Improved Visualization of Cell Nuclei in Unstained Cultured Cells

Nuclear morphology is useful in tissue culture studies in determining the presence and grade of transformed cells as well as in determining the heterogeneity of the cell population (Grogan el al. 1981, Hustin 1976, Siracky et al. 1978, Siracky 1979). The ratio of long and short nuclear axes provides a useful numerical expression of nuclear shape (Hustin 1976). Clear visualization of nuclei is critical for making the necessary measurements.     

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death ¹. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection ^2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature ⁴.Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin ^5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology ^7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin ⁹, allowed breakthroughs in the field.In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma ^10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.     

Visualization of Negative Signaling in B Cells by Quantitative Confocal Microscopy

Numerous biochemical experiments have invoked a model in which B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G (FcgammaRII) coclustering provides a dominant negative signal that blocks B-cell activation. Here, we tested this model using quantitative confocal microscopic techniques applied to ex vivo splenic B cells. We found that FcgammaRII and BCR colocalized with intact anti-Ig and that the SH2 domain-containing inositol 5'-phosphatase (SHIP) was recruited to the same site. Colocalization of BCR and SHIP was inefficient in FcgammaRII-/- but not gamma chain-/- splenic B cells. We also examined the subcellular location of a variety of enzymes and adapter proteins involved in signal transduction. Several proteins (CD19, CD22, SHP-1, and Dok) and a lipid raft marker were co-recruited to the BCR, regardless of the presence or absence of FcgammaRII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin) displayed reduced colocalization with BCR in the presence of FcgammaRII and SHIP. Colocalization of BCR and F-actin required phosphatidylinositol (PtdIns) 3-kinase and was inhibited by SHIP, because the block in BCR/F-actin colocalization was not seen in B cells of SHIP-/- animals. Furthermore, BCR internalization was inhibited with intact anti-Ig stimulation or by expression of a dominant-negative mutant form of Rac. From these results, we propose that SHIP recruitment to BCR/FcgammaRII and the resulting hydrolysis of PtdIns-3,4,5-trisphosphate prevents the appropriate spatial redistribution and activation of enzymes distal to PtdIns 3-kinase, including those that promote Rac activation, actin polymerization, and receptor internalization.     

An Improved UPLC-MS/MS Platform for Quantitative Analysis of Glycerophosphoinositol in Mammalian Cells

The glycerophosphoinositols constitute a class of biologically active lipid-derived mediators whose intracellular levels are modulated during physiological and pathological cell processes. Comprehensive assessment of the role of these compounds expands beyond the cellular biology of lipids and includes rapid and unambiguous measurement in cells and tissues. Here we describe a sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of the most abundant among these phosphoinositide derivatives in mammalian cells, the glycerophosphoinositol (GroPIns). The method has been developed in mouse Raw 264.7 macrophages with limits of quantitation at 3 ng/ml. Validation on the same cell line showed excellent response in terms of linear dynamic range (from 3 to 3,000 ng/ml), intra-day and inter-day precision (coefficient of variation ≤ 7.10%) and accuracy (between 98.1 and 109.0%) in the range 10-320 ng/ml. As proof of concept, a simplified analytical platform based on this method and external calibration was also tested on four stimulated and unstimulated cell lines, including Raw 264.7 macrophages, Jurkat T-cells, A375MM melanoma cells and rat basophilic leukemia RBL-2H3 cells. The results indicate a wide variation in GroPIns levels among different cell lines and stimulation conditions, although the measurements were always in line with the literature. No significant matrix effects were observed thus indicating that the here proposed method can be of general use for similar determinations in cells of different origin.     

Improved Procedures for Electron Microscopic Visualization of the Cytoskeleton of Cultured Cells

We have developed an improved electron microscopic procedure appropriate for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, critical point drying, and platinum/carbon coating of cultured cells and the improvements consist of modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyethylene glycol into the extraction medium; cell lysis at room temperature; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydration and drying; and uranyl acetate treatment during dehydration. As a result, we have obtained a greatly improved quality of electron microscopic images together with a high consistency of results. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia. Their polarity was distinctly revealed by decoration with myosin subfragment 1. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin, intermediate filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreported millipede-like appearance. The suggested procedure was compatible with immunogold labeling as demonstrated with an antibody to tubulin. Correlative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, producing a close resemblance between the two kinds of images.     

Visualization and Molecular Analysis of Actin Assembly in Living Cells

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.     

定量细胞化学分析大蒜油抗癌的作用

本文应用细胞化学和显微分光光度计对癌细胞的 DNA,ACP,ANAE、SDH 和G6PDH 进行定量测定,又用流式细胞计分析癌细胞 RNA 含量的变化.实验证明大蒜油能明显抑制癌细胞 DNA 与 RNA 合成,并减低五种酶的活性。表明大蒜油能影响癌细胞的核酸代谢、能量代谢及功能活动、抑制癌细胞的分裂增殖,从而起到抗癌效应.     

吴茱萸碱对黑色素瘤细胞的耐药逆转作用

黑色素瘤是目前恶性程度最高的肿瘤之一。临床上,常采用维罗非尼（PLX4032）作为晚期患者的治疗药物,但患者很快就出现了耐药。因此,如何克服耐药、提高患者生存率成为急需解决的问题。本文选用中药吴茱萸碱（EVO）与黑色素瘤A375细胞、对PLX4032耐药的A375细胞（A375/R）进行相关研究。采用CCK-8法检测发现,用EVO的细胞非毒性浓度0.5 μmol/L处理A375/R,PLX4032对A375/R细胞的半数抑制浓度（IC50）降低,逆转倍数为3.85。显示EVO能够增强黑色素瘤A375/R细胞对PLX4032的敏感性。后续实验分为对照组、EVO组、PLX组、PLX + EVO组。流式细胞仪检测结果显示,EVO组细胞凋亡率为5.88%,PLX组细胞凋亡率为17.88%,PLX + EVO组细胞凋亡率为30.28%。细胞集落结果证明,PLX4032联合EVO能够抑制A375/R细胞的克隆形成。免疫印迹法结果证明,联合用药能够上调促凋亡蛋白质Bax、胱天蛋白酶3的表达量,下调p-Akt、p-NF-κB-p65及抗凋亡蛋白Bcl-2的蛋白质水平。以上结果均表明,EVO能够有效逆转黑色素瘤A375/R细胞耐药,并且诱导细胞凋亡,抑制细胞增殖。     

吴茱萸碱对黑色素瘤细胞的耐药逆转作用

黑色素瘤是目前恶性程度最高的肿瘤之一。临床上,常采用维罗非尼（PLX4032）作为晚期患者的治疗药物,但患者很快就出现了耐药。因此,如何克服耐药、提高患者生存率成为急需解决的问题。本文选用中药吴茱萸碱（EVO）与黑色素瘤A375细胞、对PLX4032耐药的A375细胞（A375/R）进行相关研究。采用CCK-8法检测发现,用EVO的细胞非毒性浓度0.5 μmol/L处理A375/R,PLX4032对A375/R细胞的半数抑制浓度（IC50）降低,逆转倍数为3.85。显示EVO能够增强黑色素瘤A375/R细胞对PLX4032的敏感性。后续实验分为对照组、EVO组、PLX组、PLX + EVO组。流式细胞仪检测结果显示,EVO组细胞凋亡率为5.88%,PLX组细胞凋亡率为17.88%,PLX + EVO组细胞凋亡率为30.28%。细胞集落结果证明,PLX4032联合EVO能够抑制A375/R细胞的克隆形成。免疫印迹法结果证明,联合用药能够上调促凋亡蛋白质Bax、胱天蛋白酶3的表达量,下调p-Akt、p-NF-κB-p65及抗凋亡蛋白Bcl-2的蛋白质水平。以上结果均表明,EVO能够有效逆转黑色素瘤A375/R细胞耐药,并且诱导细胞凋亡,抑制细胞增殖。     

不同转移潜能膀胱癌细胞糖组相对定量分析

膀胱癌是发生在膀胱黏膜组织上的一种恶性肿瘤,是泌尿系统中最常见的恶性肿瘤,早期(非肌层浸润型膀胱癌)阶段的诊断和治疗是降低膀胱癌死亡率的最有效方式.肿瘤的发生过程与糖链表达的改变有着密切的关系,而定量分析膀胱癌发生过程中糖链的表达变化尚未有研究.本研究以2株人膀胱正常上皮细胞系(HCV29、HUCV1),1株非肌层浸润性膀胱癌细胞系(KK47),和3株浸润性膀胱癌细胞系(YTS1、J82、T24)为研究材料,应用本室建立的利用乙酰肼修饰糖链唾液酸,以及[12C6]-和[13C6]-苯胺同位素修饰糖链还原性末端技术,然后利用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS),进行膀胱上皮细胞不同病理状态的糖组相对定量分析.从6株细胞中共鉴定出52种N-连接糖链结构,并定量分析了不同类型的糖链在不同细胞中的分布差异,发现唾液酸化、岩藻糖化的N-连接糖链在膀胱癌肿瘤细胞恶化过程中呈现显著升高的趋势,同时平分型糖链和高甘露糖型N-连接糖链也呈表达升高趋势,说明这些糖链结构的表达变化与膀胱癌发生关系密切,从而有助于进一步阐明膀胱癌发生过程中糖链相关的分子机理.     

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.     

Multiple Quantitative Trait Analysis Using Bayesian Networks

Models for genome-wide prediction and association studies usually target a single phenotypic trait. However, in animal and plant genetics it is common to record information on multiple phenotypes for each individual that will be genotyped. Modeling traits individually disregards the fact that they are most likely associated due to pleiotropy and shared biological basis, thus providing only a partial, confounded view of genetic effects and phenotypic interactions. In this article we use data from a Multiparent Advanced Generation Inter-Cross (MAGIC) winter wheat population to explore Bayesian networks as a convenient and interpretable framework for the simultaneous modeling of multiple quantitative traits. We show that they are equivalent to multivariate genetic best linear unbiased prediction (GBLUP) and that they are competitive with single-trait elastic net and single-trait GBLUP in predictive performance. Finally, we discuss their relationship with other additive-effects models and their advantages in inference and interpretation. MAGIC populations provide an ideal setting for this kind of investigation because the very low population structure and large sample size result in predictive models with good power and limited confounding due to relatedness.     

Quantitative Analysis of Cell Migration Using Optical Flow

Neural crest cells exhibit dramatic migration behaviors as they populate their distant targets. Using a line of zebrafish expressing green fluorescent protein (sox10:EGFP) in neural crest cells we developed an assay to analyze and quantify cell migration as a population, and use it here to characterize in detail the subtle defects in cell migration caused by ethanol exposure during early development. The challenge was to quantify changes in the in vivo migration of all Sox10:EGFP expressing cells in the visual field of time-lapse movies. To perform this analysis we used an Optical Flow algorithm for motion detection and combined the analysis with a fit to an affine transformation. Through this analysis we detected and quantified significant differences in the cell migrations of Sox10:EGFP positive cranial neural crest populations in ethanol treated versus untreated embryos. Specifically, treatment affected migration by increasing the left-right asymmetry of the migrating cells and by altering the direction of cell movements. Thus, by applying this novel computational analysis, we were able to quantify the movements of populations of cells, allowing us to detect subtle changes in cell behaviors. Because cranial neural crest cells contribute to the formation of the frontal mass these subtle differences may underlie commonly observed facial asymmetries in normal human populations.     

Visualization and Analysis of Microtubule Dynamics Using Dual Color-Coded Display of Plus-End Labels

Investigating spatial and temporal control of microtubule dynamics in live cells is critical to understanding cell morphogenesis in development and disease. Tracking fluorescently labeled plus-end-tracking proteins over time has become a widely used method to study microtubule assembly. Here, we report a complementary approach that uses only two images of these labels to visualize and analyze microtubule dynamics at any given time. Using a simple color-coding scheme, labeled plus-ends from two sequential images are pseudocolored with different colors and then merged to display color-coded ends. Based on object recognition algorithms, these colored ends can be identified and segregated into dynamic groups corresponding to four events, including growth, rescue, catastrophe, and pause. Further analysis yields not only their spatial distribution throughout the cell but also provides measurements such as growth rate and direction for each labeled end. We have validated the method by comparing our results with ground-truth data derived from manual analysis as well as with data obtained using the tracking method. In addition, we have confirmed color-coded representation of different dynamic events by analyzing their history and fate. Finally, we have demonstrated the use of the method to investigate microtubule assembly in cells and provided guidance in selecting optimal image acquisition conditions. Thus, this simple computer vision method offers a unique and quantitative approach to study spatial regulation of microtubule dynamics in cells.     

Development of a Quantitative Polyacrylamide Gel Electrophoresis Analysis Using a Multichannel Radioactivity Counter for the Evaluation of Oligonucleotide-Bound Drug Carrier

A quantitative polyacrylamide gel electrophoresis (PAGE) analysis using a multichannel radioactivity counter was designed for the evaluation of³³P-labeled antisense oligonucleotide associated with polymeric drug carrier (nanoparticles). The proposed analytical method was first validated. The criteria of specificity, linearity, reliability, detection and quantification limits, and resolution power were determined. Results were compared to those obtained using liquid scintillation counting of crude samples or after solubilization of gel slices. The proposed method gave a better linearity and reliability than liquid scintillation counting of solubilized gel slices. In comparison with the liquid scintillation counting of crude samples, the method presented the advantage of being able to directly separate oligonucleotides differing by only one nucleotide in length. This method was applied for the separation of free oligonucleotides and oligonucleotides bound onto nanoparticles, allowing quantification of the amount of free and bound oligonucleotides without any further separation steps. Thus, because it is easy and rapid, the quantitative PAGE analysis using a multichannel radioactivity counter offers interesting possibilities for the characterization of oligonucleotide nanoparticles.     

Qualitative and Quantitative Analysis of DNA Fragmentation Using Digital Imaging

Apoptosis is an important and common pathway of cellular death. Differentiation from cellular necrosis and quantitation of apoptosis within the milieu of necrosis are analytical challenges. We describe the use of the RIT120 digital imaging software package for quantitative and qualitative analysis of apoptotic DNA ladders induced by a variety of agents, such as serum, tumor necrosis factor-α, transforming growth factor-β1, and nitric oxide. Autoradiographs of DNA ladders are densitometrically scanned to yield a set of curves with peaks corresponding to specific DNA fragments, thereby allowing quantitative subtraction of concurrent DNA degradation from necrotic death. Integration of the areas specifically under the peaks yields a quantitative measure of apoptosis. We provide a useful, rapid, and objective means to quantitate apoptosis, using relatively inexpensive hardware and software.     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻活体生殖细胞及精细胞的微丝骨架

利用绿色荧光蛋白(GFP)基因结合鼠Talin基因表达技术及水稻(Oryza sativa L.)转基因技术,筛选出表达稳定和具等位基因型的第三代转基因水稻.在其活体花粉的4个发育阶段(Ⅰ.小孢子晚期;Ⅱ.二细胞早期;Ⅲ.二细胞晚期;Ⅳ.三细胞阶段),观察了细胞内微丝骨架的分布和结构形态的变化.发现在这4个花粉发育阶段,花粉内的营养核、生殖核、生殖细胞和精细胞都在不同的发育阶段出现位移.而这些位移与微丝骨架的结构变化和运动有密切关系.在胞质中央的微丝网络以及细胞周质的网络不断变化和互动,导致营养核、生殖核或生殖细胞和精细胞的定向位移.在活体生殖细胞和精细胞内,存有一股与细胞纵轴平行排列的微丝骨架.这些微丝骨架对生殖细胞及精细胞可以提供移动的动力,这对生殖细胞或精细胞在花管内以及胚囊内的运动(包括独自游动)提供了依据.     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻活体生殖细胞及精细胞的微丝骨架

利用绿色荧光蛋白(GFP)基因结合鼠Talin基因表达技术及水稻(Oryza sativa L.)转基因技术,筛选出表达稳定和具等位基因型的第三代转基因水稻。在其活体花粉的4个发育阶段(Ⅰ．小孢子晚期;Ⅱ．二细胞早期;Ⅲ．二细胞晚期;Ⅳ．三细胞阶段),观察了细胞内微丝骨架的分布和结构形态的变化。发现在这4个花粉发育阶段,花粉内的营养核、生殖核、生殖细胞和精细胞都在不同的发育阶段出现位移。而这些位移与微丝骨架的结构变化和运动有密切关系。在胞质中央的微丝网络以及细胞周质的网络不断变化和互动,导致营养核、生殖核或生殖细胞和精细胞的定向位移。在活体生殖细胞和精细胞内,存有一股与细胞纵轴平行排列的微丝骨架。这些微丝骨架对生殖细胞及精细胞可以提供移动的动力,这对生殖细胞或精细胞在花管内以及胚囊内的运动(包括独自游动)提供了依据。