表明基于rDNA及C0t-1 DNA的荧光原位杂交核型分析技术,优于目前普遍采用的只基于rDNA的荧光原位杂交核型分析方法。结合C0t-1 DNA与25SrDNA的荧光原位杂交带型和传统的染色体的形态学标记分析方法及白菜已公布的基于rDNA分布的核型分析结果,创建了一个精确的白菜核型。