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Sample records for plant cell nuclei

  1. Plant Nuclei Move to Escape Ultraviolet-Induced DNA Damage and Cell Death1[OPEN

    PubMed Central

    Hidema, Jun; Tamura, Kentaro

    2016-01-01

    A striking feature of plant nuclei is their light-dependent movement. In Arabidopsis (Arabidopsis thaliana) leaf mesophyll cells, the nuclei move to the side walls of cells within 1 to 3 h after blue-light reception, although the reason is unknown. Here, we show that the nuclear movement is a rapid and effective strategy to avoid ultraviolet B (UVB)-induced damages. Mesophyll nuclei were positioned on the cell bottom in the dark, but sudden exposure of these cells to UVB caused severe DNA damage and cell death. The damage was remarkably reduced in both blue-light-treated leaves and mutant leaves defective in the actin cytoskeleton. Intriguingly, in plants grown under high-light conditions, the mesophyll nuclei remained on the side walls even in the dark. These results suggest that plants have two strategies for reducing UVB exposure: rapid nuclear movement against acute exposure and nuclear anchoring against chronic exposure. PMID:26681797

  2. Plant Nuclei Move to Escape Ultraviolet-Induced DNA Damage and Cell Death.

    PubMed

    Iwabuchi, Kosei; Hidema, Jun; Tamura, Kentaro; Takagi, Shingo; Hara-Nishimura, Ikuko

    2016-02-01

    A striking feature of plant nuclei is their light-dependent movement. In Arabidopsis (Arabidopsis thaliana) leaf mesophyll cells, the nuclei move to the side walls of cells within 1 to 3 h after blue-light reception, although the reason is unknown. Here, we show that the nuclear movement is a rapid and effective strategy to avoid ultraviolet B (UVB)-induced damages. Mesophyll nuclei were positioned on the cell bottom in the dark, but sudden exposure of these cells to UVB caused severe DNA damage and cell death. The damage was remarkably reduced in both blue-light-treated leaves and mutant leaves defective in the actin cytoskeleton. Intriguingly, in plants grown under high-light conditions, the mesophyll nuclei remained on the side walls even in the dark. These results suggest that plants have two strategies for reducing UVB exposure: rapid nuclear movement against acute exposure and nuclear anchoring against chronic exposure.

  3. Overexpressed TPX2 causes ectopic formation of microtubular arrays in the nuclei of acentrosomal plant cells.

    PubMed

    Petrovská, Beáta; Jerábková, Hana; Kohoutová, Lucie; Cenklová, Vera; Pochylová, Žaneta; Gelová, Zuzana; Kocárová, Gabriela; Váchová, Lenka; Kurejová, Michaela; Tomastíková, Eva; Binarová, Pavla

    2013-11-01

    TPX2 performs multiple roles in microtubule organization. Previously, it was shown that plant AtTPX2 binds AtAurora1 kinase and colocalizes with microtubules in a cell cycle-specific manner. To elucidate the function of TPX2 further, this work analysed Arabidopsis cells overexpressing AtTPX2-GFP. Distinct arrays of bundled microtubules, decorated with AtTPX2-GFP, were formed in the vicinity of the nuclear envelope and in the nuclei of overexpressing cells. The microtubular arrays showed reduced sensitivity to anti-microtubular drugs. TPX2-mediated formation of nuclear/perinuclear microtubular arrays was not specific for the transition to mitosis and occurred independently of Aurora kinase. The fibres were not observed in cells with detectable programmed cell death and, in this respect, they differed from TPX2-dependent microtubular assemblies functioning in mammalian apoptosis. Colocalization and co-purification data confirmed the interaction of importin with AtTPX2-GFP. In cells with nuclear foci of overexpressed AtTPX2-GFP, strong nuclear signals for Ran and importin diminished when microtubular arrays were assembled. This observation suggests that TPX2-mediated microtubule formation might be triggered by a Ran cycle. Collectively, the data suggest that in the acentrosomal plant cell, in conjunction with importin, overexpressed AtTPX2 reinforces microtubule formation in the vicinity of chromatin and the nuclear envelope.

  4. Multiparametric analysis, sorting, and transcriptional profiling of plant protoplasts and nuclei according to cell type.

    PubMed

    Galbraith, David W; Janda, Jaroslav; Lambert, Georgina M

    2011-01-01

    Flow cytometry has been employed for the analysis of higher plants for approximately the last 30 years. For the angiosperms, ∼500,000 species, itself a daunting number, parametric measurements enabled through the use of flow cytometers started with basic descriptors of the individual cells and their contents, and have both inspired the development of novel cytometric methods that subsequently have been applied to organisms within other kingdoms of life, and adopted cytometric methods devised for other species, particularly mammals. Higher plants offer unique challenges in terms of flow cytometric analysis, notably the facts that their organs and tissues are complex three-dimensional assemblies of different cell types, and that their individual cells are, in general, larger than those of mammals.This chapter provides an overview of the general types of parametric measurement that have been applied to plants, and provides detailed methods for selected examples based on the plant model Arabidopsis thaliana. These illustrate the use of flow cytometry for the analysis of protoplasts and nuclear DNA contents (genome size and the cell cycle). These are further integrated with measurements focusing on specific cell types, based on transgenic expression of Fluorescent Proteins (FPs), and on analysis of the spectrum of transcripts found within protoplasts and nuclei. These measurements were chosen in particular to illustrate, respectively, the issues encountered in the flow analysis and sorting of large biological cells, typified by protoplasts; how to handle flow analyses under conditions that require processing of large numbers of samples in which the individual samples contain only a very small minority of objects of interest; and how to deal with exceptionally small amounts of RNA within the sorted samples.

  5. Three-Dimensional, Live-Cell Imaging of Chromatin Dynamics in Plant Nuclei Using Chromatin Tagging Systems.

    PubMed

    Hirakawa, Takeshi; Matsunaga, Sachihiro

    2016-01-01

    In plants, chromatin dynamics spatiotemporally change in response to various environmental stimuli. However, little is known about chromatin dynamics in the nuclei of plants. Here, we introduce a three-dimensional, live-cell imaging method that can monitor chromatin dynamics in nuclei via a chromatin tagging system that can visualize specific genomic loci in living plant cells. The chromatin tagging system is based on a bacterial operator/repressor system in which the repressor is fused to fluorescent proteins. A recent refinement of promoters for the system solved the problem of gene silencing and abnormal pairing frequencies between operators. Using this system, we can detect the spatiotemporal dynamics of two homologous loci as two fluorescent signals within a nucleus and monitor the distance between homologous loci. These live-cell imaging methods will provide new insights into genome organization, development processes, and subnuclear responses to environmental stimuli in plants. PMID:27557696

  6. Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry.

    PubMed

    Wear, Emily E; Concia, Lorenzo; Brooks, Ashley M; Markham, Emily A; Lee, Tae-Jin; Allen, George C; Thompson, William F; Hanley-Bowdoin, Linda

    2016-01-01

    5-Ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that can be rapidly incorporated into replicating DNA in vivo and, subsequently, detected by using "click" chemistry to couple its terminal alkyne group to fluorescent azides such as Alexa Fluor 488. Recently, EdU incorporation followed by coupling with a fluorophore has been used to visualize newly synthesized DNA in a wide range of plant species. One particularly useful application is in flow cytometry, where two-parameter sorting can be employed to analyze different phases of the cell cycle, as defined both by total DNA content and the amount of EdU pulse-labeled DNA. This approach allows analysis of the cell cycle without the need for synchronous cell populations, which can be difficult to obtain in many plant systems. The approach presented here, which was developed for fixed, EdU-labeled nuclei, can be used to prepare analytical profiles as well as to make highly purified preparations of G1, S, or G2/M phase nuclei for molecular or biochemical analysis. We present protocols for EdU pulse labeling, tissue fixation and harvesting, nuclei preparation, and flow sorting. Although developed for Arabidopsis suspension cells and maize root tips, these protocols should be modifiable to many other plant systems. PMID:26659955

  7. Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry.

    PubMed

    Wear, Emily E; Concia, Lorenzo; Brooks, Ashley M; Markham, Emily A; Lee, Tae-Jin; Allen, George C; Thompson, William F; Hanley-Bowdoin, Linda

    2016-01-01

    5-Ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that can be rapidly incorporated into replicating DNA in vivo and, subsequently, detected by using "click" chemistry to couple its terminal alkyne group to fluorescent azides such as Alexa Fluor 488. Recently, EdU incorporation followed by coupling with a fluorophore has been used to visualize newly synthesized DNA in a wide range of plant species. One particularly useful application is in flow cytometry, where two-parameter sorting can be employed to analyze different phases of the cell cycle, as defined both by total DNA content and the amount of EdU pulse-labeled DNA. This approach allows analysis of the cell cycle without the need for synchronous cell populations, which can be difficult to obtain in many plant systems. The approach presented here, which was developed for fixed, EdU-labeled nuclei, can be used to prepare analytical profiles as well as to make highly purified preparations of G1, S, or G2/M phase nuclei for molecular or biochemical analysis. We present protocols for EdU pulse labeling, tissue fixation and harvesting, nuclei preparation, and flow sorting. Although developed for Arabidopsis suspension cells and maize root tips, these protocols should be modifiable to many other plant systems.

  8. Two parametric cell cycle analyses of plant cell suspension cultures with fragile, isolated nuclei to investigate heterogeneity in growth of batch cultivations.

    PubMed

    Haas, Christiane; Hegner, Richard; Helbig, Karsten; Bartels, Kristin; Bley, Thomas; Weber, Jost

    2016-06-01

    Plant cell suspensions are frequently considered to be heterogeneous with respect to growth in terms of progression of the cells through the cell cycle and biomass accumulation. Thus, segregated data of fractions in different cycle phases during cultivation is needed to develop robust production processes. Bromodeoxyuridine (BrdU) incorporation and BrdU-antibodies or 5-ethynyl-2'-deoxyuridine (EdU) click-it chemistry are frequently used to acquire such information. However, their use requires centrifugation steps that cannot be readily applied to sensitive cells, particularly if nuclei have to be extracted from the protective cellular milieu and envelopes for DNA analysis. Therefore, we have established a BrdU-Hoechst stain quenching protocol for analyzing nuclei directly isolated from delicate plant cell suspension cultures. After adding BrdU to test Harpagophytum procumbens cell suspension cultures the cell cycle distribution could be adequately resolved using its incorporation for the following 72 h (after which BrdU slowed biomass accumulation). Despite this limitation, the protocol allows resolution of the cell cycle distribution of cultures that cannot be analyzed using commonly applied methods due to the cells' fragility. The presented protocol enabled analysis of cycling heterogeneities in H. procumbens batch cultivations, and thus should facilitate process control of secondary metabolite production from fragile plant in vitro cultures. Biotechnol. Bioeng. 2016;113: 1244-1250. © 2015 Wiley Periodicals, Inc. PMID:26614913

  9. Two parametric cell cycle analyses of plant cell suspension cultures with fragile, isolated nuclei to investigate heterogeneity in growth of batch cultivations.

    PubMed

    Haas, Christiane; Hegner, Richard; Helbig, Karsten; Bartels, Kristin; Bley, Thomas; Weber, Jost

    2016-06-01

    Plant cell suspensions are frequently considered to be heterogeneous with respect to growth in terms of progression of the cells through the cell cycle and biomass accumulation. Thus, segregated data of fractions in different cycle phases during cultivation is needed to develop robust production processes. Bromodeoxyuridine (BrdU) incorporation and BrdU-antibodies or 5-ethynyl-2'-deoxyuridine (EdU) click-it chemistry are frequently used to acquire such information. However, their use requires centrifugation steps that cannot be readily applied to sensitive cells, particularly if nuclei have to be extracted from the protective cellular milieu and envelopes for DNA analysis. Therefore, we have established a BrdU-Hoechst stain quenching protocol for analyzing nuclei directly isolated from delicate plant cell suspension cultures. After adding BrdU to test Harpagophytum procumbens cell suspension cultures the cell cycle distribution could be adequately resolved using its incorporation for the following 72 h (after which BrdU slowed biomass accumulation). Despite this limitation, the protocol allows resolution of the cell cycle distribution of cultures that cannot be analyzed using commonly applied methods due to the cells' fragility. The presented protocol enabled analysis of cycling heterogeneities in H. procumbens batch cultivations, and thus should facilitate process control of secondary metabolite production from fragile plant in vitro cultures. Biotechnol. Bioeng. 2016;113: 1244-1250. © 2015 Wiley Periodicals, Inc.

  10. Thermal instability of cell nuclei

    NASA Astrophysics Data System (ADS)

    Warmt, Enrico; Kießling, Tobias R.; Stange, Roland; Fritsch, Anatol W.; Zink, Mareike; Käs, Josef A.

    2014-07-01

    DNA is known to be a mechanically and thermally stable structure. In its double stranded form it is densely packed within the cell nucleus and is thermo-resistant up to 70\\:^\\circ {\\rm{C}}. In contrast, we found a sudden loss of cell nuclei integrity at relatively moderate temperatures ranging from 45 to 55\\:^\\circ {\\rm{C}}. In our study, suspended cells held in an optical double beam trap were heated under controlled conditions while monitoring the nuclear shape. At specific critical temperatures, an irreversible sudden shape transition of the nuclei was observed. These temperature induced transitions differ in abundance and intensity for various normal and cancerous epithelial breast cells, which clearly characterizes different cell types. Our results show that temperatures slightly higher than physiological conditions are able to induce instabilities of nuclear structures, eventually leading to cell death. This is a surprising finding since recent thermorheological cell studies have shown that cells have a lower viscosity and are thus more deformable upon temperature increase. Since the nucleus is tightly coupled to the outer cell shape via the cytoskeleton, the force propagation of nuclear reshaping to the cell membrane was investigated in combination with the application of cytoskeletal drugs.

  11. Co-localisation studies of Arabidopsis SR splicing factors reveal different types of speckles in plant cell nuclei

    SciTech Connect

    Lorkovic, Zdravko J.; Barta, Andrea

    2008-10-15

    SR proteins are multidomain splicing factors which are important for spliceosome assembly and for regulation of alternative splicing. In mammalian nuclei these proteins localise to speckles from where they are recruited to transcription sites. By using fluorescent protein fusion technology and different experimental approaches it has been shown that Arabidopsis SR proteins, in addition to diffuse nucleoplasmic staining, localise into an irregular nucleoplasmic network resembling speckles in mammalian cells. As Arabidopsis SR proteins fall into seven conserved sub-families we investigated co-localisation of members of the different sub-families in transiently transformed tobacco protoplast. Here we demonstrate the new finding that members of different SR protein sub-families localise into distinct populations of nuclear speckles with no, partial or complete co-localisation. This is particularly interesting as we also show that these proteins do interact in a yeast two-hybrid assay as well as in pull-down and in co-immunopreciptiation assays. Our data raise the interesting possibility that SR proteins are partitioned into distinct populations of nuclear speckles to allow a more specific recruitment to the transcription/pre-mRNA processing sites of particular genes depending on cell type and developmental stage.

  12. Plant nuclei can contain extensive grooves and invaginations

    NASA Technical Reports Server (NTRS)

    Collings, D. A.; Carter, C. N.; Rink, J. C.; Scott, A. C.; Wyatt, S. E.; Allen, N. S.; Brown, C. S. (Principal Investigator)

    2000-01-01

    Plant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus.

  13. Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium.

    PubMed

    Ziemienowicz, A; Görlich, D; Lanka, E; Hohn, B; Rossi, L

    1999-03-30

    Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA-protein complexes into nuclei. Import of VirD2-ssDNA-VirE2 complexes was fast and efficient, and was shown to depended on importin alpha, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein-DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells. PMID:10097105

  14. Image analysis for neuroblastoma classification: segmentation of cell nuclei.

    PubMed

    Gurcan, Metin N; Pan, Tony; Shimada, Hiro; Saltz, Joel

    2006-01-01

    Neuroblastoma is a childhood cancer of the nervous system. Current prognostic classification of this disease partly relies on morphological characteristics of the cells from H&E-stained images. In this work, an automated cell nuclei segmentation method is developed. This method employs morphological top-hat by reconstruction algorithm coupled with hysteresis thresholding to both detect and segment the cell nuclei. Accuracy of the automated cell nuclei segmentation algorithm is measured by comparing its outputs to manual segmentation. The average segmentation accuracy is 90.24+/-5.14% PMID:17947119

  15. Direct observation of light focusing by single photoreceptor cell nuclei.

    PubMed

    Błaszczak, Zuzanna; Kreysing, Moritz; Guck, Jochen

    2014-05-01

    The vertebrate retina is inverted with respect to its optical function, which requires light to pass through the entire tissue prior to detection. The last significant barrier for photons to overcome is the outer nuclear layer formed by photoreceptor cell (PRC) nuclei. Here we experimentally characterise the optical properties of PRC nuclei using bright-field defocusing microscopy to capture near-field intensity distributions behind individual nuclei. We find that some nuclei efficiently focus incident light confirming earlier predictions based on comparative studies of chromatin organisation in nocturnal and diurnal mammals. The emergence of light focusing during the development of mouse nuclei highlights the acquired nature of the observed lens-like behaviour. Optical characterisation of these nuclei is an important first step towards an improved understanding of how light transmission through the retina is influenced by its constituents.

  16. Size-Invariant Detection of Cell Nuclei in Microscopy Images.

    PubMed

    Ram, Sundaresh; Rodriguez, Jeffrey J

    2016-07-01

    Accurate detection of individual cell nuclei in microscopy images is an essential and fundamental task for many biological studies. In particular, multivariate fluorescence microscopy is used to observe different aspects of cells in cultures. Manual detection of individual cell nuclei by visual inspection is time consuming, and prone to induce subjective bias. This makes automatic detection of cell nuclei essential for large-scale, objective studies of cell cultures. Blur, clutter, bleed-through, imaging noise and touching and partially overlapping nuclei with varying sizes and shapes make automated detection of individual cell nuclei a challenging task using image analysis. In this paper we propose a new automated method for fast and robust detection of individual cell nuclei based on their radial symmetric nature in fluorescence in-situ hybridization (FISH) images obtained via confocal microscopy. The main contributions are two-fold. 1) This work presents a more accurate cell nucleus detection system using the fast radial symmetry transform (FRST). 2) The proposed cell nucleus detection system is robust against most occlusions and variations in size and moderate shape deformations. We evaluate the performance of the proposed algorithm using precision/recall rates, Fβ-score and root-mean-squared distance (RMSD) and show that our algorithm provides improved detection accuracy compared to existing algorithms.

  17. Characterization of brain cell nuclei with decondensed chromatin.

    PubMed

    Yu, Ping; McKinney, Elizabeth C; Kandasamy, Muthugapatti M; Albert, Alexandria L; Meagher, Richard B

    2015-07-01

    Although multipotent cell types have enlarged nuclei with decondensed chromatin, this property has not been exploited to enhance the characterization of neural progenitor cell (NPC) populations in the brain. We found that mouse brain cell nuclei that expressed exceptionally high levels of the pan neuronal marker NeuN/FOX3 (NeuN-High) had decondensed chromatin relative to most NeuN-Low or NeuN-Neg (negative) nuclei. Purified NeuN-High nuclei expressed significantly higher levels of transcripts encoding markers of neurogenesis, neuroplasticity, and learning and memory (ARC, BDNF, ERG1, HOMER1, NFL/NEF1, SYT1), subunits of chromatin modifying machinery (SIRT1, HDAC1, HDAC2, HDAC11, KAT2B, KAT3A, KAT3B, KAT5, DMNT1, DNMT3A, Gadd45a, Gadd45b) and markers of NPC and cell cycle activity (BRN2, FOXG1, KLF4, c-MYC, OCT4, PCNA, SHH, SOX2) relative to neuronal NeuN-Low or to mostly non-neuronal NeuN-Neg nuclei. NeuN-High nuclei expressed higher levels of HDAC1, 2, 4, and 5 proteins. The cortex, hippocampus, hypothalamus, thalamus, and nucleus accumbens contained high percentages of large decondensed NeuN-High nuclei, while the cerebellum, and pons contained very few. NeuN-High nuclei have the properties consistent with their being derived from extremely active neurons with elevated rates of chromatin modification and/or NPC-like cells with multilineage developmental potential. The further analysis of decondensed neural cell nuclei should provide novel insights into neurobiology and neurodegenerative disease.

  18. Segmentation of touching cell nuclei using gradient flow tracking.

    PubMed

    Li, G; Liu, T; Nie, J; Guo, L; Chen, J; Zhu, J; Xia, W; Mara, A; Holley, S; Wong, S T C

    2008-07-01

    Reliable cell nuclei segmentation is an important yet unresolved problem in biological imaging studies. This paper presents a novel computerized method for robust cell nuclei segmentation based on gradient flow tracking. This method is composed of three key steps: (1) generate a diffused gradient vector flow field; (2) perform a gradient flow tracking procedure to attract points to the basin of a sink; and (3) separate the image into small regions, each containing one nucleus and nearby peripheral background, and perform local adaptive thresholding in each small region to extract the cell nucleus from the background. To show the generality of the proposed method, we report the validation and experimental results using microscopic image data sets from three research labs, with both over-segmentation and under-segmentation rates below 3%. In particular, this method is able to segment closely juxtaposed or clustered cell nuclei, with high sensitivity and specificity in different situations.

  19. Vertical uniformity of cells and nuclei in epithelial monolayers.

    PubMed

    Neelam, Srujana; Hayes, Peter Robert; Zhang, Qiao; Dickinson, Richard B; Lele, Tanmay P

    2016-01-22

    Morphological variability in cytoskeletal organization, organelle position and cell boundaries is a common feature of cultured cells. Remarkable uniformity and reproducibility in structure can be accomplished by providing cells with defined geometric cues. Cells in tissues can also self-organize in the absence of directing extracellular cues; however the mechanical principles for such self-organization are not understood. We report that unlike horizontal shapes, the vertical shapes of the cell and nucleus in the z-dimension are uniform in cells in cultured monolayers compared to isolated cells. Apical surfaces of cells and their nuclei in monolayers were flat and heights were uniform. In contrast, isolated cells, or cells with disrupted cell-cell adhesions had nuclei with curved apical surfaces and variable heights. Isolated cells cultured within micron-sized square wells displayed flat cell and nuclear shapes similar to cells in monolayers. Local disruption of nuclear-cytoskeletal linkages resulted in spatial variation in vertical uniformity. These results suggest that competition between cell-cell pulling forces that expand and shorten the vertical cell cross-section, thereby widening and flattening the nucleus, and the resistance of the nucleus to further flattening results in uniform cell and nuclear cross-sections. Our results reveal the mechanical principles of self-organized vertical uniformity in cell monolayers.

  20. Vertical uniformity of cells and nuclei in epithelial monolayers.

    PubMed

    Neelam, Srujana; Hayes, Peter Robert; Zhang, Qiao; Dickinson, Richard B; Lele, Tanmay P

    2016-01-01

    Morphological variability in cytoskeletal organization, organelle position and cell boundaries is a common feature of cultured cells. Remarkable uniformity and reproducibility in structure can be accomplished by providing cells with defined geometric cues. Cells in tissues can also self-organize in the absence of directing extracellular cues; however the mechanical principles for such self-organization are not understood. We report that unlike horizontal shapes, the vertical shapes of the cell and nucleus in the z-dimension are uniform in cells in cultured monolayers compared to isolated cells. Apical surfaces of cells and their nuclei in monolayers were flat and heights were uniform. In contrast, isolated cells, or cells with disrupted cell-cell adhesions had nuclei with curved apical surfaces and variable heights. Isolated cells cultured within micron-sized square wells displayed flat cell and nuclear shapes similar to cells in monolayers. Local disruption of nuclear-cytoskeletal linkages resulted in spatial variation in vertical uniformity. These results suggest that competition between cell-cell pulling forces that expand and shorten the vertical cell cross-section, thereby widening and flattening the nucleus, and the resistance of the nucleus to further flattening results in uniform cell and nuclear cross-sections. Our results reveal the mechanical principles of self-organized vertical uniformity in cell monolayers. PMID:26795751

  1. Vertical uniformity of cells and nuclei in epithelial monolayers

    PubMed Central

    Neelam, Srujana; Hayes, Peter Robert; Zhang, Qiao; Dickinson, Richard B.; Lele, Tanmay P.

    2016-01-01

    Morphological variability in cytoskeletal organization, organelle position and cell boundaries is a common feature of cultured cells. Remarkable uniformity and reproducibility in structure can be accomplished by providing cells with defined geometric cues. Cells in tissues can also self-organize in the absence of directing extracellular cues; however the mechanical principles for such self-organization are not understood. We report that unlike horizontal shapes, the vertical shapes of the cell and nucleus in the z-dimension are uniform in cells in cultured monolayers compared to isolated cells. Apical surfaces of cells and their nuclei in monolayers were flat and heights were uniform. In contrast, isolated cells, or cells with disrupted cell-cell adhesions had nuclei with curved apical surfaces and variable heights. Isolated cells cultured within micron-sized square wells displayed flat cell and nuclear shapes similar to cells in monolayers. Local disruption of nuclear-cytoskeletal linkages resulted in spatial variation in vertical uniformity. These results suggest that competition between cell-cell pulling forces that expand and shorten the vertical cell cross-section, thereby widening and flattening the nucleus, and the resistance of the nucleus to further flattening results in uniform cell and nuclear cross-sections. Our results reveal the mechanical principles of self-organized vertical uniformity in cell monolayers. PMID:26795751

  2. Spatial organization and correlations of cell nuclei in brain tumors.

    PubMed

    Jiao, Yang; Berman, Hal; Kiehl, Tim-Rasmus; Torquato, Salvatore

    2011-01-01

    Accepting the hypothesis that cancers are self-organizing, opportunistic systems, it is crucial to understand the collective behavior of cancer cells in their tumorous heterogeneous environment. In the present paper, we ask the following basic question: Is this self-organization of tumor evolution reflected in the manner in which malignant cells are spatially distributed in their heterogeneous environment? We employ a variety of nontrivial statistical microstructural descriptors that arise in the theory of heterogeneous media to characterize the spatial distributions of the nuclei of both benign brain white matter cells and brain glioma cells as obtained from histological images. These descriptors, which include the pair correlation function, structure factor and various nearest neighbor functions, quantify how pairs of cell nuclei are correlated in space in various ways. We map the centroids of the cell nuclei into point distributions to show that while commonly used local spatial statistics (e.g., cell areas and number of neighboring cells) cannot clearly distinguish spatial correlations in distributions of normal and abnormal cell nuclei, their salient structural features are captured very well by the aforementioned microstructural descriptors. We show that the tumorous cells pack more densely than normal cells and exhibit stronger effective repulsions between any pair of cells. Moreover, we demonstrate that brain gliomas are organized in a collective way rather than randomly on intermediate and large length scales. The existence of nontrivial spatial correlations between the abnormal cells strongly supports the view that cancer is not an unorganized collection of malignant cells but rather a complex emergent integrated system.

  3. [Selective localization of neptunium-237 in nuclei of mammalian cells].

    PubMed

    Galle, P; Boulahdour, H; Metivier, H

    1992-01-01

    After injection in the rat of soluble neptunium salt, the distribution of this element was studied at the subcellular level by electron microscopy and electron probe microanalysis. Abnormal structures have been observed by electron microscopy in the nuclei of hepatocytes, and the same structures have also been observed in the nuclei of the proximal tubules cells of the kidney. These structures are formed of clusters of very small and dense particles, several nanometers in diameter. The clusters are localized in the central part of the nuclei and they are separate from nucleoli and heterochromatin. Electron probe X-ray analysis of this cluster have shown that they contain neptunium associated with phosphorus. In the cell containing neptunium inclusions, other non specific lesions are also observed (nuclear pycnosis, mitochondrial depletion).

  4. Cloned mice derived from somatic cell nuclei.

    PubMed

    Hosaka, K; Ohi, S; Ando, A; Kobayashi, M; Sato, K

    2000-12-01

    In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum. PMID:11329940

  5. HAMLET interacts with histones and chromatin in tumor cell nuclei.

    PubMed

    Düringer, Caroline; Hamiche, Ali; Gustafsson, Lotta; Kimura, Hiroshi; Svanborg, Catharina

    2003-10-24

    HAMLET is a folding variant of human alpha-lactalbumin in an active complex with oleic acid. HAMLET selectively enters tumor cells, accumulates in their nuclei and induces apoptosis-like cell death. This study examined the interactions of HAMLET with nuclear constituents and identified histones as targets. HAMLET was found to bind histone H3 strongly and to lesser extent histones H4 and H2B. The specificity of these interactions was confirmed using BIAcore technology and chromatin assembly assays. In vivo in tumor cells, HAMLET co-localized with histones and perturbed the chromatin structure; HAMLET was found associated with chromatin in an insoluble nuclear fraction resistant to salt extraction. In vitro, HAMLET bound strongly to histones and impaired their deposition on DNA. We conclude that HAMLET interacts with histones and chromatin in tumor cell nuclei and propose that this interaction locks the cells into the death pathway by irreversibly disrupting chromatin organization.

  6. Auxetic nuclei in embryonic stem cells exiting pluripotency

    NASA Astrophysics Data System (ADS)

    Pagliara, Stefano; Franze, Kristian; McClain, Crystal R.; Wylde, George W.; Fisher, Cynthia L.; Franklin, Robin J. M.; Kabla, Alexandre J.; Keyser, Ulrich F.; Chalut, Kevin J.

    2014-06-01

    Embryonic stem cells (ESCs) self-renew in a state of naïve pluripotency in which they are competent to generate all somatic cells. It has been hypothesized that, before irreversibly committing, ESCs pass through at least one metastable transition state. This transition would represent a gateway for differentiation and reprogramming of somatic cells. Here, we show that during the transition, the nuclei of ESCs are auxetic: they exhibit a cross-sectional expansion when stretched and a cross-sectional contraction when compressed, and their stiffness increases under compression. We also show that the auxetic phenotype of transition ESC nuclei is driven at least in part by global chromatin decondensation. Through the regulation of molecular turnover in the differentiating nucleus by external forces, auxeticity could be a key element in mechanotransduction. Our findings highlight the importance of nuclear structure in the regulation of differentiation and reprogramming.

  7. Rapid Isolation of Nuclei from Cells In Vitro.

    PubMed

    Nabbi, Arash; Riabowol, Karl

    2015-08-01

    This protocol presents a rapid, efficient, and practical (REAP) method to separate nuclei from cultured cells in vitro with as little damage and contamination as possible. The REAP procedure is performed at low temperature and takes <2 min, which minimizes protein degradation, protein modification, and diffusion of soluble proteins out of the nuclear compartment while maintaining the integrity of protein complexes. A mild detergent, NP-40, is used together with mild mechanical shearing to disrupt the plasma membrane, leaving the nuclear membrane intact. The REAP method can be used with various cell lines grown in vitro and requires minimal optimization. The isolated nuclei are suitable for numerous downstream applications (e.g., western blotting, 2D gel electrophoresis, and immunoprecipitation). If desired, aliquots of whole-cell lysate and the cytoplasmic fraction can be saved for comparison. PMID:26240403

  8. Whole-mount confocal imaging of nuclei in giant feeding cells induced by root-knot nematodes in Arabidopsis.

    PubMed

    Vieira, Paulo; Engler, Gilbert; de Almeida Engler, Janice

    2012-07-01

    • Excellent visualization of nuclei was obtained here using a whole-mount procedure adapted to provide high-resolution images of large, irregularly shaped nuclei. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with the dye propidium iodide. • The method developed for standard confocal imaging was applied to large multicellular root swellings, named galls, induced in plant hosts by the root-knot nematode Meloidogyne incognita. • Here, we performed a functional analysis, and examined the nuclear structure in giant feeding cells overexpressing the cell cycle inhibitor Kip-related protein 4 (KRP4). Ectopic KRP4 expression in galls led to aberrant nuclear structure, disturbing giant cell expansion and nematode reproduction. In vivo live-cell imaging of GFP-KRP4 demonstrated that this protein co-localizes to chromosomes from prophase to late anaphase during cell cycle progression. • The data presented here suggest the involvement of KRP4 during mitotic progression in plant cells. The detailed results obtained using confocal analysis also demonstrate the potential utility of a rapid, easy-to-use clearing method for the analysis of the nuclei of certain Arabidopsis mutants and other complex plant nuclei.

  9. Plant stem cell niches.

    PubMed

    Aichinger, Ernst; Kornet, Noortje; Friedrich, Thomas; Laux, Thomas

    2012-01-01

    Multicellular organisms possess pluripotent stem cells to form new organs, replenish the daily loss of cells, or regenerate organs after injury. Stem cells are maintained in specific environments, the stem cell niches, that provide signals to block differentiation. In plants, stem cell niches are situated in the shoot, root, and vascular meristems-self-perpetuating units of organ formation. Plants' lifelong activity-which, as in the case of trees, can extend over more than a thousand years-requires that a robust regulatory network keep the balance between pluripotent stem cells and differentiating descendants. In this review, we focus on current models in plant stem cell research elaborated during the past two decades, mainly in the model plant Arabidopsis thaliana. We address the roles of mobile signals on transcriptional modules involved in balancing cell fates. In addition, we discuss shared features of and differences between the distinct stem cell niches of Arabidopsis.

  10. Optimal ultraviolet wavelength for in vivo photoacoustic imaging of cell nuclei.

    PubMed

    Yao, Da-Kang; Chen, Ruimin; Maslov, Konstantin; Zhou, Qifa; Wang, Lihong V

    2012-05-01

    In order to image noninvasively cell nuclei in vivo without staining, we have developed ultraviolet photoacoustic microscopy (UV-PAM), in which ultraviolet light excites nucleic acids in cell nuclei to produce photoacoustic waves. Equipped with a tunable laser system, the UV-PAM was applied to in vivo imaging of cell nuclei in small animals. We found that 250 nm was the optimal wavelength for in vivo photoacoustic imaging of cell nuclei. The optimal wavelength enables UV-PAM to image cell nuclei using as little as 2 nJ laser pulse energy. Besides the optimal wavelength, application of a wavelength between 245 and 275 nm can produce in vivo images of cell nuclei with specific, positive, and high optical contrast.

  11. A novel dictionary based computer vision method for the detection of cell nuclei.

    PubMed

    De Vylder, Jonas; Aelterman, Jan; Lepez, Trees; Vandewoestyne, Mado; Douterloigne, Koen; Deforce, Dieter; Philips, Wilfried

    2013-01-01

    Cell nuclei detection in fluorescent microscopic images is an important and time consuming task in a wide range of biological applications. Blur, clutter, bleed through and partial occlusion of nuclei make individual nuclei detection a challenging task for automated image analysis. This paper proposes a novel and robust detection method based on the active contour framework. Improvement over conventional approaches is achieved by exploiting prior knowledge of the nucleus shape in order to better detect individual nuclei. This prior knowledge is defined using a dictionary based approach which can be formulated as the optimization of a convex energy function. The proposed method shows accurate detection results for dense clusters of nuclei, for example, an F-measure (a measure for detection accuracy) of 0.96 for the detection of cell nuclei in peripheral blood mononuclear cells, compared to an F-measure of 0.90 achieved by state-of-the-art nuclei detection methods.

  12. Reversible mitochondrial DNA accumulation in nuclei of pluripotent stem cells.

    PubMed

    Schneider, Joel S; Cheng, Xin; Zhao, Qingshi; Underbayev, Chingiz; Gonzalez, J Patrick; Raveche, Elizabeth S; Fraidenraich, Diego; Ivessa, Andreas S

    2014-11-15

    According to the endosymbiotic hypothesis, the precursor of mitochondria invaded the precursor of eukaryotic cells, a process that began roughly 2 billion years ago. Since then, the majority of the genetic material translocated from the mitochondria to the nucleus, where now almost all mitochondrial proteins are expressed. Only a tiny amount of DNA remained in the mitochondria, known as mitochondrial DNA (mtDNA). In this study, we report that the transfer of mtDNA fragments to the nucleus of pluripotent stem cells is still ongoing. We show by in situ hybridization and agarose DNA two-dimensional gel technique that induced pluripotent stem (iPS) cells contain high levels of mtDNA in the nucleus. We found that a large proportion of the accumulated mtDNA sequences appear to be extrachromosomal. Accumulation of mtDNA in the nucleus is present not only in the iPS cells, but also in embryonic stem (ES) cells. However upon differentiation, the level of mtDNA in the nuclei of iPS and ES cells is substantially reduced. This reversible accumulation of mtDNA in the nucleus supports the notion that the nuclear copy number of mtDNA sequences may provide a novel mechanism by which chromosomal DNA is dynamically regulated in pluripotent stem cells.

  13. In vivo imaging of cell nuclei by photoacoustic microscopy without staining

    NASA Astrophysics Data System (ADS)

    Yao, Da-Kang; Chen, Ruimin; Maslov, Konstantin; Zhou, Qifa; Wang, Lihong V.

    2012-02-01

    Ultraviolet photoacoustic microscopy (UVPAM) can image cell nuclei in vivo with high contrast and resolution noninvasively without staining. Here, we used UV light at wavelengths of 210-310 nm for excitation of DNA and RNA to produce photoacoustic waves. We applied the UVPAM to in vivo imaging of cell nuclei in mouse skin, and obtained UVPAM images of the unstained cell nuclei at wavelengths of 245-282 nm as ultrasound gel was used for acoustic coupling. The largest ratio of contrast to noise was found for the images of cell nuclei at a 250 nm wavelength.

  14. Search for Elastic Coherent Neutrino Scattering off Atomic Nuclei at the Kalinin Nuclear Power Plant

    NASA Astrophysics Data System (ADS)

    Akimov, D. Yu.; Belov, V. A.; Bolozdynya, A. I.; Burenkov, A. A.; Efremenko, Yu. V.; Etenko, A. V.; Kaplin, V. A.; Khromov, A. V.; Konovalov, A. M.; Kovalenko, A. G.; Kumpan, A. V.; Melikyan, Yu. A.; Rudik, D. G.; Sosnovtsev, V. V.

    We propose to detect and study neutrino neutral elastic coherent scattering off atomic nuclei with two-phase emission detector with liquid xenon as a target medium. One of the possible experimental site is a Kalinin Nuclear Power Plant (KNPP) situated in the Russian Federation. In this paper we discuss the design of the detector and expected signals and background for this site.

  15. Development of a stained cell nuclei counting system

    NASA Astrophysics Data System (ADS)

    Timilsina, Niranjan; Moffatt, Christopher; Okada, Kazunori

    2011-03-01

    This paper presents a novel cell counting system which exploits the Fast Radial Symmetry Transformation (FRST) algorithm [1]. The driving force behind our system is a research on neurogenesis in the intact nervous system of Manduca Sexta or the Tobacco Hornworm, which was being studied to assess the impact of age, food and environment on neurogenesis. The varying thickness of the intact nervous system in this species often yields images with inhomogeneous background and inconsistencies such as varying illumination, variable contrast, and irregular cell size. For automated counting, such inhomogeneity and inconsistencies must be addressed, which no existing work has done successfully. Thus, our goal is to devise a new cell counting algorithm for the images with non-uniform background. Our solution adapts FRST: a computer vision algorithm which is designed to detect points of interest on circular regions such as human eyes. This algorithm enhances the occurrences of the stained-cell nuclei in 2D digital images and negates the problems caused by their inhomogeneity. Besides FRST, our algorithm employs standard image processing methods, such as mathematical morphology and connected component analysis. We have evaluated the developed cell counting system with fourteen digital images of Tobacco Hornworm's nervous system collected for this study with ground-truth cell counts by biology experts. Experimental results show that our system has a minimum error of 1.41% and mean error of 16.68% which is at least forty-four percent better than the algorithm without FRST.

  16. The fractionation of nuclei from mammalian cells by zonal centrifugation

    PubMed Central

    Johnston, I. R.; Mathias, A. P.; Pennington, F.; Ridge, D.

    1968-01-01

    1. Purified liver nuclei from adult rats separate into two main zones when centrifuged in the slow-speed zonal rotor. One zone contains diploid nuclei, the other tetraploid. 2. The effect of age on the pattern of rat liver ploidy was examined. Tetraploid nuclei are virtually absent from young animals. They increase in proportion steadily with age. Partial hepatectomy disturbs the pattern of ploidy. 3. The zonal centrifuge permits the separation of diploid, tetraploid, octaploid and hexadecaploid nuclei from mouse liver. 4. Rat liver nuclei are isopycnic with sucrose solutions of density 1·35 at 5°. ImagesFig. 1.PLATE 1Fig. 9. PMID:4876099

  17. Improving segmentation of 3D touching cell nuclei using flow tracking on surface meshes.

    PubMed

    Li, Gang; Guo, Lei

    2012-01-01

    Automatic segmentation of touching cell nuclei in 3D microscopy images is of great importance in bioimage informatics and computational biology. This paper presents a novel method for improving 3D touching cell nuclei segmentation. Given binary touching nuclei by the method in Li et al. (2007), our method herein consists of several steps: surface mesh reconstruction and curvature information estimation; direction field diffusion on surface meshes; flow tracking on surface meshes; and projection of surface mesh segmentation to volumetric images. The method is validated on both synthesised and real 3D touching cell nuclei images, demonstrating its validity and effectiveness.

  18. Microscopic cell nuclei segmentation based on adaptive attention window.

    PubMed

    Ko, ByoungChul; Seo, MiSuk; Nam, Jae-Yeal

    2009-06-01

    This paper presents an adaptive attention window (AAW)-based microscopic cell nuclei segmentation method. For semantic AAW detection, a luminance map is used to create an initial attention window, which is then reduced close to the size of the real region of interest (ROI) using a quad-tree. The purpose of the AAW is to facilitate background removal and reduce the ROI segmentation processing time. Region segmentation is performed within the AAW, followed by region clustering and removal to produce segmentation of only ROIs. Experimental results demonstrate that the proposed method can efficiently segment one or more ROIs and produce similar segmentation results to human perception. In future work, the proposed method will be used for supporting a region-based medical image retrieval system that can generate a combined feature vector of segmented ROIs based on extraction and patient data.

  19. Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori.

    PubMed

    Sakai, Hiroki; Yokoyama, Takeshi; Abe, Hiroaki; Fujii, Tsuguru; Suzuki, Masataka G

    2013-01-01

    In Bombyx mori, polar body nuclei are observed until 9 h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe). The heterozygous pe/+ (pe) females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/+ (pe) ) of the mother. Because the polar body nuclei had + (pe) genes in the white eggs laid by a pe/+ (pe) female, polar body nuclei participate in development and differentiate into functional cell (serosal cells). Analyses of serosal cells pigmentation indicated that ~30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26% of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25). Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos.

  20. Germ cell formation from embryonic stem cells and the use of somatic cell nuclei in oocytes.

    PubMed

    Pelosi, Emanuele; Forabosco, Antonino; Schlessinger, David

    2011-03-01

    Embryonic stem cells (ESCs) have remarkable properties of pluripotency and self-renewal, along with the retention of chromosomal integrity. Germ cells function as a kind of "transgenerational stem cells," transmitting genetic information from one generation to the next. The formation of putative primordial germ cells (PGCs) and germ cells from mouse and human ESCs (hESCs) has, in fact, been shown, and the apparent derivation of functional mouse male gametes has also been described. Additionally, investigators have successfully reprogrammed somatic nuclei into a pluripotent state by inserting them into ESCs or oocytes. This would enable the generation of ESCs genetically identical to the somatic cell donor and their use in cell therapy. However, these methodologies are still inefficient and their mechanisms poorly understood. Until full comprehension of these processes is obtained, clinical applications remain remote. Nevertheless, they represent promising tools in the future, enhancing methods of therapeutic cloning and infertility treatment.

  1. Phenotypic characteristics of hybrid cells generated by transferring neuronal nuclei into bone marrow stromal cell cytoplasts.

    PubMed

    Zhou, Zhujuan; Xu, Yan; Zhong, Qi; Zheng, Jian

    2012-02-10

    Bone marrow stromal cells (BMSCs) are promising donor cells for transplantation therapies for a variety of diseases. However, there still lack efficient ways to induce directional differentiation of BMSCs to promote their practical use in transplantation therapy. In this study, we constructed hybrid cells by transferring neuronal nuclei into BMSC cytoplasts and investigated the proliferative capacity and phenotypic characteristics of the hybrid cells. The neuronal nuclei were labeled with Hoechst 33342 before the transfer process, and the cell membrane antigen CD71 was used as a marker of BMSC cytoplasts. The BMSC cytoplasts and neuronal karyoplasts were separated by Ficoll density gradient ultracentrifugation. The hybrid cells were generated by the polyethylene glycol-mediated fusion of BMSC cytoplasts with neuronal karyoplasts. The hybrid cells exhibited Hoechst 33342 staining in their nuclei and CD71 staining on their cytomembranes, which confirmed the success of cell fusion. The hybrid cells were positive for BrdU immunostaining. Viability analysis of the cultured hybrid cells by the MTT assay demonstrated their proliferative ability. Immunocytochemical staining revealed the expression of the neuron-specific markers NeuN and MAP2 in the third passage hybrid cells, which indicated their neuronal phenotypic characteristics. The results demonstrated that the hybrid cells produced by fusing neuronal karyoplasts with BMSC cytoplasts had proliferative capability and expressed the neuron-specific markers. Further study is required to investigate the phenotype of the hybrid cells both structurally and functionally.

  2. Plant Stem Cells.

    PubMed

    Greb, Thomas; Lohmann, Jan U

    2016-09-12

    Among the trending topics in the life sciences, stem cells have received a fair share of attention in the public debate - mostly in connection with their potential for biomedical application and therapies. While the promise of organ regeneration and the end of cancer have captured our imagination, it has gone almost unnoticed that plant stem cells represent the ultimate origin of much of the food we eat, the oxygen we breathe, as well the fuels we burn. Thus, plant stem cells may be ranked among the most important cells for human well-being. Research by many labs in the last decades has uncovered a set of independent stem cell systems that fulfill the specialized needs of plant development and growth in four dimensions. Surprisingly, the cellular and molecular design of these systems is remarkably similar, even across diverse species. In some long-lived plants, such as trees, plant stem cells remain active over hundreds or even thousands of years, revealing the exquisite precision in the underlying control of proliferation, self-renewal and differentiation. In this minireview, we introduce the basic features of the three major plant stem cell systems building on these facts, highlight their modular design at the level of cellular layout and regulatory underpinnings and briefly compare them with their animal counterparts. PMID:27623267

  3. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells.

    PubMed

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-09-30

    Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery.

  4. Towards automated segmentation of cells and cell nuclei in nonlinear optical microscopy.

    PubMed

    Medyukhina, Anna; Meyer, Tobias; Schmitt, Michael; Romeike, Bernd F M; Dietzek, Benjamin; Popp, Jürgen

    2012-11-01

    Nonlinear optical (NLO) imaging techniques based e.g. on coherent anti-Stokes Raman scattering (CARS) or two photon excited fluorescence (TPEF) show great potential for biomedical imaging. In order to facilitate the diagnostic process based on NLO imaging, there is need for an automated calculation of quantitative values such as cell density, nucleus-to-cytoplasm ratio, average nuclear size. Extraction of these parameters is helpful for the histological assessment in general and specifically e.g. for the determination of tumor grades. This requires an accurate image segmentation and detection of locations and boundaries of cells and nuclei. Here we present an image processing approach for the detection of nuclei and cells in co-registered TPEF and CARS images. The algorithm developed utilizes the gray-scale information for the detection of the nuclei locations and the gradient information for the delineation of the nuclear and cellular boundaries. The approach reported is capable for an automated segmentation of cells and nuclei in multimodal TPEF-CARS images of human brain tumor samples. The results are important for the development of NLO microscopy into a clinically relevant diagnostic tool.

  5. Towards automated segmentation of cells and cell nuclei in nonlinear optical microscopy.

    PubMed

    Medyukhina, Anna; Meyer, Tobias; Schmitt, Michael; Romeike, Bernd F M; Dietzek, Benjamin; Popp, Jürgen

    2012-11-01

    Nonlinear optical (NLO) imaging techniques based e.g. on coherent anti-Stokes Raman scattering (CARS) or two photon excited fluorescence (TPEF) show great potential for biomedical imaging. In order to facilitate the diagnostic process based on NLO imaging, there is need for an automated calculation of quantitative values such as cell density, nucleus-to-cytoplasm ratio, average nuclear size. Extraction of these parameters is helpful for the histological assessment in general and specifically e.g. for the determination of tumor grades. This requires an accurate image segmentation and detection of locations and boundaries of cells and nuclei. Here we present an image processing approach for the detection of nuclei and cells in co-registered TPEF and CARS images. The algorithm developed utilizes the gray-scale information for the detection of the nuclei locations and the gradient information for the delineation of the nuclear and cellular boundaries. The approach reported is capable for an automated segmentation of cells and nuclei in multimodal TPEF-CARS images of human brain tumor samples. The results are important for the development of NLO microscopy into a clinically relevant diagnostic tool. PMID:22811013

  6. Cell nuclei segmentation in fluorescence microscopy images using inter- and intra-region discriminative information.

    PubMed

    Song, Yang; Cai, Weidong; Feng, David Dagan; Chen, Mei

    2013-01-01

    Automated segmentation of cell nuclei in microscopic images is critical to high throughput analysis of the ever increasing amount of data. Although cell nuclei are generally visually distinguishable for human, automated segmentation faces challenges when there is significant intensity inhomogeneity among cell nuclei or in the background. In this paper, we propose an effective method for automated cell nucleus segmentation using a three-step approach. It first obtains an initial segmentation by extracting salient regions in the image, then reduces false positives using inter-region feature discrimination, and finally refines the boundary of the cell nuclei using intra-region contrast information. This method has been evaluated on two publicly available datasets of fluorescence microscopic images with 4009 cells, and has achieved superior performance compared to popular state of the art methods using established metrics.

  7. Cell type-specific affinity purification of nuclei for chromatin profiling in whole animals.

    PubMed

    Steiner, Florian A; Henikoff, Steven

    2015-01-01

    Analyzing cell differentiation during development in a complex organism requires the analysis of expression and chromatin profiles in individual cell types. Our laboratory has developed a simple and generally applicable strategy to purify specific cell types from whole organisms for simultaneous analysis of chromatin and expression. The method, termed INTACT for Isolation of Nuclei TAgged in specific Cell Types, depends on the expression of an affinity-tagged nuclear envelope protein in the cell type of interest. These nuclei can be affinity-purified from the total pool of nuclei and used as a source for RNA and chromatin. The method serves as a simple and scalable alternative to FACS sorting or laser capture microscopy to circumvent the need for expensive equipment and specialized skills. This chapter provides detailed protocols for the cell-type specific purification of nuclei from Caenorhabditis elegans.

  8. Agrobacterium tumefaciens Gene Transfer: How a Plant Pathogen Hacks the Nuclei of Plant and Nonplant Organisms.

    PubMed

    Bourras, Salim; Rouxel, Thierry; Meyer, Michel

    2015-10-01

    Agrobacterium species are soilborne gram-negative bacteria exhibiting predominantly a saprophytic lifestyle. Only a few of these species are capable of parasitic growth on plants, causing either hairy root or crown gall diseases. The core of the infection strategy of pathogenic Agrobacteria is a genetic transformation of the host cell, via stable integration into the host genome of a DNA fragment called T-DNA. This genetic transformation results in oncogenic reprogramming of the host to the benefit of the pathogen. This unique ability of interkingdom DNA transfer was largely used as a tool for genetic engineering. Thus, the artificial host range of Agrobacterium is continuously expanding and includes plant and nonplant organisms. The increasing availability of genomic tools encouraged genome-wide surveys of T-DNA tagged libraries, and the pattern of T-DNA integration in eukaryotic genomes was studied. Therefore, data have been collected in numerous laboratories to attain a better understanding of T-DNA integration mechanisms and potential biases. This review focuses on the intranuclear mechanisms necessary for proper targeting and stable expression of Agrobacterium oncogenic T-DNA in the host cell. More specifically, the role of genome features and the putative involvement of host's transcriptional machinery in relation to the T-DNA integration and effects on gene expression are discussed. Also, the mechanisms underlying T-DNA integration into specific genome compartments is reviewed, and a theoretical model for T-DNA intranuclear targeting is presented. PMID:26151736

  9. A robust procedure for distinctively visualizing zebrafish retinal cell nuclei under bright field light microscopy.

    PubMed

    Fu, Jinling; Fang, Wei; Zou, Jian; Sun, Ming; Lathrop, Kira; Su, Guanfang; Wei, Xiangyun

    2013-03-01

    To simultaneously visualize individual cell nuclei and tissue morphologies of the zebrafish retina under bright field light microscopy, it is necessary to establish a procedure that specifically and sensitively stains the cell nuclei in thin tissue sections. This necessity arises from the high nuclear density of the retina and the highly decondensed chromatin of the cone photoreceptors, which significantly reduces their nuclear signals and makes nuclei difficult to distinguish from possible high cytoplasmic background staining. Here we optimized a procedure that integrates JB4 plastic embedding and Feulgen reaction for visualizing zebrafish retinal cell nuclei under bright field light microscopy. This method produced highly specific nuclear staining with minimal cytoplasmic background, allowing us to distinguish individual retinal nuclei despite their tight packaging. The nuclear staining is also sensitive enough to distinguish the euchromatin from heterochromatin in the zebrafish cone nuclei. In addition, this method could be combined with in situ hybridization to simultaneously visualize the cell nuclei and mRNA expression patterns. With its superb specificity and sensitivity, this method may be extended to quantify cell density and analyze global chromatin organization throughout the retina or other tissues.

  10. Foci of Entotic Nuclei in Different Grades of Noninherited Renal Cell Cancers.

    PubMed

    Kong, Yuke; Liang, Yaojun; Wang, Jianqin

    2015-02-01

    We report here an intriguing pattern in nuclear appearance of renal clear cell cancer. In low grade clear cell cancer, detailed examination showed that in many cells, two or more nuclei were within the confines of a single cell membrane. This likely resulted from a cell being contained within its neighboring cell. Consequently, this resulted in appearance of multicellularity. This appearance of the nuclei were not associated with mitotic figures, suggesting that these did not result from nuclear fission. Additionally, the cells containing this nuclei did not show any evidence of cytokinesis including equatorial tapering, suggesting that the process may have resulted from cytokinesis failure. In some sections of higher grade clear cell cancer, these appearance were higher, though we did not observe any frank syncytium formation. On careful observation, there were isolated events of fusion of nuclei within a single cell in different grades of renal cell cancers. There occurrence was more frequent in higher grades of clear cell renal cancer and metastatic clear cell carcinoma. These features were also demonstrable in multiple fields of lower grades of clear cell carcinoma. This phenomenon of entosis may contribute to aneuploidy and tumor progression to dysplastic stages and genomic instability in renal cancers. Future studies are aimed at delineating the cell-cell boundaries and the mechanism contributing to this observation, either from peripheral cell engulfing or failure of cytosolic division for cell separation.

  11. Observation of DNA and protein distributions in mammalian cell nuclei using STXM

    NASA Astrophysics Data System (ADS)

    Ohigashi, Takuji; Ito, Atsushi; Shinohara, Kunio; Tone, Shigenobu; Kado, Masataka; Inagaki, Yuichi; Wang, Yu-Fu; Kosugi, Nobuhiro

    2016-01-01

    A whole A549 cell and isolated nuclei of HeLa S3 cells in the apoptotic process were investigated by using a scanning transmission X-ray microscope (STXM) in the UVSOR Synchrotron (Okazaki, Japan). Near edge X-ray absorption fine structures (NEXAFS) of DNA and histone in the N K-edge region were measured as reference and their distribution in the nuclei was determined by using these reference spectra. The four stages of the apoptosis were successfully distinguished.

  12. Segmentation of whole cells and cell nuclei from 3-D optical microscope images using dynamic programming.

    PubMed

    McCullough, D P; Gudla, P R; Harris, B S; Collins, J A; Meaburn, K J; Nakaya, M A; Yamaguchi, T P; Misteli, T; Lockett, S J

    2008-05-01

    Communications between cells in large part drive tissue development and function, as well as disease-related processes such as tumorigenesis. Understanding the mechanistic bases of these processes necessitates quantifying specific molecules in adjacent cells or cell nuclei of intact tissue. However, a major restriction on such analyses is the lack of an efficient method that correctly segments each object (cell or nucleus) from 3-D images of an intact tissue specimen. We report a highly reliable and accurate semi-automatic algorithmic method for segmenting fluorescence-labeled cells or nuclei from 3-D tissue images. Segmentation begins with semi-automatic, 2-D object delineation in a user-selected plane, using dynamic programming (DP) to locate the border with an accumulated intensity per unit length greater that any other possible border around the same object. Then the two surfaces of the object in planes above and below the selected plane are found using an algorithm that combines DP and combinatorial searching. Following segmentation, any perceived errors can be interactively corrected. Segmentation accuracy is not significantly affected by intermittent labeling of object surfaces, diffuse surfaces, or spurious signals away from surfaces. The unique strength of the segmentation method was demonstrated on a variety of biological tissue samples where all cells, including irregularly shaped cells, were accurately segmented based on visual inspection.

  13. Nuclear proteins of quiescent Xenopus laevis cells inhibit DNA replication in intact and permeabilized nuclei

    PubMed Central

    1996-01-01

    Quiescent cells from adult vertebrate liver and contact-inhibited or serum-deprived tissue cultures are active metabolically but do not carry out nuclear DNA replication and cell division. Replication of intact nuclei isolated from either quiescent Xenopus liver or cultured Xenopus A6 cells in quiescence was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with approximately 100% efficiency in the same extracts. Permeabilization of nuclei from quiescent Xenopus liver or cultured Xenopus epithelial A6 cells did not facilitate efficient replication in egg extracts. Moreover, replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei; in contrast, complementary-strand synthesis on single-stranded DNA templates in egg extracts was not affected. Inhibition was specific to endogenous molecules localized preferentially in quiescent as opposed to proliferating cell nuclei, and was not due to suppression of cdk2 kinase activity. Extracts of Xenopus liver nuclei also inhibited growth of sperm nuclei formed in egg extracts. However, the rate and extent of decondensation of sperm chromatin in egg extracts were not affected. The formation of prereplication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when liver nuclear extracts were added to metaphase egg extracts before or immediately after Ca++ ion-induced transition to interphase. A plausible interpretation of our data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in Xenopus cells, both in vivo and in cultured cells, perhaps by negatively regulating positive modulators of the replication machinery. PMID:8655587

  14. Formalinized chicken red cell nuclei as a simple antigen for standardized antinuclear factor determination

    PubMed Central

    Veen, J. H. Ten; Feltkamp, T. E. W.

    1969-01-01

    Chicken red cell nuclei treated with formalin appear to be specific and easy to handle nuclear antigens for antinuclear factor determination. They can be kept without cooling or freezing for at least 6 months, and probably considerably longer. As these nuclei can easily be obtained in great amounts it appears feasible to develop an international nuclear standard antigen for standardization in immunofluorescence. They might also be applied as a nuclear antigen in automated antinuclear factor determination. PMID:4904069

  15. Incorporation of mammalian actin into microfilaments in plant cell nucleus

    PubMed Central

    Paves, Heiti; Truve, Erkki

    2004-01-01

    Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area. PMID:15102327

  16. In vitro assays predictive of telomerase inhibitory effect of G-quadruplex ligands in cell nuclei.

    PubMed

    Yaku, Hidenobu; Murashima, Takashi; Miyoshi, Daisuke; Sugimoto, Naoki

    2014-03-13

    G-quadruplex-binding and telomerase-inhibiting capacities of G-quadruplex ligands were examined under a cell nuclei-mimicking condition including excess double-stranded DNA (λ DNA) and molecular crowding cosolute (PEG 200). Under the cell nuclei-mimicking condition, a cationic porphyrin (TMPyP4) did not bind to the G-quadruplex despite the high affinity (Ka = 3.6 × 10(6) M(-1)) under a diluted condition without λ DNA and PEG 200. Correspondingly, TMPyP4 inhibited telomerase activity under the diluted condition (IC50 = 1.6 μM) but not under the cell nuclei-mimicking condition. In contrast, the Ka and IC50 values of an anionic copper phthalocyanine (Cu-APC) under the diluted (2.8 × 10(4) M(-1) and 0.86 μM) and the cell nuclei-mimicking (2.8 × 10(4) M(-1) and 2.1 μM) conditions were similar. In accordance with these results, 10 μM TMPyP4 did not affect the proliferation of HeLa cells, while Cu-APC efficiently inhibited the proliferation (IC50 = 1.4 μM). These results show that the cell nuclei-mimicking condition is effective to predict capacities of G-quadruplex ligands in the cell. In addition, the antiproliferative effect of Cu-APC on normal cells was smaller than that on HeLa cells, indicating that the cell nuclei-mimicking condition is also useful to predict side effects of ligands.

  17. In vitro assays predictive of telomerase inhibitory effect of G-quadruplex ligands in cell nuclei.

    PubMed

    Yaku, Hidenobu; Murashima, Takashi; Miyoshi, Daisuke; Sugimoto, Naoki

    2014-03-13

    G-quadruplex-binding and telomerase-inhibiting capacities of G-quadruplex ligands were examined under a cell nuclei-mimicking condition including excess double-stranded DNA (λ DNA) and molecular crowding cosolute (PEG 200). Under the cell nuclei-mimicking condition, a cationic porphyrin (TMPyP4) did not bind to the G-quadruplex despite the high affinity (Ka = 3.6 × 10(6) M(-1)) under a diluted condition without λ DNA and PEG 200. Correspondingly, TMPyP4 inhibited telomerase activity under the diluted condition (IC50 = 1.6 μM) but not under the cell nuclei-mimicking condition. In contrast, the Ka and IC50 values of an anionic copper phthalocyanine (Cu-APC) under the diluted (2.8 × 10(4) M(-1) and 0.86 μM) and the cell nuclei-mimicking (2.8 × 10(4) M(-1) and 2.1 μM) conditions were similar. In accordance with these results, 10 μM TMPyP4 did not affect the proliferation of HeLa cells, while Cu-APC efficiently inhibited the proliferation (IC50 = 1.4 μM). These results show that the cell nuclei-mimicking condition is effective to predict capacities of G-quadruplex ligands in the cell. In addition, the antiproliferative effect of Cu-APC on normal cells was smaller than that on HeLa cells, indicating that the cell nuclei-mimicking condition is also useful to predict side effects of ligands. PMID:24328194

  18. Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation.

    PubMed

    Thilly, William G; Gostjeva, Elena V; Koledova, Vera V; Zukerberg, Lawrence R; Chung, Daniel; Fomina, Janna N; Darroudi, Firouz; Stollar, B David

    2014-01-01

    Bell shaped nuclei of metakaryotic cells double their DNA content during and after symmetric and asymmetric amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells. A parsimonious hypothesis was tested that the two anti-parallel strands of each chromatid DNA helix were first segregated as ssDNA-containing complexes into sister nuclei then copied to recreate a dsDNA genome. Metakaryotic nuclei that were treated during amitosis with RNase A and stained with acridine orange or fluorescent antibody to ssDNA revealed large amounts of ssDNA. Without RNase treatment metakaryotic nuclei in amitosis stained strongly with an antibody complex specific to dsRNA/DNA. Images of amitotic figures co-stained with dsRNA/DNA antibody and DAPI indicated that the entire interphase dsDNA genome (B-form helices) was transformed into two dsRNA/DNA genomes (A-form helices) that were segregated in the daughter cell nuclei then retransformed into dsDNA. As this process segregates DNA strands of opposite polarity in sister cells it hypothetically offers a sequential switching mechanism within the diverging stem cell lineages of development.

  19. Lysis gradient centrifugation: a flexible method for the isolation of nuclei from primary cells.

    PubMed

    Katholnig, Karl; Poglitsch, Marko; Hengstschläger, Markus; Weichhart, Thomas

    2015-01-01

    The isolation of nuclei from eukaryotic cells is essential for studying the composition and the dynamic changes of the nuclear proteome to gain insight into the mechanisms of gene expression and cell signalling. Primary cells are particularly challenging for standard nuclear isolation protocols due to low protein content, sample degradation, or nuclear clumping. Here, we describe a rapid and flexible protocol for the isolation of clean and intact nuclei, which results in the recovery of 90-95 % highly pure nuclei. The method, called lysis gradient centrifugation (LGC), is based on an iso-osmolar discontinuous iodixanol-based density gradient including a detergent-containing lysis layer. A single low g-force centrifugation step enables mild cell lysis and prevents extensive contact of the nuclei with the cytoplasmic environment. This fast method shows high reproducibility due to the relatively little cell manipulation required by the investigator. Further advantages are the low amount of starting material required, easy parallel processing of multiple samples, and isolation of nuclei and cytoplasm at the same time from the same sample.

  20. Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation

    PubMed Central

    Thilly, William G; Gostjeva, Elena V; Koledova, Vera V; Zukerberg, Lawrence R; Chung, Daniel; Fomina, Janna N; Darroudi, Firouz; Stollar, B David

    2014-01-01

    Bell shaped nuclei of metakaryotic cells double their DNA content during and after symmetric and asymmetric amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells. A parsimonious hypothesis was tested that the two anti-parallel strands of each chromatid DNA helix were first segregated as ssDNA-containing complexes into sister nuclei then copied to recreate a dsDNA genome. Metakaryotic nuclei that were treated during amitosis with RNase A and stained with acridine orange or fluorescent antibody to ssDNA revealed large amounts of ssDNA. Without RNase treatment metakaryotic nuclei in amitosis stained strongly with an antibody complex specific to dsRNA/DNA. Images of amitotic figures co-stained with dsRNA/DNA antibody and DAPI indicated that the entire interphase dsDNA genome (B-form helices) was transformed into two dsRNA/DNA genomes (A-form helices) that were segregated in the daughter cell nuclei then retransformed into dsDNA. As this process segregates DNA strands of opposite polarity in sister cells it hypothetically offers a sequential switching mechanism within the diverging stem cell lineages of development. PMID:24418910

  1. Rapid 3-D delineation of cell nuclei for high-content screening platforms.

    PubMed

    Gertych, Arkadiusz; Ma, Zhaoxuan; Tajbakhsh, Jian; Velásquez-Vacca, Adriana; Knudsen, Beatrice S

    2016-02-01

    High-resolution three-dimensional (3-D) microscopy combined with multiplexing of fluorescent labels allows high-content analysis of large numbers of cell nuclei. The full automation of 3-D screening platforms necessitates image processing algorithms that can accurately and robustly delineate nuclei in images with little to no human intervention. Imaging-based high-content screening was originally developed as a powerful tool for drug discovery. However, cell confluency, complexity of nuclear staining as well as poor contrast between nuclei and background result in slow and unreliable 3-D image processing and therefore negatively affect the performance of studying a drug response. Here, we propose a new method, 3D-RSD, to delineate nuclei by means of 3-D radial symmetries and test it on high-resolution image data of human cancer cells treated by drugs. The nuclei detection performance was evaluated by means of manually generated ground truth from 2351 nuclei (27 confocal stacks). When compared to three other nuclei segmentation methods, 3D-RSD possessed a better true positive rate of 83.3% and F-score of 0.895±0.045 (p-value=0.047). Altogether, 3D-RSD is a method with a very good overall segmentation performance. Furthermore, implementation of radial symmetries offers good processing speed, and makes 3D-RSD less sensitive to staining patterns. In particular, the 3D-RSD method performs well in cell lines, which are often used in imaging-based HCS platforms and are afflicted by nuclear crowding and overlaps that hinder feature extraction.

  2. Comparative analysis of the nucleosome structure of cell nuclei by small-angle neutron scattering

    NASA Astrophysics Data System (ADS)

    Isaev-Ivanov, V. V.; Lebedev, D. V.; Lauter, H.; Pantina, R. A.; Kuklin, A. I.; Islamov, A. Kh.; Filatov, M. V.

    2010-05-01

    The nucleosome structure in native nuclei of normal (chicken erythrocyte and rat leukocyte nuclei) and anomalously proliferating (the human cervical adenocarcinoma cell line HeLa and the Chinese hamster fibroblast cell line A238) cells has been investigated using small-angle neutron scattering. The experimental results obtained allow one to make the inference that the parameters of the nucleosome structure for the chicken erythrocyte and rat leukocyte nuclei (on average over the nucleus) are close to the universally accepted values and that the distance distribution function is bimodal. The bimodality of the distance distribution function reflects a narrow distribution of distances between nucleosomes (on average over the nucleus) at the fibril level of the chromatin organization. The histone core of the nucleosome structure in the nuclei of the HeLa and A238 cells (on average over the nucleus) is considerably less compact than that in the chicken erythrocyte and rat leukocyte nuclei, and the distance distribution function does not exhibit indications of the bimodality.

  3. Finding splitting lines for touching cell nuclei with a shortest path algorithm.

    PubMed

    Bai, Xiangzhi; Wang, Peng; Sun, Changming; Zhang, Yu; Zhou, Fugen; Meng, Cai

    2015-08-01

    A shortest path-based algorithm is proposed in this paper to find splitting lines for touching cell nuclei. First, an initial splitting line is obtained through the distance transform of a marker image and the watershed algorithm. The initial splitting line is then separated into different line segments as necessary, and the endpoint positions of these line segments are adjusted to the concave points on the contour. Finally, a shortest path algorithm is used to find the accurate splitting line between the starting-point and the end-point, and the final split can be achieved by the contour of the touching cell nuclei and the splitting lines. Comparisons of experimental results show that the proposed algorithm is effective for segmentation of different types of touching cell nuclei.

  4. Development of porcine tetraploid somatic cell nuclear transfer embryos is influenced by oocyte nuclei.

    PubMed

    Fu, Bo; Liu, Di; Ma, Hong; Guo, Zhen-Hua; Wang, Liang; Li, Zhong-Qiu; Peng, Fu-Gang; Bai, Jing

    2016-02-01

    Cloning efficiency in mammalian systems remains low because reprogramming of donor cells is frequently incomplete. Nuclear factors in the oocyte are removed by enucleation, and this removal may adversely affect reprogramming efficiency. Here, we investigated the role of porcine oocyte nuclear factors during reprogramming. We introduced somatic cell nuclei into intact MII oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. We then examined the influence of the oocyte nucleus on tetraploid SCNT embryo development by assessing characteristics including pronucleus formation, cleavage rate, and blastocyst formation. Overall, tetraploid SCNT embryos have a higher developmental competence than do standard diploid SCNT embryos. Therefore, we have established an embryonic model in which a fetal fibroblast nucleus and an oocyte metaphase II plate coexist. Tetraploid SCNT represents a new research platform that is potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming.

  5. Glioma grading using cell nuclei morphologic features in digital pathology images

    NASA Astrophysics Data System (ADS)

    Reza, Syed M. S.; Iftekharuddin, Khan M.

    2016-03-01

    This work proposes a computationally efficient cell nuclei morphologic feature analysis technique to characterize the brain gliomas in tissue slide images. In this work, our contributions are two-fold: 1) obtain an optimized cell nuclei segmentation method based on the pros and cons of the existing techniques in literature, 2) extract representative features by k-mean clustering of nuclei morphologic features to include area, perimeter, eccentricity, and major axis length. This clustering based representative feature extraction avoids shortcomings of extensive tile [1] [2] and nuclear score [3] based methods for brain glioma grading in pathology images. Multilayer perceptron (MLP) is used to classify extracted features into two tumor types: glioblastoma multiforme (GBM) and low grade glioma (LGG). Quantitative scores such as precision, recall, and accuracy are obtained using 66 clinical patients' images from The Cancer Genome Atlas (TCGA) [4] dataset. On an average ~94% accuracy from 10 fold crossvalidation confirms the efficacy of the proposed method.

  6. [Alpha-lipoic acid triggers elimination of cells with abnormal nuclei in human carcinoma epidermoid cell line].

    PubMed

    Kisurina-Evgen'eva, O P; Onishchenko, G E

    2010-01-01

    The skin is usually exposed to adverse environmental conditions that may cause pathological cell proliferation and cellular transformations leading to the formation of malignant cells. Antioxidants may affect these processes and induce the elimination of transformed cell. The purpose of this work was to investigate the effect of alfa-lipoic acid on human carcinoma epidermoid cell line A431. Our results showed that alfa-lipoic acid induced inhibition of cell proliferation or stimulated apoptotic cell death. Cells with abnormal nuclei were eliminated by apoptosis. Electron microscopy showed that survived cells had typical for control cells shape and organization of the nuclei, organization of the cytoplasm and organelles. Thus, alfa-lipoic acid not only triggered apoptosis of carcinoma cells, but it may also activate the mechanism of elimination of cells with abnormal chromosome number.

  7. Imaging Nuclear Morphology and Organization in Cleared Plant Tissues Treated with Cell Cycle Inhibitors.

    PubMed

    de Souza Junior, José Dijair Antonino; de Sa, Maria Fatima Grossi; Engler, Gilbert; Engler, Janice de Almeida

    2016-01-01

    Synchronization of root cells through chemical treatment can generate a large number of cells blocked in specific cell cycle phases. In plants, this approach can be employed for cell suspension cultures and plant seedlings. To identify plant cells in the course of the cell cycle, especially during mitosis in meristematic tissues, chemical inhibitors can be used to block cell cycle progression. Herein, we present a simplified and easy-to-apply protocol to visualize mitotic figures, nuclei morphology, and organization in whole Arabidopsis root apexes. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with DAPI. The protocol allows carrying out bulk analysis of nuclei and cell cycle phases in root cells and will be valuable to investigate mutants like overexpressing lines of genes disturbing the plant cell cycle.

  8. Identification of feline panleukopenia virus proteins expressed in Purkinje cell nuclei of cats with cerebellar hypoplasia.

    PubMed

    Poncelet, Luc; Héraud, Céline; Springinsfeld, Marie; Ando, Kunie; Kabova, Anna; Beineke, Andreas; Peeters, Dominique; Op De Beeck, Anne; Brion, Jean-Pierre

    2013-06-01

    Parvoviruses depend on initiation of host cell division for their replication. Undefined parvoviral proteins have been detected in Purkinje cells of the cerebellum after experimental feline panleukopenia virus (FPV) infection of neonatal kittens and in naturally occurring cases of feline cerebellar hypoplasia. In this study, a parvoviral protein in the nucleus of Purkinje cells of kittens with cerebellar hypoplasia was shown by immunoprecipitation to be the FPV viral capsid protein VP2. In PCR-confirmed, FPV-associated feline cerebellar hypoplasia, expression of the FPV VP2 protein was demonstrated by immunohistochemistry in Purkinje cell nuclei in 4/10 cases and expression of the FPV non-structural protein NS1 was demonstrated in Purkinje cell nuclei in 5/10 cases. Increased nuclear ERK1 expression was observed in several Purkinje cells in 1/10 kittens. No expression of the G1 and S mitotic phase marker proliferating cell nuclear antigen (PCNA) was evident in Purkinje cell nuclei. These results support the hypothesis that FPV is able to proceed far into its replication cycle in post-mitotic Purkinje cells.

  9. The role of p53 in the trafficking of copper-64 to tumor cell nuclei.

    PubMed

    Eiblmaier, Martin; Meyer, Laura A; Anderson, Carolyn J

    2008-01-01

    Copper-64 (T(1/2) = 12.7 h; beta(+): 17.8%, beta(-): 41%) has applications in both positron emission tomography (PET) imaging and targeted radiotherapy of cancer. Copper-64 radiopharmaceuticals have shown tumor growth inhibition with a relatively low radiation dose in animal models; however, the mechanism of cytotoxicity has not been fully elucidated. Here, we report an investigation on the potential role of the tumor suppressor protein p53 in trafficking (64)Cu to tumor cell nuclei. Two EGFR expressing human colorectal cell lines (HCT 116 +/+ and HCT 116 -/-) that are positive or negative for p53 expression respectively, were used to compare internalization and nuclear localization of [(64)Cu]copper acetate and of (64)Cu-DOTA-cetuximab, a monoclonal anti-EGFR antibody. [(64)Cu]copper acetate uptake into cells was similar between the two cell lines during a 24 h time course. In contrast, the uptake of [(64)Cu]copper acetate in the nuclei of HCT 116 +/+ cells was significantly higher than in HCT 116 -/- cells (p < 0.0001) at 24 h. There was no difference in receptor binding, receptor-mediated internalization, and efflux of (64)Cu-DOTA-cetuximab between the two HCT 116 cells lines. However, nuclear localization of (64)Cu-DOTA-cetuximab showed increased uptake in the nuclei of HCT 116 +/+ cells as early as 4 h. These data demonstrate that (64)Cu is delivered to tumor cell nuclei in a p53 positive cell line in significantly greater amounts than in p53 negative cells by both non-specific and receptor-mediated uptake mechanisms.

  10. Fluorescence-activated sorting of fixed nuclei: a general method for studying nuclei from specific cell populations that preserves post-translational modifications.

    PubMed

    Marion-Poll, Lucile; Montalban, Enrica; Munier, Annie; Hervé, Denis; Girault, Jean-Antoine

    2014-04-01

    Long-lasting brain alterations that underlie learning and memory are triggered by synaptic activity. How activity can exert long-lasting effects on neurons is a major question in neuroscience. Signalling pathways from cytoplasm to nucleus and the resulting changes in transcription and epigenetic modifications are particularly relevant in this context. However, a major difficulty in their study comes from the cellular heterogeneity of brain tissue. A promising approach is to directly purify identified nuclei. Using mouse striatum we have developed a rapid and efficient method for isolating cell type-specific nuclei from fixed adult brain (fluorescence-activated sorting of fixed nuclei; FAST-FIN). Animals are quickly perfused with a formaldehyde fixative that stops enzymatic reactions and maintains the tissue in the state it was at the time of death, including nuclear localisation of soluble proteins such as GFP and differences in nuclear size between cell types. Tissue is subsequently dissociated with a Dounce homogeniser and nuclei prepared by centrifugation in an iodixanol density gradient. The purified fixed nuclei can then be immunostained with specific antibodies and analysed or sorted by flow cytometry. Simple criteria allow distinction of neurons and non-neuronal cells. Immunolabelling and transgenic mice that express fluorescent proteins can be used to identify specific cell populations, and the nuclei from these populations can be efficiently isolated, even rare cell types such as parvalbumin-expressing interneurons. FAST-FIN allows the preservation and study of dynamic and labile post-translational protein modifications. It should be applicable to other tissues and species, and allow study of DNA and its modifications.

  11. Fluorescent Magnesium Nanocomplex in Protein Scaffold for Cell Nuclei Imaging Application

    SciTech Connect

    Pandya, Alok; Tripathi, Apritam; Purohit, Rahul; Singh, Sanjay; Nandasiri, Manjula I.; Karakoti, Ajay S.; Singh, Surinder P.; Shanker, Rishi

    2015-10-27

    Here in, we report a facile strategy for the synthesis of water-soluble ultra-fine blue emitting fluorescent Magnesium nanoparticles-protein complex (MgNC). This MgNC is demonstrated to exhibit excellent photo stability and biocompatibility. It was also observed that MgNC stain cell nuclei with high specifcity.

  12. Multiple nuclei tracking using integer programming for quantitative cancer cell cycle analysis.

    PubMed

    Li, Fuhai; Zhou, Xiaobo; Ma, Jinwen; Wong, Stephen T C

    2010-01-01

    Automated cell segmentation and tracking are critical for quantitative analysis of cell cycle behavior using time-lapse fluorescence microscopy. However, the complex, dynamic cell cycle behavior poses new challenges to the existing image segmentation and tracking methods. This paper presents a fully automated tracking method for quantitative cell cycle analysis. In the proposed tracking method, we introduce a neighboring graph to characterize the spatial distribution of neighboring nuclei, and a novel dissimilarity measure is designed based on the spatial distribution, nuclei morphological appearance, migration, and intensity information. Then, we employ the integer programming and division matching strategy, together with the novel dissimilarity measure, to track cell nuclei. We applied this new tracking method for the tracking of HeLa cancer cells over several cell cycles, and the validation results showed that the high accuracy for segmentation and tracking at 99.5% and 90.0%, respectively. The tracking method has been implemented in the cell-cycle analysis software package, DCELLIQ, which is freely available. PMID:19643704

  13. Refractometry of melanocyte cell nuclei using optical scatter images recorded by digital Fourier microscopy

    NASA Astrophysics Data System (ADS)

    Seet, Katrina Y. T.; Nieminen, Timo A.; Zvyagin, Andrei V.

    2009-07-01

    The cell nucleus is the dominant optical scatterer in the cell. Neoplastic cells are characterized by cell nucleus polymorphism and polychromism-i.e., the nuclei exhibits an increase in the distribution of both size and refractive index. The relative size parameter, and its distribution, is proportional to the product of the nucleus size and its relative refractive index and is a useful discriminant between normal and abnormal (cancerous) cells. We demonstrate a recently introduced holographic technique, digital Fourier microscopy (DFM), to provide a sensitive measure of this relative size parameter. Fourier holograms were recorded and optical scatter of individual scatterers were extracted and modeled with Mie theory to determine the relative size parameter. The relative size parameter of individual melanocyte cell nuclei were found to be 16.5+/-0.2, which gives a cell nucleus refractive index of 1.38+/-0.01 and is in good agreement with previously reported data. The relative size parameters of individual malignant melanocyte cell nuclei are expected to be greater than 16.5.

  14. Improved automatic detection and segmentation of cell nuclei in histopathology images.

    PubMed

    Al-Kofahi, Yousef; Lassoued, Wiem; Lee, William; Roysam, Badrinath

    2010-04-01

    Automatic segmentation of cell nuclei is an essential step in image cytometry and histometry. Despite substantial progress, there is a need to improve accuracy, speed, level of automation, and adaptability to new applications. This paper presents a robust and accurate novel method for segmenting cell nuclei using a combination of ideas. The image foreground is extracted automatically using a graph-cuts-based binarization. Next, nuclear seed points are detected by a novel method combining multiscale Laplacian-of-Gaussian filtering constrained by distance-map-based adaptive scale selection. These points are used to perform an initial segmentation that is refined using a second graph-cuts-based algorithm incorporating the method of alpha expansions and graph coloring to reduce computational complexity. Nuclear segmentation results were manually validated over 25 representative images (15 in vitro images and 10 in vivo images, containing more than 7400 nuclei) drawn from diverse cancer histopathology studies, and four types of segmentation errors were investigated. The overall accuracy of the proposed segmentation algorithm exceeded 86%. The accuracy was found to exceed 94% when only over- and undersegmentation errors were considered. The confounding image characteristics that led to most detection/segmentation errors were high cell density, high degree of clustering, poor image contrast and noisy background, damaged/irregular nuclei, and poor edge information. We present an efficient semiautomated approach to editing automated segmentation results that requires two mouse clicks per operation.

  15. Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.

    PubMed

    Hancock, Ronald; Hadj-Sahraoui, Yasmina

    2009-10-23

    Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v) or 70 kDa dextran (35% w/v) to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 microM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II) were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox that the small

  16. Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.

    PubMed

    Hancock, Ronald; Hadj-Sahraoui, Yasmina

    2009-01-01

    Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v) or 70 kDa dextran (35% w/v) to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 microM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II) were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox that the small

  17. Graft derived cells with double nuclei in the penumbral region of experimental brain trauma.

    PubMed

    Horváth, Eszter M; Lacza, Zsombor; Csordás, Attila; Szabó, Csaba; Kollai, Márk; Busija, David W

    2006-04-01

    Recent in vitro studies showed that stem cells might fuse with mature cells or each other; however, there is no in vivo evidence for this phenomenon in the cerebral cortex. Our goal was to find evidence for cell fusion in a model of traumatic brain injury followed by grafting of embryonic cortical cells. Cold lesion protocol was applied to induce lesion of the motor cortex in adult male rats. Six days later we grafted a suspension of freshly isolated rat brain cortical cells of early embryonic stage (E14) into the penumbra area of the lesion. The grafted cell nuclei were labelled with bromodeoxyuridine (BrDU). Six days after transplantation 4,328 BrDU positive cells were observed in nine animals. 89.5% of these cells had cytoplasmic staining probably representing dead or phagocyted grafted cells. Ten percent of surviving BrDU positive cells had only one BrDU positive nucleus and negative cytoplasm, while 0.5% had two distinct nuclei, one was unlabelled and one was BrDU positive. These cells were similar in appearance and size to the astrocytes in the vicinity and expressed the astocyte specific glial fibrillaly acidic protein. Thus, these cells showed a possible sign of cell fusion in the penumbral region of the injured brain. PMID:16377084

  18. A nonemissive iridium(III) complex that specifically lights-up the nuclei of living cells.

    PubMed

    Li, Chunyan; Yu, Mengxiao; Sun, Yun; Wu, Yongquan; Huang, Chunhui; Li, Fuyou

    2011-07-27

    A nonemissive cyclometalated iridium(III) solvent complex, without conjugation with a cell-penetrating molecular transporter, [Ir(ppy)(2)(DMSO)(2)](+)PF(6)(-) (LIr1), has been developed as a first reaction-based fluorescence-turn-on agent for the nuclei of living cells. LIr1 can rapidly and selectively light-up the nuclei of living cells over fixed cells, giving rise to a significant luminescence enhancement (200-fold), and shows very low cytotoxicity at the imaging concentration (incubation time <10 min, LIr1 concentration 10 μM). More importantly, in contrast to the reported nuclear stains that are based on luminescence enhancement through interaction with nucleic acids, complex LIr1 as a nuclear stain has a reaction-based mode of action, which relies on its rapid reaction with histidine/histidine-containing proteins. Cellular uptake of LIr1 has been investigated in detail under different conditions, such as at various temperatures, with hypertonic treatment, and in the presence of metabolic and endocytic inhibitors. The results have indicated that LIr1 permeates the outer and nuclear membranes of living cells through an energy-dependent entry pathway within a few minutes. As determined by an inductively coupled plasma atomic emission spectroscopy (ICP-AEC), LIr1 is accumulated in the nuclei of living cells and converted into an intensely emissive adduct. Such novel reaction-based nuclear staining for visualizing exclusively the nuclei of living cells with a significant luminescence enhancement may extend the arsenal of currently available fluorescent stains for specific staining of cellular compartments.

  19. Estrogen- and progestin-receptor complexes and their interaction with rat liver cell nuclei during ontogenesis

    SciTech Connect

    Konoplya, E.F.; Luksha, G.L.; Savateev, S.K.; Naumov, A.D.

    1986-11-10

    Steroid-receptor complexes (SRC) of estrogens and progestins have been isolated from the rat liver and purified 1500-2000-fold. Both in the state of the initial cytosol and purified 2000-fold, the SRC were characterized by gel filtration on Sephadex G-100 and by DEAE-cellulose chromatography. The purified SRC from the rat liver were used for binding to isolated liver cell nuclei from rats of different ages (1.5 months, 6 months, 12 months, 24 months). The maximum binding of SRC of progestins and estrogens from the rat liver is observed with homologous nuclei of 1.5-month-old rats. With age the binding of SRC by the nuclei decreases progressively and reaches a minimum by 24 months. The detected differences in the binding of SRC by the nuclei of cells of animals of different ages, in their opinion, may lie at the basis of changes in the functioning of the organism under the influence of hormones at different stages of ontogenesis.

  20. Automatic Segmentation of Cell Nuclei in Bladder and Skin Tissue for Karyometric Analysis

    PubMed Central

    Korde, Vrushali R.; Bartels, Hubert; Barton, Jennifer; Ranger-Moore, James

    2010-01-01

    Objective To automatically segment cell nuclei in histology images of bladder and skin tissue for karyometric analysis. Study Design The four main steps in the program were as follows: median filtering and thresholding, segmentation, categorizing, and cusp correction. This robust segmentation technique used properties of the image histogram to optimally select a threshold and create closed four-way chain code nuclear segmentations. Each cell nucleus segmentation was treated as an individual object whose properties of segmentation quality were used for criteria to classify each nucleus as: throw away, salvageable, or good. An erosion/dilation procedure and re-thresholding were performed on salvageable nuclei to correct cusps. Results Ten bladder histology images were segmented both by hand and using this automatic segmentation algorithm. The automatic segmentation resulted in a sensitivity of 76.4%, defined as the percentage of hand segmented nuclei that were automatically segmented with good quality. The median proportional difference between hand and automatic segmentations over 42 nuclei each with 95 features used in karyometric analysis was 1.6%. The same procedure was performed on 10 skin histology images with a sensitivity of 83.0% and median proportional difference of 2.6%. Conclusion The close agreement in karyometric features with hand segmentation shows that automated segmentation can be used for analysis of bladder and skin histology images. PMID:19402384

  1. Occurrence of amitotic division of trophoblast cell nuclei in blastocysts of the western spotted skunk (Spilogale putorius latifrons).

    PubMed

    Isakova, Galina K; Mead, Rodney A

    2004-01-01

    A cytogenetic examination of spreaded cells of diapausing and early activated blastocysts obtained from 7 female western spotted skunks was performed. Mitosis was not observed in 1626 cells obtained from 9 diapausing blastocysts; however, 12 (1.5%) figures of diploid mitosis were seen in 851 cells from 5 early activated embryos. Diameter of the cell nuclei varied from 4 to 29 microm during diapause, and from 5 to 40 microm in activated blastocyst, and the heterogeneity in nuclear size was significantly different between diapausing and activated embryos (P<0.01). About 80% of nuclei from diapausing blastocysts measured 9 to 16 microm, whereas a similar percentage of nuclei from activated blastocysts ranged from 15 to 27 microm. Many enlarged nuclei exhibited morphological features characteristic of mammalian polytene (i.e. endopolyploid with polytenic organization of chromosomes) trophoblast cells. The number of silver stained nucleoli in all the nuclei did not exceed 2, which corresponds to the number of nucleolus organizers in the diploid karyotype in this species of skunk and suggests the polytene organization of chromosomes in enlarged nuclei. About 10% of large interphase nuclei were observed to undergo amitosis, i.e. direct division by constriction. The resulting nuclear fragments in diapausing blastocysts usually had normal morphology and active nucleoli. In activated embryos, nearly 15% of amitotically divided nuclei appeared to be dividing into fragments of unequal size, one of which had normal cell nuclear morphology and extremely large silver positive nucleoli, and the other fragment exhibited signs of cell death. We interpret these data as indicating that 1) amitotic division of trophoblast endopolyploid cell nuclei in the skunk blastocysts may generate new trophoblast cells which contribute to increased cell number during both diapause and activation stages, and 2) activation of blastocysts after diapause is related to the production of trophoblast

  2. Fluorimetric measurements and chromatin condensation patterns of nuclei from 3T3 cells throughout G1.

    PubMed

    Moser, G C; Fallon, R J; Meiss, H K

    1981-02-01

    Using two cytological methods based on nuclear morphology, quinacrine dihydrochloride (QDH) staining and premature chromosome condensation (PCC), it has been possible to identify cell cycle positions within G1 of growing and arrested 3T3 cells. The fluorescent intensity of QDH-stained interphase cells appears to decrease as the cells pass from mitosis to S phase. Likewise, the length and thickness of prematurely condensed chromatids can be related to the cells; position within the G1 period. Data are presented that deal with three interrelated topics: 1) We determined by fluorometric measurements of nuclei from 3T3 cells that the visual observation of the decrease in QDH fluorescence during G1 reflects an actual decrease in total fluorescence and not a dispersion of the fluorescent chromatin in a larger nuclear area. 2) We correlated the results obtained by QDH staining with those of PCC on the same cell samples blocked in G1 by different conditions. Serum-starved and contact-inhibited cell nuclei had the highest intensity, hydroxyurea-treated ones had the lowest intensity, while that of isoleucine-deprived cells was in between. The same relative order of G1 positions was obtained based on PCC morphology. Thus, both methods monitor the state of chromatin condensation and can be used to identify cell cycle position within G1. 3) We showed with both methods that the states of chromatin resulting from the various G1 blocking conditions differ from each other.

  3. DNA-dependent targeting of cell nuclei by a lupus autoantibody.

    PubMed

    Weisbart, Richard H; Chan, Grace; Jordaan, Gwen; Noble, Philip W; Liu, Yanfeng; Glazer, Peter M; Nishimura, Robert N; Hansen, James E

    2015-07-09

    A nuclear-penetrating lupus anti-DNA autoantibody, 3E10, has been found to inhibit DNA repair and selectively kill certain cancer cells that are highly vulnerable to DNA damage. In addition, a 3E10 single chain variable fragment (scFv) has been developed for use as a delivery vehicle to carry therapeutic cargo proteins into cell nuclei. A greater understanding of the mechanism by which 3E10 penetrates cell nuclei is needed to help determine the scope of its potential therapeutic applications. Here we show that the presence of extracellular DNA significantly enhances the nuclear uptake of 3E10 scFv. In addition, we find that 3E10 scFv preferentially localizes into tumor cell nuclei in vivo, likely due to increased DNA in the local environment released from ischemic and necrotic regions of tumor. These data provide insight into the mechanism of nuclear penetration by 3E10 and demonstrate the potential for use of 3E10 in therapeutic approaches to diseases ranging from malignancy to ischemic conditions such as stroke.

  4. Lifeact and Utr230 induce distinct actin assemblies in cell nuclei.

    PubMed

    Du, Jing; Fan, Yan-Lei; Chen, Tai-Lin; Feng, Xi-Qiao

    2015-11-01

    Nuclear actin assembly in somatic cells has been an enigma for a long time. Recently, with the advancement of novel F-actin probes, researchers have started to uncover this mystery. In this study, we investigated the actin dynamics in somatic cell nuclei using two probes: Lifeact and Utr230. Surprisingly, we observed that both Lifeact and Utr230 significantly interfered with actin dynamics in cell nuclei. Moreover, these two probes induced distinct patterns of nuclear actin assembly. While Lifeact induced filamentous actin assembly in cell nuclei, Utr230 led to various patterns of actin aggregates, including fibers, small puncta, and large patches. Moreover, the interference of actin dynamics by Lifeact was limited to nuclear actin, while Utr230 induced actin aggregation in both the nucleus and cytoplasm. Using time-lapse microscopy, we found that Lifeact-induced actin fibers remained steady over hours of observation, indicating a deficiency of nuclear F-actin reorganization. These results suggest that Lifeact and Utr230 both interfere with nuclear actin dynamics but with distinct mechanisms. This is an important finding for research on nuclear actin assembly and highlights the potential value of these two probes for exploring the native mechanisms underlying nuclear actin dynamics, which appear to be altered in the presence of these probes.

  5. DNA-dependent targeting of cell nuclei by a lupus autoantibody

    PubMed Central

    Weisbart, Richard H.; Chan, Grace; Jordaan, Gwen; Noble, Philip W.; Liu, Yanfeng; Glazer, Peter M.; Nishimura, Robert N.; Hansen, James E.

    2015-01-01

    A nuclear-penetrating lupus anti-DNA autoantibody, 3E10, has been found to inhibit DNA repair and selectively kill certain cancer cells that are highly vulnerable to DNA damage. In addition, a 3E10 single chain variable fragment (scFv) has been developed for use as a delivery vehicle to carry therapeutic cargo proteins into cell nuclei. A greater understanding of the mechanism by which 3E10 penetrates cell nuclei is needed to help determine the scope of its potential therapeutic applications. Here we show that the presence of extracellular DNA significantly enhances the nuclear uptake of 3E10 scFv. In addition, we find that 3E10 scFv preferentially localizes into tumor cell nuclei in vivo, likely due to increased DNA in the local environment released from ischemic and necrotic regions of tumor. These data provide insight into the mechanism of nuclear penetration by 3E10 and demonstrate the potential for use of 3E10 in therapeutic approaches to diseases ranging from malignancy to ischemic conditions such as stroke. PMID:26156563

  6. Plant cell walls to ethanol.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conversion of plant cell walls to ethanol constitutes generation 2 bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation, and separation. Ultimately, it is desired to combine as man...

  7. Application and Quantitative Validation of Computer-Automated Three-Dimensional Counting of Cell Nuclei

    NASA Astrophysics Data System (ADS)

    Shain, William; Kayali, Soraya; Szarowski, Donald; Davis-Cox, Margaret; Ancin, Hakan; Bhattacharjya, Anoop K.; Roysam, Badrinath; Turner, James N.

    1999-03-01

    This study provides a quantitative validation of qualitative automated three-dimensional (3-D) analysis methods reported earlier. It demonstrates the applicability and quantitative accuracy of our method to detect, characterize, and count Feulgen stained cell nuclei in two tissues (hippocampus and testes). A laser-scanned confocal light microscope was used to record 3-D images i which our algorithms automatically identified individual nuclei from the optical sections given an estimate of minimum nuclear size. The hippocampal data sets were also manually counted independently by five trained observers using the STERECON 3-D image reconstruction system. The automated and manual counts were compared. A nucleus-by-nucleus comparison of the manual and automated counts verified that the automated analysis was accurate and reproducible, and permitted additional quantitative analyses not available from manual methods. The algorithms also identified subpopulations of nuclei within the hippocampal samples, and haploid and diploid nuclei in the testes. Our methods were shown to be repeatable, accurate, and more consistent than manual counting.

  8. Cell nuclei and cytoplasm joint segmentation using the sliding band filter.

    PubMed

    Quelhas, Pedro; Marcuzzo, Monica; Mendonça, Ana Maria; Campilho, Aurélio

    2010-08-01

    Microscopy cell image analysis is a fundamental tool for biological research. In particular, multivariate fluorescence microscopy is used to observe different aspects of cells in cultures. It is still common practice to perform analysis tasks by visual inspection of individual cells which is time consuming, exhausting and prone to induce subjective bias. This makes automatic cell image analysis essential for large scale, objective studies of cell cultures. Traditionally the task of automatic cell analysis is approached through the use of image segmentation methods for extraction of cells' locations and shapes. Image segmentation, although fundamental, is neither an easy task in computer vision nor is it robust to image quality changes. This makes image segmentation for cell detection semi-automated requiring frequent tuning of parameters. We introduce a new approach for cell detection and shape estimation in multivariate images based on the sliding band filter (SBF). This filter's design makes it adequate to detect overall convex shapes and as such it performs well for cell detection. Furthermore, the parameters involved are intuitive as they are directly related to the expected cell size. Using the SBF filter we detect cells' nucleus and cytoplasm location and shapes. Based on the assumption that each cell has the same approximate shape center in both nuclei and cytoplasm fluorescence channels, we guide cytoplasm shape estimation by the nuclear detections improving performance and reducing errors. Then we validate cell detection by gathering evidence from nuclei and cytoplasm channels. Additionally, we include overlap correction and shape regularization steps which further improve the estimated cell shapes. The approach is evaluated using two datasets with different types of data: a 20 images benchmark set of simulated cell culture images, containing 1000 simulated cells; a 16 images Drosophila melanogaster Kc167 dataset containing 1255 cells, stained for DNA and

  9. Cell nuclei attributed relational graphs for efficient representation and classification of gastric cancer in digital histopathology

    NASA Astrophysics Data System (ADS)

    Sharma, Harshita; Zerbe, Norman; Heim, Daniel; Wienert, Stephan; Lohmann, Sebastian; Hellwich, Olaf; Hufnagl, Peter

    2016-03-01

    This paper describes a novel graph-based method for efficient representation and subsequent classification in histological whole slide images of gastric cancer. Her2/neu immunohistochemically stained and haematoxylin and eosin stained histological sections of gastric carcinoma are digitized. Immunohistochemical staining is used in practice by pathologists to determine extent of malignancy, however, it is laborious to visually discriminate the corresponding malignancy levels in the more commonly used haematoxylin and eosin stain, and this study attempts to solve this problem using a computer-based method. Cell nuclei are first isolated at high magnification using an automatic cell nuclei segmentation strategy, followed by construction of cell nuclei attributed relational graphs of the tissue regions. These graphs represent tissue architecture comprehensively, as they contain information about cell nuclei morphology as vertex attributes, along with knowledge of neighborhood in the form of edge linking and edge attributes. Global graph characteristics are derived and ensemble learning is used to discriminate between three types of malignancy levels, namely, non-tumor, Her2/neu positive tumor and Her2/neu negative tumor. Performance is compared with state of the art methods including four texture feature groups (Haralick, Gabor, Local Binary Patterns and Varma Zisserman features), color and intensity features, and Voronoi diagram and Delaunay triangulation. Texture, color and intensity information is also combined with graph-based knowledge, followed by correlation analysis. Quantitative assessment is performed using two cross validation strategies. On investigating the experimental results, it can be concluded that the proposed method provides a promising way for computer-based analysis of histopathological images of gastric cancer.

  10. Segmentation of densely populated cell nuclei from confocal image stacks using 3D non-parametric shape priors.

    PubMed

    Ong, Lee-Ling S; Wang, Mengmeng; Dauwels, Justin; Asada, H Harry

    2014-01-01

    An approach to jointly estimate 3D shapes and poses of stained nuclei from confocal microscopy images, using statistical prior information, is presented. Extracting nuclei boundaries from our experimental images of cell migration is challenging due to clustered nuclei and variations in their shapes. This issue is formulated as a maximum a posteriori estimation problem. By incorporating statistical prior models of 3D nuclei shapes into level set functions, the active contour evolutions applied on the images is constrained. A 3D alignment algorithm is developed to build the training databases and to match contours obtained from the images to them. To address the issue of aligning the model over multiple clustered nuclei, a watershed-like technique is used to detect and separate clustered regions prior to active contour evolution. Our method is tested on confocal images of endothelial cells in microfluidic devices, compared with existing approaches.

  11. Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover

    SciTech Connect

    Bergmann, Olaf; Zdunek, Sofia; Alkass, Kanar; Druid, Henrik; Bernard, Samuel; Frisen, Jonas

    2011-01-15

    Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously {sup 14}C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that {sup 14}C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans.

  12. Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status.

    PubMed

    Raulf, Alexandra; Horder, Hannes; Tarnawski, Laura; Geisen, Caroline; Ottersbach, Annika; Röll, Wilhelm; Jovinge, Stefan; Fleischmann, Bernd K; Hesse, Michael

    2015-05-01

    Even though the mammalian heart has been investigated for many years, there are still uncertainties in the fields of cardiac cell biology and regeneration with regard to exact fractions of cardiomyocytes (CMs) at different developmental stages, their plasticity after cardiac lesion and also their basal turnover rate. A main shortcoming is the accurate identification of CM and the demonstration of CM division. Therefore, an in vivo model taking advantage of a live reporter-based identification of CM nuclei and their cell cycle status is needed. In this technical report, we describe the generation and characterization of embryonic stem cells and transgenic mice expressing a fusion protein of human histone 2B and the red fluorescence protein mCherry under control of the CM specific αMHC promoter. This fluorescence label allows unequivocal identification and quantitation of CM nuclei and nuclearity in isolated cells and native tissue slices. In ventricles of adults, we determined a fraction of <20 % CMs and binucleation of 77-90 %, while in atria a CM fraction of 30 % and a binucleation index of 14 % were found. We combined this transgenic system with the CAG-eGFP-anillin transgene, which identifies cell division and established a novel screening assay for cell cycle-modifying substances in isolated, postnatal CMs. Our transgenic live reporter-based system enables reliable identification of CM nuclei and determination of CM fractions and nuclearity in heart tissue. In combination with CAG-eGFP-anillin-mice, the cell cycle status of CMs can be monitored in detail enabling screening for proliferation-inducing substances in vitro and in vivo.

  13. The basal position of nuclei is one pre-requisite for asymmetric cell divisions in the early mouse embryo.

    PubMed

    Ajduk, Anna; Biswas Shivhare, Sourima; Zernicka-Goetz, Magdalena

    2014-08-15

    The early mouse embryo undertakes two types of cell division: symmetric that gives rise to the trophectoderm and then placenta or asymmetric that gives rise to inner cells that generate the embryo proper. Although cell division orientation is important, the mechanism regulating it has remained unclear. Here, we identify the relationship between the plane of cell division and the position of the nucleus and go towards identifying the mechanism behind it. We first find that as the 8-cell embryo progresses through the cell cycle, the nuclei of most - but not all - cells move from apical to more basal positions, in a microtubule- and kinesin-dependent manner. We then find that all asymmetric divisions happen when nuclei are located basally and, in contrast, all cells, in which nuclei remain apical, divide symmetrically. To understand the potential mechanism behind this, we determine the effects of modulating expression of Cdx2, a transcription factor key for trophectoderm formation and cell polarity. We find that increased expression of Cdx2 leads to an increase in a number of apical nuclei, whereas down-regulation of Cdx2 leads to more nuclei moving basally, which explains a previously identified relationship between Cdx2 and cell division orientation. Finally, we show that down-regulation of aPKC, involved in cell polarity, decreases the number of apical nuclei and doubles the number of asymmetric divisions. These results suggest a model in which the mutual interdependence of Cdx2 and cell polarity affects the cytoskeleton-dependent positioning of nuclei and, in consequence, the plane of cell division in the early mouse embryo.

  14. Accurate Automatic Detection of Densely Distributed Cell Nuclei in 3D Space

    PubMed Central

    Tokunaga, Terumasa; Kanamori, Manami; Teramoto, Takayuki; Jang, Moon Sun; Kuge, Sayuri; Ishihara, Takeshi; Yoshida, Ryo; Iino, Yuichi

    2016-01-01

    To measure the activity of neurons using whole-brain activity imaging, precise detection of each neuron or its nucleus is required. In the head region of the nematode C. elegans, the neuronal cell bodies are distributed densely in three-dimensional (3D) space. However, no existing computational methods of image analysis can separate them with sufficient accuracy. Here we propose a highly accurate segmentation method based on the curvatures of the iso-intensity surfaces. To obtain accurate positions of nuclei, we also developed a new procedure for least squares fitting with a Gaussian mixture model. Combining these methods enables accurate detection of densely distributed cell nuclei in a 3D space. The proposed method was implemented as a graphical user interface program that allows visualization and correction of the results of automatic detection. Additionally, the proposed method was applied to time-lapse 3D calcium imaging data, and most of the nuclei in the images were successfully tracked and measured. PMID:27271939

  15. Accurate Automatic Detection of Densely Distributed Cell Nuclei in 3D Space.

    PubMed

    Toyoshima, Yu; Tokunaga, Terumasa; Hirose, Osamu; Kanamori, Manami; Teramoto, Takayuki; Jang, Moon Sun; Kuge, Sayuri; Ishihara, Takeshi; Yoshida, Ryo; Iino, Yuichi

    2016-06-01

    To measure the activity of neurons using whole-brain activity imaging, precise detection of each neuron or its nucleus is required. In the head region of the nematode C. elegans, the neuronal cell bodies are distributed densely in three-dimensional (3D) space. However, no existing computational methods of image analysis can separate them with sufficient accuracy. Here we propose a highly accurate segmentation method based on the curvatures of the iso-intensity surfaces. To obtain accurate positions of nuclei, we also developed a new procedure for least squares fitting with a Gaussian mixture model. Combining these methods enables accurate detection of densely distributed cell nuclei in a 3D space. The proposed method was implemented as a graphical user interface program that allows visualization and correction of the results of automatic detection. Additionally, the proposed method was applied to time-lapse 3D calcium imaging data, and most of the nuclei in the images were successfully tracked and measured. PMID:27271939

  16. Structure Tensor Based Analysis of Cells and Nuclei Organization in Tissues.

    PubMed

    Zhang, Wenxing; Fehrenbach, Jérôme; Desmaison, Annaïck; Lobjois, Valérie; Ducommun, Bernard; Weiss, Pierre

    2016-01-01

    Extracting geometrical information from large 2D or 3D biomedical images is important to better understand fundamental phenomena such as morphogenesis. We address the problem of automatically analyzing spatial organization of cells or nuclei in 2D or 3D images of tissues. This problem is challenging due to the usually low quality of microscopy images as well as their typically large sizes. The structure tensor is a simple and robust descriptor that was developed to analyze textures orientation. Contrarily to segmentation methods which rely on an object based modeling of images, the structure tensor considers the sample at a macroscopic scale, like a continuous medium. We show that this tool allows quantifying two important features of nuclei in tissues: their privileged orientation as well as the ratio between the length of their main axes. A quantitative evaluation of the method is provided for synthetic and real 2D and 3D images. As an application, we analyze the nuclei orientation and anisotropy on multicellular tumor spheroids cryosections. This analysis reveals that cells are elongated in a privileged direction that is parallel to the spheroid boundary. A MATLAB toolbox and an Icy plugin are available to use the proposed method.

  17. Accurate Automatic Detection of Densely Distributed Cell Nuclei in 3D Space.

    PubMed

    Toyoshima, Yu; Tokunaga, Terumasa; Hirose, Osamu; Kanamori, Manami; Teramoto, Takayuki; Jang, Moon Sun; Kuge, Sayuri; Ishihara, Takeshi; Yoshida, Ryo; Iino, Yuichi

    2016-06-01

    To measure the activity of neurons using whole-brain activity imaging, precise detection of each neuron or its nucleus is required. In the head region of the nematode C. elegans, the neuronal cell bodies are distributed densely in three-dimensional (3D) space. However, no existing computational methods of image analysis can separate them with sufficient accuracy. Here we propose a highly accurate segmentation method based on the curvatures of the iso-intensity surfaces. To obtain accurate positions of nuclei, we also developed a new procedure for least squares fitting with a Gaussian mixture model. Combining these methods enables accurate detection of densely distributed cell nuclei in a 3D space. The proposed method was implemented as a graphical user interface program that allows visualization and correction of the results of automatic detection. Additionally, the proposed method was applied to time-lapse 3D calcium imaging data, and most of the nuclei in the images were successfully tracked and measured.

  18. Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells.

    PubMed

    Yamada, Mitsutoshi; Johannesson, Bjarki; Sagi, Ido; Burnett, Lisa Cole; Kort, Daniel H; Prosser, Robert W; Paull, Daniel; Nestor, Michael W; Freeby, Matthew; Greenberg, Ellen; Goland, Robin S; Leibel, Rudolph L; Solomon, Susan L; Benvenisty, Nissim; Sauer, Mark V; Egli, Dieter

    2014-06-26

    The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

  19. Rapid isolation of nuclei from living immune cells by a single centrifugation through a multifunctional lysis gradient.

    PubMed

    Poglitsch, Marko; Katholnig, Karl; Säemann, Marcus D; Weichhart, Thomas

    2011-10-28

    Due to their low protein content and limited nuclear detergent stability, primary human immune cells such as monocytes or T lymphocytes represent a great challenge for standard nuclear isolation protocols. Nuclei clumping during the multiple centrifugation steps or contamination of isolated nuclei with cytoplasmic proteins due to membrane lysis is a frequently observed problem. Here we describe a versatile and novel method for the isolation of clean and intact nuclei from primary human monocytes, which can be applied for virtually any cell type. Living cells were applied on an iso-osmolar discontinuous iodixanol-based density gradient including a detergent-containing lysis layer. Mild cell lysis as well as efficient washing of the nuclei was performed during the course of one single low g-force centrifugation step. The isolation procedure, which we call lysis gradient centrifugation (LGC), results in the recovery of 90-95% of highly pure nuclei. This easy and highly reproducible procedure allows an effective preparation of nuclei and the cytoplasm in only 15 min with the ability to handle as little as one million cells per sample and easy parallel processing of multiple samples.

  20. Differential effects of growth temperature on ice nuclei active at different temperatures that are produced by cells of Pseudomonas syringae.

    PubMed

    Gurian-Sherman, D; Lindow, S E

    1995-04-01

    The temperature at which ice-nucleating bacteria are grown causes differences of 100- to 10,000-fold in the fraction of cells that nucleate ice at a given temperature (ice nucleation frequency). Ice nucleation frequencies of cells of Pseudomonas syringae grown at temperatures that ranged from 9 to 33 degrees C were examined in order to more accurately characterize physiological effects on ice nuclei active at temperatures of from about -2 to -10 degrees C, the temperature range for this phenotype. Large differences in ice nucleation frequency occurred at all but the lowest assay temperatures in cells of P. syringae grown in the temperature range of 15 to 33 degrees C. These differences in ice nucleation frequency may be attributed, at least in part, to post-translational factors. Because other studies have indicated that ice nuclei active at the lowest assay temperatures may reflect the amount of ice nucleation protein produced, while higher nucleation temperatures reflect aggregates of this ice nucleation protein, data was normalized to the frequency of ice nuclei active at the lowest ice nucleation temperatures (which also correspond to the most abundant nuclei). This was done in order to develop a baseline of comparison for cells grown at different temperatures that more clearly shows possible post-translational effects such as aggregation of the nucleation protein. After this normalization was performed, and in contrast to the results noted above, the number of ice nuclei in cells grown at 9, 15, and 20 degrees C that were active at different assay temperatures was very similar. Differences in ice nucleation frequency that occurred over all assay temperatures in cells grown between 9 and 20 degrees C may be attributed to differences in the total number of nuclei present in the population of cells. The large effects of growth temperature on nucleation frequency have important implications for estimating numbers of ice nucleating bacteria in environmental samples

  1. Germ cell mutagenesis in medaka fish after exposures to high-energy cosmic ray nuclei: A human model

    NASA Astrophysics Data System (ADS)

    Shimada, Atsuko; Shima, Akihiro; Nojima, Kumie; Seino, Yo; Setlow, Richard B.

    2005-04-01

    Astronauts beyond the Earth's orbit are exposed to high-energy cosmic-ray nuclei with high values of linear energy transfer (LET), resulting in much more biological damage than from x-rays or -rays and may result in mutations and cancer induction. The relative biological effectiveness of these nuclei depends on the LET, rising to as high as 50 at LET values of 100-200 keV/µm. An endpoint of concern is germ cell mutations passed on to offspring, arising from exposure to these nuclei. A vertebrate model for germ cell mutation is Medaka fish (Oryzias latipes). We exposed wild type males to doses of 1 GeV per nucleon Fe nuclei or to 290 MeV per nucleon C nuclei. They were mated to females with recessive mutations at five-color loci. The transparent embryos from >100 days of mating (representing exposed sperm, spermatids, or spermatogonia) were observed so as to detect dominant lethal mutations and total color mutations, even though the embryos might not hatch. The relative number of mutant embryos as a function of dose were compared with those induced by -rays. The relative biological effectiveness values for dominant lethal mutations and total color mutations for exposed sperm and spermatids were 1.3-2.1 for exposure to C nuclei and 1.5-3.0 for exposure to Fe nuclei. (The spermatogonial data were uncertain.) These low values, and the negligible number of viable mutations, compared with those for mutations in somatic cells and for neoplastic transformation, indicate that germ cell mutations arising from exposures to cosmic ray nuclei are not a significant hazard to astronauts. astronaut hazards | linear energy transfer | relative biological effect

  2. Germ cell mutagenesis in medaka fish after exposures to high-energy cosmic ray nuclei: A human model.

    PubMed

    Shimada, Atsuko; Shima, Akihiro; Nojima, Kumie; Seino, Yo; Setlow, Richard B

    2005-04-26

    Astronauts beyond the Earth's orbit are exposed to high-energy cosmic-ray nuclei with high values of linear energy transfer (LET), resulting in much more biological damage than from x-rays or gamma-rays and may result in mutations and cancer induction. The relative biological effectiveness of these nuclei depends on the LET, rising to as high as approximately 50 at LET values of approximately 100-200 keV/microm. An endpoint of concern is germ cell mutations passed on to offspring, arising from exposure to these nuclei. A vertebrate model for germ cell mutation is Medaka fish (Oryzias latipes). We exposed wild type males to doses of 1 GeV per nucleon Fe nuclei or to 290 MeV per nucleon C nuclei. They were mated to females with recessive mutations at five-color loci. The transparent embryos from >100 days of mating (representing exposed sperm, spermatids, or spermatogonia) were observed so as to detect dominant lethal mutations and total color mutations, even though the embryos might not hatch. The relative number of mutant embryos as a function of dose were compared with those induced by gamma-rays. The relative biological effectiveness values for dominant lethal mutations and total color mutations for exposed sperm and spermatids were 1.3-2.1 for exposure to C nuclei and 1.5-3.0 for exposure to Fe nuclei. (The spermatogonial data were uncertain.) These low values, and the negligible number of viable mutations, compared with those for mutations in somatic cells and for neoplastic transformation, indicate that germ cell mutations arising from exposures to cosmic ray nuclei are not a significant hazard to astronauts. PMID:15829584

  3. Study of RNA Polymerase II Clustering inside Live-Cell Nuclei Using Bayesian Nanoscopy.

    PubMed

    Chen, Xuanze; Wei, Mian; Zheng, M Mocarlo; Zhao, Jiaxi; Hao, Huiwen; Chang, Lei; Xi, Peng; Sun, Yujie

    2016-02-23

    Nanoscale spatiotemporal clustering of RNA polymerase II (Pol II) plays an important role in transcription regulation. However, dynamics of individual Pol II clusters in live-cell nuclei has not been measured directly, prohibiting in-depth understanding of their working mechanisms. In this work, we studied the dynamics of Pol II clustering using Bayesian nanoscopy in live mammalian cell nuclei. With 50 nm spatial resolution and 4 s temporal resolution, Bayesian nanoscopy allows direct observation of the assembly and disassembly dynamics of individual Pol II clusters. The results not only provide quantifications of Pol II clusters but also shed light on the understanding of cluster formation and regulation. Our study suggests that transcription factories form on-demand and recruit Pol II molecules in their pre-elongation phase. The assembly and disassembly of individual Pol II clusters take place asynchronously. Overall, the methods developed herein are also applicable to studying a wide realm of real-time nanometer-scale nuclear processes in live cells.

  4. Human mitochondrial transcription factor A functions in both nuclei and mitochondria and regulates cancer cell growth

    SciTech Connect

    Han, Bin; Izumi, Hiroto; Yasuniwa, Yoshihiro; Akiyama, Masaki; Yamaguchi, Takahiro; Fujimoto, Naohiro; Matsumoto, Tetsuro; Wu, Bin; Tanimoto, Akihide; Sasaguri, Yasuyuki; Kohno, Kimitoshi

    2011-04-29

    Highlights: {yields} Mitochondrial transcription factor A (mtTFA) localizes in nuclei and binds tightly to the nuclear chromatin. {yields} mtTFA contains two putative nuclear localization signals (NLS) in the HMG-boxes. {yields} Overexpression of mtTFA enhances the growth of cancer cells, whereas downregulation of mtTFA inhibits their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). {yields} Knockdown of mtTFA expression induces p21-dependent G1 cell cycle arrest. -- Abstract: Mitochondrial transcription factor A (mtTFA) is one of the high mobility group protein family and is required for both transcription from and maintenance of mitochondrial genomes. However, the roles of mtTFA have not been extensively studied in cancer cells. Here, we firstly reported the nuclear localization of mtTFA. The proportion of nuclear-localized mtTFA varied among different cancer cells. Some mtTFA binds tightly to the nuclear chromatin. DNA microarray and chromatin immunoprecipitation assays showed that mtTFA can regulate the expression of nuclear genes. Overexpression of mtTFA enhanced the growth of cancer cell lines, whereas downregulation of mtTFA inhibited their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). Knockdown of mtTFA expression induced p21-dependent G1 cell cycle arrest. These results imply that mtTFA functions in both nuclei and mitochondria to promote cell growth.

  5. Automatic segmentation of cell nuclei in Feulgen-stained histological sections of prostate cancer and quantitative evaluation of segmentation results.

    PubMed

    Nielsen, Birgitte; Albregtsen, Fritz; Danielsen, Håvard E

    2012-07-01

    Digital image analysis of cell nuclei is useful to obtain quantitative information for the diagnosis and prognosis of cancer. However, the lack of a reliable automatic nuclear segmentation is a limiting factor for high-throughput nuclear image analysis. We have developed a method for automatic segmentation of nuclei in Feulgen-stained histological sections of prostate cancer. A local adaptive thresholding with an object perimeter gradient verification step detected the nuclei and was combined with an active contour model that featured an optimized initialization and worked within a restricted region to improve convergence of the segmentation of each nucleus. The method was tested on 30 randomly selected image frames from three cases, comparing the results from the automatic algorithm to a manual delineation of 924 nuclei. The automatic method segmented a few more nuclei compared to the manual method, and about 73% of the manually segmented nuclei were also segmented by the automatic method. For each nucleus segmented both manually and automatically, the accuracy (i.e., agreement with manual delineation) was estimated. The mean segmentation sensitivity/specificity were 95%/96%. The results from the automatic method were not significantly different from the ground truth provided by manual segmentation. This opens the possibility for large-scale nuclear analysis based on automatic segmentation of nuclei in Feulgen-stained histological sections.

  6. Bovine ooplasm partially remodels primate somatic nuclei following somatic cell nuclear transfer.

    PubMed

    Wang, Kai; Beyhan, Zeki; Rodriguez, Ramon M; Ross, Pablo J; Iager, Amy E; Kaiser, German G; Chen, Ying; Cibelli, Jose B

    2009-03-01

    Interspecies somatic cell nuclear transfer (iSCNT) has the potential to become a useful tool to address basic questions about the nucleus-cytoplasm interactions between species. It has also been proposed as an alternative for the preservation of endangered species and to derive autologous embryonic stem cells. Using chimpanzee/ bovine iSCNT as our experimental model we studied the early epigenetic events that take place soon after cell fusion until embryonic genome activation (EGA). Our analysis suggested partial EGA in iSCNT embryos at the eight-cell stage, as indicated by Br-UTP incorporation and expression of chimpanzee embryonic genes. Oct4, Stella, Crabp1, CCNE2, CXCL6, PTGER4, H2AFZ, c-MYC, KLF4, and GAPDH transcripts were expressed, while Nanog, Glut1, DSC2, USF2, Adrbk1, and Lin28 failed to be activated. Although development of iSCNT embryos did not progress beyond the 8- to 16-cell stage, chromatin remodeling events, monitored by H3K27 methylation, H4K5 acetylation, and global DNA methylation, were similar in both intra- and interspecies SCNT embryos. However, bisulfite sequencing indicated incomplete demethylation of Oct4 and Nanog promoters in eight-cell iSCNT embryos. ATP production levels were significantly higher in bovine SCNT embryos than in iSCNT embryos, TUNEL assays did not reveal any difference in the apoptotic status of the nuclei from both types of embryos. Collectively, our results suggest that bovine ooplasm can partially remodel chimpanzee somatic nuclei, and provides insight into some of the current barriers iSCNT must overcome if further embryonic development is to be expected. PMID:19196039

  7. Hit rates and radiation doses to nuclei of bone lining cells from alpha-particle-emitting radionuclides

    NASA Technical Reports Server (NTRS)

    Polig, E.; Jee, W. S.; Kruglikov, I. L.

    1992-01-01

    Factors relating the local concentration of a bone-seeking alpha-particle emitter to the mean hit rate have been determined for nuclei of bone lining cells using a Monte Carlo procedure. Cell nuclei were approximated by oblate spheroids with dimensions and location taken from a previous histomorphometric study. The Monte Carlo simulation is applicable for planar and diffuse labels at plane or cylindrical bone surfaces. Additionally, the mean nuclear dose per hit, the dose mean per hit, the mean track segment length and its second moment, the percentage of stoppers, and the frequency distribution of the dose have been determined. Some basic features of the hit statistics for bone lining cells have been outlined, and the consequences of existing standards of radiation protection with regard to the hit frequency to cell nuclei are discussed.

  8. ADAMTS-1 Is Found in the Nuclei of Normal and Tumoral Breast Cells

    PubMed Central

    Silva, Suély V.; Lima, Maíra A.; Cella, Nathalie; Jaeger, Ruy G.

    2016-01-01

    Proteins secreted in the extracellular matrix microenvironment (ECM) by tumor cells are involved in cell adhesion, motility, intercellular communication and invasion. The tumor microenvironment is expansively modified and remodeled by proteases, resulting in important changes in both cell-cell and cell-ECM interactions and in the generation of new signals from the cell surface. Metalloproteinases belonging to the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family have been implicated in tissue remodeling events observed in cancer development, growth and progression. Here we investigated the subcellular localization of ADAMTS-1 in normal-like (MCF10-A) and tumoral (MCF7 and MDA-MB-231) human breast cells. ADAMTS-1 is a secreted protease found in the extracellular matrix. However, in this study we show for the first time that ADAMTS-1 is also present in the nuclei and nucleoli of the three mammary cell lines studied here. Our findings indicate that ADAMTS-1 has proteolytic functions in the nucleus through its interaction with aggrecan substrate. PMID:27764205

  9. Condensin I and II behaviour in interphase nuclei and cells undergoing premature chromosome condensation.

    PubMed

    Zhang, Tao; Paulson, James R; Bakhrebah, Muhammed; Kim, Ji Hun; Nowell, Cameron; Kalitsis, Paul; Hudson, Damien F

    2016-05-01

    Condensin is an integral component of the mitotic chromosome condensation machinery, which ensures orderly segregation of chromosomes during cell division. In metazoans, condensin exists as two complexes, condensin I and II. It is not yet clear what roles these complexes may play outside mitosis, and so we have examined their behaviour both in normal interphase and in premature chromosome condensation (PCC). We find that a small fraction of condensin I is retained in interphase nuclei, and our data suggests that this interphase nuclear condensin I is active in both gene regulation and chromosome condensation. Furthermore, live cell imaging demonstrates condensin II dramatically increases on G1 nuclei following completion of mitosis. Our PCC studies show condensins I and II and topoisomerase II localise to the chromosome axis in G1-PCC and G2/M-PCC, while KIF4 binding is altered. Individually, condensins I and II are dispensable for PCC. However, when both are knocked out, G1-PCC chromatids are less well structured. Our results define new roles for the condensins during interphase and provide new information about the mechanism of PCC.

  10. Transcription is Associated with Z-DNA Formation in Metabolically Active Permeabilized Mammalian Cell Nuclei

    NASA Astrophysics Data System (ADS)

    Wittig, Burghardt; Dorbic, Tomislav; Rich, Alexander

    1991-03-01

    Mammalian cells have been encapsulated in agarose microbeads, and from these cells metabolically active permeabilized nuclei were prepared. Previously, we showed that biotin-labeled monoclonal antibodies against Z-DNA can be diffused into the nuclei and, over a specific concentration range, they will bind to Z-DNA within the nucleus in a concentration-independent manner. By using radiolabeled streptavidin, we showed that the amount of Z-DNA antibody bound is related to the torsional strain of the DNA in the nucleus. Relaxation of the DNA results in a decrease of Z-DNA formation, whereas increasing torsional strain through inhibiting topoisomerase I results in increased Z-DNA formation. Here we measure the influence of RNA transcription and DNA replication. Transcription is associated with a substantial increase in the binding of anti-Z-DNA antibodies, paralleling the increased level of RNA synthesized as the level of ribonucleoside triphosphate in the medium is increased. DNA replication yields smaller increases in the binding of Z-DNA antibodies. Stopping RNA transcription with inhibitors results in a large loss of Z-DNA antibody binding, whereas only a small decrease is associated with inhibition of DNA replication.

  11. An entropy-based automated cell nuclei segmentation and quantification: application in analysis of wound healing process.

    PubMed

    Oswal, Varun; Belle, Ashwin; Diegelmann, Robert; Najarian, Kayvan

    2013-01-01

    The segmentation and quantification of cell nuclei are two very significant tasks in the analysis of histological images. Accurate results of cell nuclei segmentation are often adapted to a variety of applications such as the detection of cancerous cell nuclei and the observation of overlapping cellular events occurring during wound healing process in the human body. In this paper, an automated entropy-based thresholding system for segmentation and quantification of cell nuclei from histologically stained images has been presented. The proposed translational computation system aims to integrate clinical insight and computational analysis by identifying and segmenting objects of interest within histological images. Objects of interest and background regions are automatically distinguished by dynamically determining 3 optimal threshold values for the 3 color components of an input image. The threshold values are determined by means of entropy computations that are based on probability distributions of the color intensities of pixels and the spatial similarity of pixel intensities within neighborhoods. The effectiveness of the proposed system was tested over 21 histologically stained images containing approximately 1800 cell nuclei, and the overall performance of the algorithm was found to be promising, with high accuracy and precision values.

  12. Neutron capture nuclei-containing carbon nanoparticles for destruction of cancer cells.

    PubMed

    Hwang, Kuo Chu; Lai, Po Dong; Chiang, Chi-Shiun; Wang, Pei-Jen; Yuan, Chiun-Jye

    2010-11-01

    HeLa cells were incubated with neutron capture nuclei (boron-10 and gadolinium)-containing carbon nanoparticles, followed by irradiation of slow thermal neutron beam. Under a neutron flux of 6 x 10(11) n/cm(2) (or 10 min irradiation at a neutron flux of 1 x 10(9) n/cm(2) s), the percentages of acute cell death at 8 h after irradiation are 52, 55, and 28% for HeLa cells fed with BCo@CNPs, GdCo@CNPs, and Co@CNPs, respectively. The proliferation capability of the survived HeLa cells was also found to be significantly suppressed. At 48 h after neutron irradiation, the cell viability further decreases to 35 +/- 5% as compared to the control set receiving the same amount of neutron irradiation dose but in the absence of carbon nanoparticles. This work demonstrates "proof-of-concept" examples of neutron capture therapy using (10)B-, (157)Gd-, and (59)Co-containing carbon nanoparticles for effective destruction of cancer cells. It will also be reported the preparation and surface functionalization of boron or gadolinium doped core-shell cobalt/carbon nanoparticles (BCo@CNPs, GdCo@CNPs and Co@CNPs) using a modified DC pulsed arc discharge method, and their characterization by various spectroscopic measurements, including TEM, XRD, SQUID, FT-IR, etc. Tumor cell targeting ability was introduced by surface modification of these carbon nanoparticles with folate moieties.

  13. Morphology of Nurse Cell Nuclei Induced by Meloidodera floridensis: A Graphics Application.

    PubMed

    Mundo-Ocampo, M; Greene, M; Flaxman, M; Baldwin, J G

    1993-12-01

    The highly irregular distribution of nuclear material in the host nurse cell induced by Meloidodera floridensis has made it difficult to interpret the number of nuclei from two-dimensional micrographs alone. The primary goal of this investigation was to determine the distribution of nuclear material from a three-dimensional solid surface model of the nurse cell nucleus. This model demonstrated the continuity of nuclear material as a single highly irregular nucleus. Custom computer graphics programs were written to accept digitized tracings of nuclear material. From these digitized tracings, a wireframe or polygonalized mesh model was generated. The model was shaded, colored, rotated, and analyzed. This technique provides controlled transparency of the model to display nucleoli within the nucleus. Photographs of the computer screen, color printouts, and video recordings were used to record final results. These refined computer graphic tools have a range of applications in nematode host-parasite relationships, ontogeny, and morphology.

  14. Single Molecule Localization Microscopy of Mammalian Cell Nuclei on the Nanoscale

    PubMed Central

    Szczurek, Aleksander; Xing, Jun; Birk, Udo J.; Cremer, Christoph

    2016-01-01

    Nuclear texture analysis is a well-established method of cellular pathology. It is hampered, however, by the limits of conventional light microscopy (ca. 200 nm). These limits have been overcome by a variety of super-resolution approaches. An especially promising approach to chromatin texture analysis is single molecule localization microscopy (SMLM) as it provides the highest resolution using fluorescent based methods. At the present state of the art, using fixed whole cell samples and standard DNA dyes, a structural resolution of chromatin in the 50–100 nm range is obtained using SMLM. We highlight how the combination of localization microscopy with standard fluorophores opens the avenue to a plethora of studies including the spatial distribution of DNA and associated proteins in eukaryotic cell nuclei with the potential to elucidate the functional organization of chromatin. These views are based on our experience as well as on recently published research in this field. PMID:27446198

  15. Single Molecule Localization Microscopy of Mammalian Cell Nuclei on the Nanoscale.

    PubMed

    Szczurek, Aleksander; Xing, Jun; Birk, Udo J; Cremer, Christoph

    2016-01-01

    Nuclear texture analysis is a well-established method of cellular pathology. It is hampered, however, by the limits of conventional light microscopy (ca. 200 nm). These limits have been overcome by a variety of super-resolution approaches. An especially promising approach to chromatin texture analysis is single molecule localization microscopy (SMLM) as it provides the highest resolution using fluorescent based methods. At the present state of the art, using fixed whole cell samples and standard DNA dyes, a structural resolution of chromatin in the 50-100 nm range is obtained using SMLM. We highlight how the combination of localization microscopy with standard fluorophores opens the avenue to a plethora of studies including the spatial distribution of DNA and associated proteins in eukaryotic cell nuclei with the potential to elucidate the functional organization of chromatin. These views are based on our experience as well as on recently published research in this field. PMID:27446198

  16. Regulation of Water in Plant Cells

    ERIC Educational Resources Information Center

    Kowles, Richard V.

    2010-01-01

    Cell water relationships are important topics to be included in cell biology courses. Differences exist in the control of water relationships in plant cells relative to control in animal cells. One important reason for these differences is that turgor pressure is a consideration in plant cells. Diffusion and osmosis are the underlying factors…

  17. Regio- and stereoselectivities in plant cell biotransformation

    SciTech Connect

    Hamada, H.

    1995-12-01

    The ability of plant cultured cells to convert foreign substrates into more useful substances is of considerable interest. Therefore I have studied biotransformation of foreign substrate by plant cell suspension cultures. In this presentation, I report regio- and stereoselectivities in biotransformation of steroids and indole alkaloids and taxol by plant (tobacco, periwinkle, moss, orchid) cell suspension cultures.

  18. Characterization of adenovirus type 2 transcriptional complexes isolated from infected HeLa cell nuclei.

    PubMed Central

    Wilhelm, J; Brison, O; Kedinger, C; Chambon, P

    1976-01-01

    HeLa cell nuclei, isolated 17 h after infection with human adenovirus type 2 (Ad2), were treated with 200 mM ammonium sulfate. The extract (S200 fraction) contained 50 to 70% of the nonintegrated Ad2 DNA, which was in the form of nucleoprotein complexes. These complexes contained native, intact Ad2 DNA (with the exception of replicative intermediates) and could be partially purified and resolved by velocity gradient centrifugation. Using high-salt (200 mM ammonium sulfate) incubation conditions, more than 95% of the nuclear RNA polymerase activity belonged to class B. About 45% of the class B enzyme molecules bound to DNA in the nuclei (those "engaged" in RNA synthesis) were released from the nuclei in the form of Ad2 transcriptional complexes by treatment with 200 mM ammonium sulfate. At least 90% of the RNA synthesized in high salt in the nuclei or in the S200 fraction was Ad2 specific, and essentially all of this RNA was complementary to the l strand of Ad2 DNA. These findings are compatible with what is known about Ad2-specific RNA synthesis in vivo. The analysis of the RNA synthesized from partially purified transcriptional complexes supports the contention that its transcription is almost entirely asymmetric, and that the asymmetry observed in vivo is not a consequence of the rapid degradation of h-strand transcripts. The RNA synthesized in vitro in the absence of detectable RNase activity sedimented with a maximum size of 35 to 40S. Less than 5% of the nuclear or the S200 fraction RNA polymerase activity was class C when assayed under non-reinitiating conditions. Although much of the RNA synthesized by the class C enzyme was Ad2 specific, 5.5S virus-associated RNA was not the predominant product. The isolation of Ad2 DNA transcriptional complexes provides an attractive system for further characterizing the Ad2 DNA template used for transcription and for studying the regulation of the expression of the Ad2 genome during the productive infection cycle. PMID

  19. Necrotic and apoptotic cells serve as nuclei for calcification on osteoblastic differentiation of human mesenchymal stem cells in vitro.

    PubMed

    Fujita, Hirofumi; Yamamoto, Masanao; Ogino, Tetsuya; Kobuchi, Hirotsugu; Ohmoto, Naoko; Aoyama, Eriko; Oka, Takashi; Nakanishi, Tohru; Inoue, Keiji; Sasaki, Junzo

    2014-01-01

    A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase-3 activation increased in this culture. A pan-caspase inhibitor (Z-VAD-FMK) and anti-oxidants (Tiron and n-acetylcysteine) inhibited osteogenic culture-induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co-localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro.

  20. Isolation of cell nuclei in microchannels by short-term chemical treatment via two-step carrier medium exchange.

    PubMed

    Toyama, Kaori; Yamada, Masumi; Seki, Minoru

    2012-08-01

    Separation/purification of nuclei from cells is a critical process required for medical and biochemical research applications. Here, we report a flow-through microfluidic device for isolating cell nuclei by selectively digesting the cell membrane by using the concept of hydrodynamic filtration (HDF). When a cell suspension is continuously introduced into a microchannel (main channel) possessing multiple side channels, cells flow through the main channel, whereas the carrier medium of the cells is drained through the side channels. Introductions of a cell treatment solution containing a surfactant and a washing buffer enable the two-step exchange of the carrier-medium and the cell treatment by the surfactant for a short span of time. The precise control of the treatment time by changing the flow rate and/or the size of the microchannel enables the selective digestion of cell membranes, resulting in the isolation of cell nuclei after separation from membrane debris and cytoplasmic components according to size. We examined several surfactant molecules and demonstrated that Triton X-100 exhibited high efficiency regarding nucleus isolation for both adherent (HeLa) and nonadherent (JM) cells, with a recovery ratio of ~80 %. In addition, the isolation efficiency was evaluated by western blotting. The presented flow-through microfluidic cell-nucleus separator may be a useful tool for general biological applications, because of its simplicity in operation, high reproducibility, and accuracy.

  1. Three-dimensional imaging of interphase cell nuclei with laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Boecker, Wilfried; Radtke, Thomas; Streffer, Christian

    1998-10-01

    During the past decade 3D image processing has become an important key component in biological research mainly due to two different developments. The first is based on an optical instrument, the so-called confocal laser scanning microscope, allowing optical sectioning of the biological specimen. The second is a biological preparatory method, the so-called FISH-technique (Fluorescence-In-Situ- Hybridization), allowing labeling of certain cellular and sub-cellular compartments with highly specific fluorescent dyes. Both methods make it possible to investigate the 3D biological framework within cells and nuclei. Image acquisition with confocal laser scanning microscopy must deal with different limits of resolution along and across the optical axis. Although lateral resolution is about 0.7 times better than in non-confocal arrangements, axial resolution is more than 3 - 4 times poorer than that of the lateral (depending on the pinhole size). For 3D reconstruction it is desirable to improve axial resolution in order to provide nearly identical image information across the 3D specimen space. This presentation will give an overview of some of the most popular restoration and deblurring algorithms used in 3D image microscopy. After 3D image restoration, segmentation of certain details of the cell structure is usually the next step in image processing. We compared two different kinds of algorithms for segmentation of chromosome territories in interphase cell nuclei. One is based on Mathematical Morphology, the other on Split & Merge methods. The segmented image regions provided the basis for chromosome domain reconstruction as well as for regional localization for subsequent quantitative measurements. As a result the chromatin density within certain chromosome domains as well as some terminal DNA sequences (telomere signals) could be measured.

  2. Detection and segmentation of cell nuclei in virtual microscopy images: a minimum-model approach.

    PubMed

    Wienert, Stephan; Heim, Daniel; Saeger, Kai; Stenzinger, Albrecht; Beil, Michael; Hufnagl, Peter; Dietel, Manfred; Denkert, Carsten; Klauschen, Frederick

    2012-01-01

    Automated image analysis of cells and tissues has been an active research field in medical informatics for decades but has recently attracted increased attention due to developments in computer and microscopy hardware and the awareness that scientific and diagnostic pathology require novel approaches to perform objective quantitative analyses of cellular and tissue specimens. Model-based approaches use a priori information on cell shape features to obtain the segmentation, which may introduce a bias favouring the detection of cell nuclei only with certain properties. In this study we present a novel contour-based "minimum-model" cell detection and segmentation approach that uses minimal a priori information and detects contours independent of their shape. This approach avoids a segmentation bias with respect to shape features and allows for an accurate segmentation (precision = 0.908; recall = 0.859; validation based on ∼8000 manually-labeled cells) of a broad spectrum of normal and disease-related morphological features without the requirement of prior training.

  3. Reprogramming of two somatic nuclei in the same ooplasm leads to pluripotent embryonic stem cells.

    PubMed

    Pfeiffer, Martin J; Esteves, Telma C; Balbach, Sebastian T; Araúzo-Bravo, Marcos J; Stehling, Martin; Jauch, Anna; Houghton, Franchesca D; Schwarzer, Caroline; Boiani, Michele

    2013-11-01

    The conversion of the nuclear program of a somatic cell from a differentiated to an undifferentiated state can be accomplished by transplanting its nucleus to an enucleated oocyte (somatic cell nuclear transfer [SCNT]) in a process termed "reprogramming." This process achieves pluripotency and occasionally also totipotency. Exploiting the obstacle of tetraploidy to full development in mammals, we show that mouse ooplasts transplanted with two somatic nuclei simultaneously (double SCNT) support preimplantation development and derivation of novel tetraploid SCNT embryonic stem cells (tNT-ESCs). Although the double SCNT embryos do not recapitulate the expression pattern of the pluripotency-associated gene Oct4 in fertilized embryos, derivative tNT-ESCs have characteristics of genuine pluripotency: in vitro they differentiate into neurons, cardiomyocytes, and endodermal cells; in vivo, tNT-ESCs form teratomas, albeit at reduced rates compared to diploid counterparts. Global transcriptome analysis revealed only few specific alterations, for example, in the quantitative expression of gastrulation-associated genes. In conclusion, we have shown that the oocyte's reprogramming capacity is in excess of a single nucleus and that double nucleus-transplanted embryos and derivative ESCs are very similar to their diploid counterparts. These results have key implications for reprogramming studies based on pluripotency: while reprogramming in the tetraploid state was known from fusion-mediated reprogramming and from fetal and adult hepatocyte-derived induced pluripotent stem cells, we have now accomplished it with enucleated oocytes.

  4. The Oncogenic Transforming Potential of the Passage of Single α Particles through Mammalian Cell Nuclei

    NASA Astrophysics Data System (ADS)

    Miller, Richard C.; Randers-Pehrson, Gerhard; Geard, Charles R.; Hall, Eric J.; Brenner, David J.

    1999-01-01

    Domestic, low-level exposure to radon gas is considered a major environmental lung-cancer hazard involving DNA damage to bronchial cells by α particles from radon progeny. At domestic exposure levels, the relevant bronchial cells are very rarely traversed by more than one α particle, whereas at higher radon levels--at which epidemiological studies in uranium miners allow lung-cancer risks to be quantified with reasonable precision--these bronchial cells are frequently exposed to multiple α -particle traversals. Measuring the oncogenic transforming effects of exactly one α particle without the confounding effects of multiple traversals has hitherto been unfeasible, resulting in uncertainty in extrapolations of risk from high to domestic radon levels. A technique to assess the effects of single α particles uses a charged-particle microbeam, which irradiates individual cells or cell nuclei with predefined exact numbers of particles. Although previously too slow to assess the relevant small oncogenic risks, recent improvements in throughput now permit microbeam irradiation of large cell numbers, allowing the first oncogenic risk measurements for the traversal of exactly one α particle through a cell nucleus. Given positive controls to ensure that the dosimetry and biological controls were comparable, the measured oncogenicity from exactly one α particle was significantly lower than for a Poisson-distributed mean of one α particle, implying that cells traversed by multiple α particles contribute most of the risk. If this result applies generally, extrapolation from high-level radon risks (involving cellular traversal by multiple α particles) may overestimate low-level (involving only single α particles) radon risks.

  5. Computer vision approach to morphometric feature analysis of basal cell nuclei for evaluating malignant potentiality of oral submucous fibrosis.

    PubMed

    Muthu Rama Krishnan, M; Pal, Mousumi; Paul, Ranjan Rashmi; Chakraborty, Chandan; Chatterjee, Jyotirmoy; Ray, Ajoy K

    2012-06-01

    This research work presents a quantitative approach for analysis of histomorphometric features of the basal cell nuclei in respect to their size, shape and intensity of staining, from surface epithelium of Oral Submucous Fibrosis showing dysplasia (OSFD) to that of the Normal Oral Mucosa (NOM). For all biological activity, the basal cells of the surface epithelium form the proliferative compartment and therefore their morphometric changes will spell the intricate biological behavior pertaining to normal cellular functions as well as in premalignant and malignant status. In view of this, the changes in shape, size and intensity of staining of the nuclei in the basal cell layer of the NOM and OSFD have been studied. Geometric, Zernike moments and Fourier descriptor (FD) based as well as intensity based features are extracted for histomorphometric pattern analysis of the nuclei. All these features are statistically analyzed along with 3D visualization in order to discriminate the groups. Results showed increase in the dimensions (area and perimeter), shape parameters and decreasing mean nuclei intensity of the nuclei in OSFD in respect to NOM. Further, the selected features are fed to the Bayesian classifier to discriminate normal and OSFD. The morphometric and intensity features provide a good sensitivity of 100%, specificity of 98.53% and positive predicative accuracy of 97.35%. This comparative quantitative characterization of basal cell nuclei will be of immense help for oral onco-pathologists, researchers and clinicians to assess the biological behavior of OSFD, specially relating to their premalignant and malignant potentiality. As a future direction more extensive study involving more number of disease subjects is observed.

  6. Embryogenic plant cells in microgravity

    NASA Technical Reports Server (NTRS)

    Krikorian, Abraham D.

    1991-01-01

    In view of circumstantial evidence for the role of gravity (g) in shaping the embryo environment, normal embryo development may not occur reliably and efficiently in the microgravity environment of space. Attention must accordingly be given to those aspects of higher plant reproductive biology in space environments required for the production of viable embryos in a 'seed to seed to seed' experiment. It is suggested that cultured cells can be grown to be morphogenetically competent, and can be evaluated as to their ability to simulate embryogenic events usually associated with fertilized eggs in the embryo sac of the ovule in the ovary.

  7. Individual nuclei differ in their sensitivity to the cytoplasmic inducers of DNA synthesis: implications for the origin of cell cycle variability.

    PubMed

    Hola, M; Howard, M; Nawaz, F N; Castleden, S; Brooks, R F

    1996-12-15

    Nuclei of multinucleate cells generally initiate DNA synthesis simultaneously, suggesting that the timing of DNA synthesis depends upon the appearance of a cytoplasmic signal. In contrast, intact nuclei from quiescent mammalian cells initiate DNA synthesis asynchronously in cell-free extracts of Xenopus eggs, despite the common environment. Here we show that the two nuclei of permeabilized binucleate cells enter DNA synthesis coordinately in egg extracts, as they do in vivo, with different pairs of nuclei initiating replication at different times. This indicates that the two nuclei of a binucleate cell are identical in their sensitivity to the inducers of DNA synthesis in egg extracts; this sensitivity varies in general between the nuclei of unrelated cells. The asynchrony of DNA synthesis shown by unrelated nuclei in egg extracts is therefore not an artifact of the cell-free system but a reflection of genuine differences preexisting within the intact cell. Evidence that these differences between nuclei are responsible for a substantial fraction of G1 variability in living cells is presented.

  8. Pathogen Tactics to Manipulate Plant Cell Death.

    PubMed

    Mukhtar, M Shahid; McCormack, Maggie E; Argueso, Cristiana T; Pajerowska-Mukhtar, Karolina M

    2016-07-11

    Cell death is a vital process for multicellular organisms. Programmed cell death (PCD) functions in a variety of processes including growth, development, and immune responses for homeostasis maintenance. In particular, plants and animals utilize PCD to control pathogen invasion and infected cell populations. Despite some similarity, there are a number of key differences between how these organisms initiate and regulate cell death. In contrast to animals, plants are sessile, lack a circulatory system, and have additional cellular structures, including cell walls and chloroplasts. Plant cells have the autonomous ability to induce localized cell death using conserved eukaryotic pathways as well as unique plant-specific pathways. Thus, in order to successfully infect host cells, pathogens must subvert immune responses and avoid detection to prevent PCD and allow infection. Here we discuss the roles of cell death in plant immune responses and the tactics pathogens utilize to avert cell death. PMID:27404256

  9. Moringa oleifera Lam. seed extract prevents fat diet induced oxidative stress in mice and protects liver cell-nuclei from hydroxyl radical mediated damage.

    PubMed

    Das, Nilanjan; Ganguli, Debdutta; Dey, Sanjit

    2015-12-01

    High fat diet (HFD) prompts metabolic pattern inducing reactive oxygen species (ROS) production in mitochondria thereby triggering multitude of chronic disorders in human. Antioxidants from plant sources may be an imperative remedy against this disorder. However, it requires scientific validation. In this study, we explored if (i) Moringa oleifera seed extract (MoSE) can neutralize ROS generated in HFD fed mice; (ii) protect cell-nuclei damage developed by Fenton reaction in vitro. Swiss mice were fed with HFD to develop oxidative stress model (HFD group). Other groups were control, seed extract alone treated, and MoSE simultaneously (HS) treated. Treatment period was of 15 days. Antioxidant enzymes with tissue nitrite content (TNC) and lipid peroxidation (LPO) were estimated from liver homogenate. HS group showed significantly higher (P < 0.05) superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) activity, and ferric reducing antioxidant power (FRAP) compared to only HFD fed group. Further, TNC and LPO decreased significantly (P < 0.05) in HS group compared to HFD fed group. MoSE also protected hepatocytes nuclei from the hydroxyl radicals generated by Fenton reaction. MoSE was found to be polyphenol rich with potent reducing power, free radicals and hydroxyl radicals scavenging activity. Thus, MoSE exhibited robust antioxidant prospective to neutralize ROS developed in HFD fed mice and also protected the nuclei damage from hydroxyl radicals. Hence, it can be used as herbal medication against HFD induced ROS mediated disorders.

  10. Moringa oleifera Lam. seed extract prevents fat diet induced oxidative stress in mice and protects liver cell-nuclei from hydroxyl radical mediated damage.

    PubMed

    Das, Nilanjan; Ganguli, Debdutta; Dey, Sanjit

    2015-12-01

    High fat diet (HFD) prompts metabolic pattern inducing reactive oxygen species (ROS) production in mitochondria thereby triggering multitude of chronic disorders in human. Antioxidants from plant sources may be an imperative remedy against this disorder. However, it requires scientific validation. In this study, we explored if (i) Moringa oleifera seed extract (MoSE) can neutralize ROS generated in HFD fed mice; (ii) protect cell-nuclei damage developed by Fenton reaction in vitro. Swiss mice were fed with HFD to develop oxidative stress model (HFD group). Other groups were control, seed extract alone treated, and MoSE simultaneously (HS) treated. Treatment period was of 15 days. Antioxidant enzymes with tissue nitrite content (TNC) and lipid peroxidation (LPO) were estimated from liver homogenate. HS group showed significantly higher (P < 0.05) superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) activity, and ferric reducing antioxidant power (FRAP) compared to only HFD fed group. Further, TNC and LPO decreased significantly (P < 0.05) in HS group compared to HFD fed group. MoSE also protected hepatocytes nuclei from the hydroxyl radicals generated by Fenton reaction. MoSE was found to be polyphenol rich with potent reducing power, free radicals and hydroxyl radicals scavenging activity. Thus, MoSE exhibited robust antioxidant prospective to neutralize ROS developed in HFD fed mice and also protected the nuclei damage from hydroxyl radicals. Hence, it can be used as herbal medication against HFD induced ROS mediated disorders. PMID:26742324

  11. Reprogramming of plant cells induced by 6b oncoproteins from the plant pathogen Agrobacterium.

    PubMed

    Ito, Masaki; Machida, Yasunori

    2015-05-01

    Reprogramming of plant cells is an event characterized by dedifferentiation, reacquisition of totipotency, and enhanced cell proliferation, and is typically observed during formation of the callus, which is dependent on plant hormones. The callus-like cell mass, called a crown gall tumor, is induced at the sites of infection by Agrobacterium species through the expression of hormone-synthesizing genes encoded in the T-DNA region, which probably involves a similar reprogramming process. One of the T-DNA genes, 6b, can also by itself induce reprogramming of differentiated cells to generate tumors and is therefore recognized as an oncogene acting in plant cells. The 6b genes belong to a group of Agrobacterium T-DNA genes, which include rolB, rolC, and orf13. These genes encode proteins with weakly conserved sequences and may be derived from a common evolutionary origin. Most of these members can modify plant growth and morphogenesis in various ways, in most cases without affecting the levels of plant hormones. Recent studies have suggested that the molecular function of 6b might be to modify the patterns of transcription in the host nuclei, particularly by directly targeting the host transcription factors or by changing the epigenetic status of the host chromatin through intrinsic histone chaperone activity. In light of the recent findings on zygotic resetting of nucleosomal histone variants in Arabidopsis thaliana, one attractive idea is that acquisition of totipotency might be facilitated by global changes of epigenetic status, which might be induced by replacement of histone variants in the zygote after fertilization and in differentiated cells upon stimulation by plant hormones as well as by expression of the 6b gene. PMID:25694001

  12. Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling

    PubMed Central

    Steiner, Florian A.; Talbert, Paul B.; Kasinathan, Sivakanthan; Deal, Roger B.; Henikoff, Steven

    2012-01-01

    An understanding of developmental processes requires knowledge of transcriptional and epigenetic landscapes at the level of tissues and ultimately individual cells. However, obtaining tissue- or cell-type-specific expression and chromatin profiles for animals has been challenging. Here we describe a method for purifying nuclei from specific cell types of animal models that allows simultaneous determination of both expression and chromatin profiles. The method is based on in vivo biotin-labeling of the nuclear envelope and subsequent affinity purification of nuclei. We describe the use of the method to isolate nuclei from muscle of adult Caenorhabditis elegans and from mesoderm of Drosophila melanogaster embryos. As a case study, we determined expression and nucleosome occupancy profiles for affinity-purified nuclei from C. elegans muscle. We identified hundreds of genes that are specifically expressed in muscle tissues and found that these genes are depleted of nucleosomes at promoters and gene bodies in muscle relative to other tissues. This method should be universally applicable to all model systems that allow transgenesis and will make it possible to determine epigenetic and expression profiles of different tissues and cell types. PMID:22219512

  13. Overlapping cell nuclei segmentation using a spatially adaptive active physical model.

    PubMed

    Plissiti, Marina E; Nikou, Christophoros

    2012-11-01

    A method for the segmentation of overlapping nuclei is presented, which combines local characteristics of the nuclei boundary and a priori knowledge about the expected shape of the nuclei. A deformable model whose behavior is driven by physical principles is trained on images containing a single nuclei, and attributes of the shapes of the nuclei are expressed in terms of modal analysis. Based on the estimated modal distribution and driven by the image characteristics, we develop a framework to detect and describe the unknown nuclei boundaries in images containing two overlapping nuclei. The problem of the estimation of an accurate nucleus boundary in the overlapping areas is successfully addressed with the use of appropriate weight parameters that control the contribution of the image force in the total energy of the deformable model. The proposed method was evaluated using 152 images of conventional Pap smears, each containing two overlapping nuclei. Comparisons with other segmentation methods indicate that our method produces more accurate nuclei boundaries which are closer to the ground truth.

  14. [Transport of RNA from rat liver cell nuclei in vitro. Effect of superoxide dismutase on the release of rapidly labeled RNA from isolated nuclei].

    PubMed

    Koen, Ia M; Peskin, A V; Baru, V A; Fedorchenko, V V; Zbarskiĭ, I B; Konstantinov, A A

    1978-12-01

    Purified superoxide dismutase from beaf and rat liver cytosol was found to inhibit in vitro a release of the newly synthesized poly(A)-containing RNA from isolated hepatocyte nuclei in a cell-free system. The inhibition was concentration-dependent. Similar effect was observed with Cu2+ and coppertyrosine complex, which possess SOD-like type catalytic activity. The effectiveness of the complex and of Cu2+ however was an order smaller than that of SOD. The inhibitory effects of SOD and the two other copper-containing compounds could be abolished by potassium cyanide and reduced glutathione as far as by gomologous cytosol. Catalase failed to effect the RNA release. Although serum albumin itself did not affect release of RNA it was capable to abolish the inhibitory effects of Cu2+ and of copper-tyrosine, but not that of SOD. Possible mechanisms for the inhibitory effect of SOD on RNA transfer across the nuclear envelope are discussed. PMID:743506

  15. [Morpho-functional status of large-cell hypothalamic nuclei following chronic exposure to wide-range electromagnetic impulses].

    PubMed

    Popov, S S; Dolzhanov, A Ia; Zuev, V G

    2001-01-01

    In a experiment with white nubilous male rats the morphofuntional state of secretory neurons (SN) of the large-cell hypothalamic nuclei (LCHN) was evaluated with the morphological, morphometric and statistical methods following electromagnetic exposure (500, 100 and 50 impulses once a week) inducing in the animal body the averaged current densities of 1.5 kA/m2. It was stated that 100 EMI a week called forth opposite LCHN reactions, that is suppression of synthesis and elimination of SN neuro-secret in supraoptic nuclei and their activation in the paraventricular nuclei. LCHN reaction to the other frequencies of EM impulses was uniform and included rearrangement of both neurosecretory centers for a lower functional activity. Decrease in the number of effectively functioning SN due to the EM exposure may point to a dead-aptive effect of this factor.

  16. Natural Paradigms of Plant Cell Wall Degradation

    SciTech Connect

    Wei, H.; Xu, Q.; Taylor, L. E.; Baker, J. O.; Tucker, M. P.; Ding, S. Y.

    2009-01-01

    Natural processes of recycling carbon from plant cell walls are slow but very efficient, generally involving microbial communities and their secreted enzymes. Efficient combinations of microbial communities and enzymes act in a sequential and synergistic manner to degrade plant cell walls. Recent understanding of plant cell wall ultra-structure, as well as the carbon metabolism, ATP production, and ecology of participating microbial communities, and the biochemical properties of their cellulolytic enzymes have led to new perspectives on saccharification of biomass. Microbial communities are dynamic functions of the chemical and structural compositions of plant cell wall components. The primitive 'multicellularity' exhibited by certain cellulolytic microorganisms may play a role in facilitating cell-cell communication and cell-plant cell wall-substrate interaction.

  17. Plant cell gravisensitivity and adaptation to microgravity.

    PubMed

    Kordyum, E L

    2014-01-01

    A short overview on the effects of real and simulated microgravity on certain cell components and processes, including new information obtained recently, is presented. Attention is focused on the influence of real and simulated microgravity on plant cells that are not specialised to gravity perception and on seed formation. The paper considers the possibility of full adaptation of plants to microgravity, and suggests some questions for future plant research in order to make decisions on fundamental and applied problems of plant space biology.

  18. Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

    PubMed Central

    Wang, Xiaozhu; Takebayashi, Shin-ichiro; Bernardin, Evans; Gilbert, David M.; Chella, Ravindran

    2012-01-01

    We have developed a novel method for genetic characterization of single cells by integrating microfluidic stretching of chromosomal DNA and fiber fluorescence in situ hybridization (FISH). In this method, individually isolated cell nuclei were immobilized in a microchannel. Chromosomal DNA was released from the nuclei and stretched by a pressure-driven flow. We analyzed and optimized flow conditions to generate a millimeter-long band of stretched DNA from each nucleus. Telomere fiber FISH was successfully performed on the stretched chromosomal DNA. Individual telomere fiber FISH signals from single cells could be resolved and their lengths measured, demonstrating the ability of the method to quantify genetic features at the level of single cells. PMID:22231286

  19. Organization of transcribed and nontranscribed chromatin in isolated nuclei of Zea mays root cells.

    PubMed

    Greimers, R; Deltour, R

    1981-02-01

    A slightly modified technique of Miller was applied to nuclei isolated from roots of germinating corn embryos. Under the conditions used, the extranucleolar chromatin was easily spread but the nucleolar chromatin often remained aggregated. The nontranscribed chromatin regions consisted of long deoxyribonucleoprotein (DNP) fibrils with the typical "beads-on-a-string" appearance of nucleosomal arrangement. When the EDTA concentration of the spreading medium was increased, linear arrangement of nucleosomes, 20 nm and 40-50 nm DNP granules were observed on the same DNP fibril. The supranucleosomal granules were also closely packed without any apparent order. Independently of the EDTA concentration, numerous non-nucleolar ribonucleoprotein (RNP) fibrils with a knobby structure were seen laterally bound to DNP fibrils. These knobby RNP fibrils (transcripts) were highly folded and appeared in "bunch-of-grapes" configurations. Each RNP knob measured 23 nm in diameter and the longest of these RNP fibrils were estimated to reach 1.5 micron. The RNP fibrils were often solitary and never exceeded 8 per 10 micron of the DNP axis. Distribution, morphology, and size of these transcripts distinguished them from the nucleolar "Christmas-tree"-like figures, which were rarely observed with our material. These non-nucleolar transcriptional units may correspond to genes coding for the heterodisperse nuclear RNA (hnRNA) and the knobs of 23 nm could be related to informofers or hnRNA particles. Up to now, the visualization of transcriptional units for higher plant tissues have never been reported.

  20. Refractive index of plant cell walls

    NASA Technical Reports Server (NTRS)

    Gausman, H. W.; Allen, W. A.; Escobar, D. E.

    1974-01-01

    Air was replaced with media of higher refractive indices by vacuum infiltration in leaves of cucumber, blackeye pea, tomato, and string bean plants, and reflectance of noninfiltrated and infiltrated leaves was spectrophotometrically measured. Infiltrated leaves reflected less light than noninfiltrated leaves over the 500-2500-nm wavelength interval because cell wall-air interfaces were partly eliminated. Minimal reflectance should occur when the average refractive index of plant cell walls was matched by the infiltrating fluid. Although refractive indices that resulted in minimal reflectance differed among the four plant genera, an average value of 1.425 approximates the refractive index of plant cell walls for the four plant genera.

  1. DNA (deoxyribonucleic acid) synthesis following microinjection of heterologous sperm and somatic cell nuclei into hamster oocytes

    SciTech Connect

    Naish, S.J.; Perreault, S.D.; Zirkin, B.R.

    1987-01-01

    The authors investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in /sup 3/H-thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. The authors conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is present.

  2. What can plants do for cell biology?

    PubMed Central

    Bezanilla, Magdalena

    2013-01-01

    Historically, cell biologists studied organisms that represented a reasonable sampling of life's diversity, whereas recently research has narrowed into a few model systems. As a result, the cells of plants have been relatively neglected. Here I choose three examples to illustrate how plants have been informative and could be even more so. Owing to their ease of imaging and genetic tractability, multicellular plant model systems provide a unique opportunity to address long-standing questions in cell biology. PMID:23943803

  3. A difunctional squarylium indocyanine dye distinguishes dead cells through diverse staining of the cell nuclei/membranes.

    PubMed

    Li, Jie; Guo, Kunru; Shen, Jie; Yang, Wantai; Yin, Meizhen

    2014-04-01

    Functionalized fluorescent dyes have attracted great interest for the specific staining of subcellular organelles in multicellular organisms. A novel nanometer-sized water-soluble multi-functional squarylium indocyanine dye (D1) that contains four primary amines is synthesized. The dye exhibits good photostability, non-toxicity and biocompatibility. Isothermal titration calorimetry demonstrates that an affinity between D1 and DNA is higher than that between D1 and analogue of phospholipids. Analysis of circular dichroism spectra indicates that D1 targets to the DNA minor groove and aggregates to a helix. Because of the distinct affinity between the dye and subcellular organelles, the dye exhibits difunctional abilities to label the cell nuclei in fixed cells/tissue and the cell membranes in live cells/tissue. By combination of the two staining capabilities, the dye is further explored as a specific marker to distinguish apoptotic cells in live cells/tissue. The research opens a new way to design novel multifunctional dyes for life science applications.

  4. Plant cell walls to ethanol.

    PubMed

    Jordan, Douglas B; Bowman, Michael J; Braker, Jay D; Dien, Bruce S; Hector, Ronald E; Lee, Charles C; Mertens, Jeffrey A; Wagschal, Kurt

    2012-03-01

    Conversion of plant cell walls to ethanol constitutes second generation bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation and separation. Ultimately, it is desirable to combine as many of the biochemical steps as possible in a single organism to achieve CBP (consolidated bioprocessing). A commercially ready CBP organism is currently unreported. Production of second generation bioethanol is hindered by economics, particularly in the cost of pretreatment (including waste management and solvent recovery), the cost of saccharification enzymes (particularly exocellulases and endocellulases displaying kcat ~1 s-1 on crystalline cellulose), and the inefficiency of co-fermentation of 5- and 6-carbon monosaccharides (owing in part to redox cofactor imbalances in Saccharomyces cerevisiae). PMID:22329798

  5. Plant cell walls to ethanol.

    PubMed

    Jordan, Douglas B; Bowman, Michael J; Braker, Jay D; Dien, Bruce S; Hector, Ronald E; Lee, Charles C; Mertens, Jeffrey A; Wagschal, Kurt

    2012-03-01

    Conversion of plant cell walls to ethanol constitutes second generation bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation and separation. Ultimately, it is desirable to combine as many of the biochemical steps as possible in a single organism to achieve CBP (consolidated bioprocessing). A commercially ready CBP organism is currently unreported. Production of second generation bioethanol is hindered by economics, particularly in the cost of pretreatment (including waste management and solvent recovery), the cost of saccharification enzymes (particularly exocellulases and endocellulases displaying kcat ~1 s-1 on crystalline cellulose), and the inefficiency of co-fermentation of 5- and 6-carbon monosaccharides (owing in part to redox cofactor imbalances in Saccharomyces cerevisiae).

  6. Thymoquinone causes multiple effects, including cell death, on dividing plant cells.

    PubMed

    Hassanien, Sameh E; Ramadan, Ahmed M; Azeiz, Ahmed Z Abdel; Mohammed, Rasha A; Hassan, Sabah M; Shokry, Ahmed M; Atef, Ahmed; Kamal, Khalid B H; Rabah, Samar; Sabir, Jamal S M; Abuzinadah, Osama A; El-Domyati, Fotouh M; Martin, Gregory B; Bahieldin, Ahmed

    2013-01-01

    Thymoquinone (TQ) is a major constituent of Nigella sativa oil with reported anti-oxidative activity and anti-inflammatory activity in animal cells. It also inhibits proliferation and induces programmed cell death (apoptosis) in human skin cancer cells. The present study sought to detect the influence of TQ on dividing cells of three plant systems and on expression of Bcl2-associated athanogene-like (BAG-like) genes that might be involved during the process of cell death. BAG genes are known for the regulation of diverse physiological processes in animals, including apoptosis, tumorigenesis, stress responses, and cell division. Synthetic TQ at 0.1mg/mL greatly reduced wheat seed germination rate, whereas 0.2mg/mL completely inhibited germination. An Evans blue assay revealed moderate cell death in the meristematic zone of Glycine max roots after 1h of TQ treatment (0.2mg/mL), with severe cell death occurring in this zone after 2h of treatment. Light microscopy of TQ-treated (0.2mg/mL) onion hairy root tips for 1h revealed anti-mitotic activity and also cell death-associated changes, including nuclear membrane disruption and nuclear fragmentation. Transmission electron microscopy of TQ-treated cells (0.2mg/mL) for 1h revealed shrinkage of the plasma membrane, leakage of cell lysate, degradation of cell walls, enlargement of vacuoles and condensation of nuclei. Expression of one BAG-like gene, previously associated with cell death, was induced 20 min after TQ treatment in Glycine max root tip cells. Thus, TQ has multiple effects, including cell death, on dividing plant cells and plants may serve as a useful system to further investigate the mechanisms underlying the response of eukaryotic cells to TQ.

  7. Plant stem cells as innovation in cosmetics.

    PubMed

    Moruś, Martyna; Baran, Monika; Rost-Roszkowska, Magdalena; Skotnicka-Graca, Urszula

    2014-01-01

    The stem cells thanks to their ability of unlimited division number or transformation into different cell types creating organs, are responsible for regeneration processes. Depending on the organism in which the stem cells exists, they divide to the plant or animal ones. The later group includes the stem cells existing in both embryo's and adult human's organs. It includes, among others, epidermal stem cells, located in the hair follicle relieves and also in its basal layers, and responsible for permanent regeneration of the epidermis. Temporary science looks for method suitable for stimulation of the epidermis stem cells, amongst the other by delivery of e.g., growth factors for proliferation that decrease with the age. One of the methods is the use of the plant cell culture technology, including a number of methods that should ensure growth of plant cells, issues or organs in the environment with the microorganism-free medium. It uses abilities of the different plant cells to dedifferentiation into stem cells and coming back to the pluripotent status. The extracts obtained this way from the plant stem cells are currently used for production of both common or professional care cosmetics. This work describes exactly impact of the plant stem cell extract, coming from one type of the common apple tree (Uttwiler Spätlauber) to human skin as one of the first plant sorts, which are used in cosmetology and esthetic dermatology.

  8. Production of calves by transfer of nuclei from cultured inner cell mass cells.

    PubMed

    Sims, M; First, N L

    1994-06-21

    We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days. PMID:8016127

  9. Production of calves by transfer of nuclei from cultured inner cell mass cells.

    PubMed

    Sims, M; First, N L

    1994-06-21

    We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days.

  10. Production of calves by transfer of nuclei from cultured inner cell mass cells.

    PubMed Central

    Sims, M; First, N L

    1994-01-01

    We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days. Images PMID:8016127

  11. Nuquantus: Machine learning software for the characterization and quantification of cell nuclei in complex immunofluorescent tissue images

    PubMed Central

    Gross, Polina; Honnorat, Nicolas; Varol, Erdem; Wallner, Markus; Trappanese, Danielle M.; Sharp, Thomas E.; Starosta, Timothy; Duran, Jason M.; Koller, Sarah; Davatzikos, Christos; Houser, Steven R.

    2016-01-01

    Determination of fundamental mechanisms of disease often hinges on histopathology visualization and quantitative image analysis. Currently, the analysis of multi-channel fluorescence tissue images is primarily achieved by manual measurements of tissue cellular content and sub-cellular compartments. Since the current manual methodology for image analysis is a tedious and subjective approach, there is clearly a need for an automated analytical technique to process large-scale image datasets. Here, we introduce Nuquantus (Nuclei quantification utility software) - a novel machine learning-based analytical method, which identifies, quantifies and classifies nuclei based on cells of interest in composite fluorescent tissue images, in which cell borders are not visible. Nuquantus is an adaptive framework that learns the morphological attributes of intact tissue in the presence of anatomical variability and pathological processes. Nuquantus allowed us to robustly perform quantitative image analysis on remodeling cardiac tissue after myocardial infarction. Nuquantus reliably classifies cardiomyocyte versus non-cardiomyocyte nuclei and detects cell proliferation, as well as cell death in different cell classes. Broadly, Nuquantus provides innovative computerized methodology to analyze complex tissue images that significantly facilitates image analysis and minimizes human bias. PMID:27005843

  12. Nuquantus: Machine learning software for the characterization and quantification of cell nuclei in complex immunofluorescent tissue images

    NASA Astrophysics Data System (ADS)

    Gross, Polina; Honnorat, Nicolas; Varol, Erdem; Wallner, Markus; Trappanese, Danielle M.; Sharp, Thomas E.; Starosta, Timothy; Duran, Jason M.; Koller, Sarah; Davatzikos, Christos; Houser, Steven R.

    2016-03-01

    Determination of fundamental mechanisms of disease often hinges on histopathology visualization and quantitative image analysis. Currently, the analysis of multi-channel fluorescence tissue images is primarily achieved by manual measurements of tissue cellular content and sub-cellular compartments. Since the current manual methodology for image analysis is a tedious and subjective approach, there is clearly a need for an automated analytical technique to process large-scale image datasets. Here, we introduce Nuquantus (Nuclei quantification utility software) - a novel machine learning-based analytical method, which identifies, quantifies and classifies nuclei based on cells of interest in composite fluorescent tissue images, in which cell borders are not visible. Nuquantus is an adaptive framework that learns the morphological attributes of intact tissue in the presence of anatomical variability and pathological processes. Nuquantus allowed us to robustly perform quantitative image analysis on remodeling cardiac tissue after myocardial infarction. Nuquantus reliably classifies cardiomyocyte versus non-cardiomyocyte nuclei and detects cell proliferation, as well as cell death in different cell classes. Broadly, Nuquantus provides innovative computerized methodology to analyze complex tissue images that significantly facilitates image analysis and minimizes human bias.

  13. How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection.

    PubMed

    Jiráková, Klára; Kulda, Jaroslav; Nohýnková, Eva

    2012-05-01

    Differentiation into infectious cysts (encystation) and multiplication of pathogenic trophozoites after hatching from the cyst (excystation) are fundamental processes in the life cycle of the human intestinal parasite Giardia intestinalis. During encystation, a bi-nucleated trophozoite transforms to a dormant tetra-nucleated cyst enveloped by a protective cyst wall. Nuclear division during encystation is not followed by cytokinesis. In contrast to the well-studied mechanism of cyst wall formation, information on nuclei behavior is incomplete and basic cytological data are lacking. Here we present evidence that (1) the nuclei divide by semi-open mitosis during early encystment; (2) the daughter nuclei coming from different parent nuclei are always arranged in pairs; (3) in both pairs, the nuclei are interconnected via bridges formed by fusion of their nuclear envelopes; (4) each interconnected nuclear pair is associated with one basal body tetrad of the undivided diplomonad mastigont; and (5) the interconnection between nuclei persists through the cyst stage being a characteristic feature of encysted Giardia. Based on the presented results, a model of nuclei behavior during Giardia differentiation is proposed.

  14. [Genetic regulation of plant shoot stem cells].

    PubMed

    Al'bert, E V; Ezhova, T A

    2013-02-01

    This article describes the main features of plant stem cells and summarizes the results of studies of the genetic control of stem cell maintenance in the apical meristem of the shoot. It is demonstrated that the WUS-CLV gene system plays a key role in the maintenance of shoot apical stem cells and the formation of adventitious buds and somatic embryos. Unconventional concepts of plant stem cells are considered.

  15. Segmentation of cervical cell nuclei in high-resolution microscopic images: A new algorithm and a web-based software framework.

    PubMed

    Bergmeir, Christoph; García Silvente, Miguel; Benítez, José Manuel

    2012-09-01

    In order to automate cervical cancer screening tests, one of the most important and longstanding challenges is the segmentation of cell nuclei in the stained specimens. Though nuclei of isolated cells in high-quality acquisitions often are easy to segment, the problem lies in the segmentation of large numbers of nuclei with various characteristics under differing acquisition conditions in high-resolution scans of the complete microscope slides. We implemented a system that enables processing of full resolution images, and proposes a new algorithm for segmenting the nuclei under adequate control of the expert user. The system can work automatically or interactively guided, to allow for segmentation within the whole range of slide and image characteristics. It facilitates data storage and interaction of technical and medical experts, especially with its web-based architecture. The proposed algorithm localizes cell nuclei using a voting scheme and prior knowledge, before it determines the exact shape of the nuclei by means of an elastic segmentation algorithm. After noise removal with a mean-shift and a median filtering takes place, edges are extracted with a Canny edge detection algorithm. Motivated by the observation that cell nuclei are surrounded by cytoplasm and their shape is roughly elliptical, edges adjacent to the background are removed. A randomized Hough transform for ellipses finds candidate nuclei, which are then processed by a level set algorithm. The algorithm is tested and compared to other algorithms on a database containing 207 images acquired from two different microscope slides, with promising results.

  16. Pathological modifications of plant stem cell destiny

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In higher plants, the shoot apex contains undifferentiated stem cells that give rise to various tissues and organs. The fate of these stem cells determines the pattern of plant growth as well as reproduction; and such fate is genetically preprogrammed. We found that a bacterial infection can derai...

  17. Segmentation of cytoplasm and nuclei of abnormal cells in cervical cytology using global and local graph cuts.

    PubMed

    Zhang, Ling; Kong, Hui; Chin, Chien Ting; Liu, Shaoxiong; Chen, Zhi; Wang, Tianfu; Chen, Siping

    2014-07-01

    Automation-assisted reading (AAR) techniques have the potential to reduce errors and increase productivity in cervical cancer screening. The sensitivity of AAR relies heavily on automated segmentation of abnormal cervical cells, which is handled poorly by current segmentation algorithms. In this paper, a global and local scheme based on graph cut approach is proposed to segment cervical cells in images with a mix of healthy and abnormal cells. For cytoplasm segmentation, the multi-way graph cut is performed globally on the a* channel enhanced image, which can be effective when the image histogram presents a non-bimodal distribution. For segmentation of nuclei, especially when they are abnormal, we propose to use graph cut adaptively and locally, which allows the combination of intensity, texture, boundary and region information. Two concave points-based approaches are integrated to split the touching-nuclei. As part of an ongoing clinical trial, preliminary validation results obtained from 21 cervical cell images with non-ideal imaging condition and pathology show that our segmentation method achieved 93% accuracy for cytoplasm, and 88.4% F-measure for abnormal nuclei, outperforming state of the art methods in terms of accuracy. Our method has the potential to improve the sensitivity of AAR in screening for cervical cancer.

  18. Cell wall, cytoskeleton, and cell expansion in higher plants.

    PubMed

    Bashline, Logan; Lei, Lei; Li, Shundai; Gu, Ying

    2014-04-01

    To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.

  19. Time-dependent reduction of structural complexity of the buccal epithelial cell nuclei after treatment with silver nanoparticles.

    PubMed

    Pantic, I; Paunovic, J; Perovic, M; Cattani, C; Pantic, S; Suzic, S; Nesic, D; Basta-Jovanovic, G

    2013-12-01

    Recent studies have suggested that silver nanoparticles (AgNPs) may affect cell DNA structure in in vitro conditions. In this paper, we present the results indicating that AgNPs change nuclear complexity properties in isolated human epithelial buccal cells in a time-dependent manner. Epithelial buccal cells were plated in special tissue culture chamber / slides and were kept at 37°C in an RPMI 1640 cell culture medium supplemented with L-glutamine. The cells were treated with colloidal silver nanoparticles suspended in RPMI 1640 medium at the concentration 15 mg L⁻¹. Digital micrographs of the cell nuclei in a sample of 30 cells were created at five different time steps: before the treatment (controls), immediately after the treatment, as well as 15 , 30 and 60 min after the treatment with AgNPs. For each nuclear structure, values of fractal dimension, lacunarity, circularity, as well as parameters of grey level co-occurrence matrix (GLCM) texture, were determined. The results indicate time-dependent reduction of structural complexity in the cell nuclei after the contact with AgNPs. These findings further suggest that AgNPs, at concentrations present in today's over-the-counter drug products, might have significant effects on the cell genetic material.

  20. Development of the serotonergic cells in murine raphe nuclei and their relations with rhombomeric domains.

    PubMed

    Alonso, Antonia; Merchán, Paloma; Sandoval, Juan E; Sánchez-Arrones, Luisa; Garcia-Cazorla, Angels; Artuch, Rafael; Ferrán, José L; Martínez-de-la-Torre, Margaret; Puelles, Luis

    2013-09-01

    The raphe nuclei represent the origin of central serotonergic projections. The literature distinguishes seven nuclei grouped into rostral and caudal clusters relative to the pons. The boundaries of these nuclei have not been defined precisely enough, particularly with regard to developmental units, notably hindbrain rhombomeres. We hold that a developmental point of view considering rhombomeres may explain observed differences in connectivity and function. There are twelve rhombomeres characterized by particular genetic profiles, and each develops between one and four distinct serotonergic populations. We have studied the distribution of the conventional seven raphe nuclei among these twelve units. To this aim, we correlated 5-HT-immunoreacted neurons with rhombomeric boundary landmarks in sagittal mouse brain sections at different developmental stages. Furthermore, we performed a partial genoarchitectonic analysis of the developing raphe nuclei, mapping all known serotonergic differentiation markers, and compared these results, jointly with others found in the literature, with our map of serotonin-containing populations, in order to examine regional variations in correspondence. Examples of regionally selective gene patterns were identified. As a result, we produced a rhombomeric classification of some 45 serotonergic populations, and suggested a corresponding modified terminology. Only a minor rostral part of the dorsal raphe nucleus lies in the midbrain. Some serotonergic neurons were found in rhombomere 4, contrary to the conventional assumption that it lacks such neurons. We expect that our reclassification of raphe nuclei may be useful for causal analysis of their differential molecular specification, as well as for studies of differential connectivity and function.

  1. Rapid and Semi-Automated Extraction of Neuronal Cell Bodies and Nuclei from Electron Microscopy Image Stacks

    PubMed Central

    Holcomb, Paul S.; Morehead, Michael; Doretto, Gianfranco; Chen, Peter; Berg, Stuart; Plaza, Stephen; Spirou, George

    2016-01-01

    Connectomics—the study of how neurons wire together in the brain—is at the forefront of modern neuroscience research. However, many connectomics studies are limited by the time and precision needed to correctly segment large volumes of electron microscopy (EM) image data. We present here a semi-automated segmentation pipeline using freely available software that can significantly decrease segmentation time for extracting both nuclei and cell bodies from EM image volumes. PMID:27259933

  2. Release of cell-free ice nuclei from Halomonas elongata expressing the ice nucleation gene inaZ of Pseudomonas syringae.

    PubMed

    Tegos, G; Vargas, C; Perysinakis, A; Koukkou, A I; Christogianni, A; Nieto, J J; Ventosa, A; Drainas, C

    2000-11-01

    Release of ice nuclei in the growth medium of recombinant Halomonas elongata cells expressing the inaZ gene of Pseudomonas syringae was studied in an attempt to produce cell-free active ice nuclei for biotechnological applications. Cell-free ice nuclei were not retained by cellulose acetate filters of 0.2 microm pore size. Highest activity of cell-free ice nuclei was obtained when cells were grown in low salinity (0.5-5% NaCl, w/v). Freezing temperature threshold, estimated to be below -7 degrees C indicating class C nuclei, was not affected by medium salinity. Their density, as estimated by Percoll density centrifugation, was 1.018 +/- 0.002 gml(-1) and they were found to be free of lipids. Ice nuclei are released in the growth medium of recombinant H. elongata cells probably because of inefficient anchoring of the ice-nucleation protein aggregates in the outer membrane. The ice+ recombinant H. elongata cells could be useful for future use as a source of active cell-free ice nucleation protein. PMID:11119152

  3. The Fluorescence Methods to Study Neurotransmitters (Biomediators) in Plant Cells.

    PubMed

    Roshchina, Victoria V

    2016-05-01

    Fluorescence as a parameter for analysis of intracellular binding and localization of neurotransmitters also named biomediators (acetylcholine and biogenic amines such as catecholamines, serotonin, histamine) as well as their receptors in plant cells has been estimated basing on several world publications and own experiments of the author. The subjects of the consideration were 1. application of reagents forming fluorescent products (for catecholamines - glyoxylic acid, for histamine - formaldehyde or ortho-phthalic aldehyde) to show the presence and binding of the compounds in cells, 2. binding of their fluorescent agonists and antagonists with cell, 3. effects of the compounds, their agonists and antagonists on autofluorescence, 4. action of external factors on the accumulation of the compounds in cells. How neurotransmitters can bind to certain cellular compartments has been shown on intact individual cells (vegetative microspores, pollens, secretory cells) and isolated organelles. The staining with reagents on biogenic amines leads to the appearance blue or blue-green emission on the surface and excretions of intact cells as well in some DNA-containing organelles within cells. The difference between autofluorescence and histochemically induced fluorescence may reflect the occurrence and amount of biogenic amines in the cells studied. Ozone and salinity as external factors can regulate the emission of intact cells related to biogenic amines. After the treatment of isolated cellular organelles with glyoxylic acid blue emission with maximum 460-475 nm was seen in nuclei and chloroplasts (in control variants in this spectral region the noticeable emission was absent) and very expressive fluorescence (more than twenty times as compared to control) in the vacuoles. After exposure to ortho-phthalic aldehyde blue emission was more noticeable in nuclei and chloroplasts. Fluorescent agonists (muscarine, 6,7-diOHATN, BODIPY-dopamine or BODIPY-5HT) or antagonists (d

  4. Skin reaction and antibody responses in guinea-pigs sensitized to human leukaemia cells or their nuclei in combination with Bacillus Calmette-Guérin.

    PubMed

    Barnes, R M; Lewis, C M; Pegrum, G D; Prince, G H

    1976-12-01

    Guinea-pigs sensitized by subcutaneous injection of chronic lymphatic leukaemia (CLL) cells combined with Bacillus Calmette-Guérin (BCG) displayed good skin reacitons 24 and 48 h after challenge with CLL cells. Equally good responses were also demonstrated using nuclei from the leukaemic cells in combination with BCG. These reactions were significantly greater than those produced in the same manner but without BCG. Sera form the animals were examined for the presence of antibodies against CLL cells by cytotoxicity and immunofluorescence techniques. Only samples from guinea-pigs innoculated with CLL cells were found to contain significant antibodies. Histological examination showed that whereas leukaemic cells persisted at the sensitizing injection site leukaemic cell nuclei could not be visualized. It is suggested that because leukaemic cell nuclei in combination with BCG are able to induce good skin reactivity without provoking a vigorous humoral antibody response they may have possible advantages over leukaemic cells when used for immunotherapy.

  5. Differential GABAergic and glycinergic inputs of inhibitory interneurons and Purkinje cells to principal cells of the cerebellar nuclei.

    PubMed

    Husson, Zoé; Rousseau, Charly V; Broll, Ilja; Zeilhofer, Hanns Ulrich; Dieudonné, Stéphane

    2014-07-01

    The principal neurons of the cerebellar nuclei (CN), the sole output of the olivo-cerebellar system, receive a massive inhibitory input from Purkinje cells (PCs) of the cerebellar cortex. Morphological evidence suggests that CN principal cells are also contacted by inhibitory interneurons, but the properties of this connection are unknown. Using transgenic, tracing, and immunohistochemical approaches in mice, we show that CN interneurons form a large heterogeneous population with GABA/glycinergic phenotypes, distinct from GABAergic olive-projecting neurons. CN interneurons are found to contact principal output neurons, via glycine receptor (GlyR)-enriched synapses, virtually devoid of the main GABA receptor (GABAR) subunits α1 and γ2. Those clusters account for 5% of the total number of inhibitory receptor clusters on principal neurons. Brief optogenetic stimulations of CN interneurons, through selective expression of channelrhodopsin 2 after viral-mediated transfection of the flexed gene in GlyT2-Cre transgenic mice, evoked fast IPSCs in principal cells. GlyR activation accounted for 15% of interneuron IPSC amplitude, while the remaining current was mediated by activation of GABAR. Surprisingly, small GlyR clusters were also found at PC synapses onto principal CN neurons in addition to α1 and γ2 GABAR subunits. However, GlyR activation was found to account for <3% of the PC inhibitory synaptic currents evoked by electrical stimulation. This work establishes CN glycinergic neurons as a significant source of inhibition to CN principal cells, forming contacts molecularly distinct from, but functionally similar to, Purkinje cell synapses. Their impact on CN output, motor learning, and motor execution deserves further investigation.

  6. Volume increase and spatial shifts of chromosome territories in nuclei of radiation-induced polyploidizing tumour cells.

    PubMed

    Schwarz-Finsterle, Jutta; Scherthan, Harry; Huna, Anda; González, Paula; Mueller, Patrick; Schmitt, Eberhard; Erenpreisa, Jekaterina; Hausmann, Michael

    2013-08-30

    The exposure of tumour cells to high doses of ionizing radiation can induce endopolyploidization as an escape route from cell death. This strategy generally results in mitotic catastrophe during the first few days after irradiation. However, some cells escape mitotic catastrophe, polyploidize and attempt to undergo genome reduction and de-polyploidization in order to create new, viable para-diploid tumour cell sub-clones. In search for the consequences of ionizing radiation induced endopolyploidization, genome and chromosome architecture in nuclei of polyploid tumour cells, and sub-nuclei after division of bi- or multi-nucleated cells were investigated during 7 days following irradiation. Polyploidization was induced in p53-function deficient HeLa cells by exposure to 10Gy of X-irradiation. Chromosome territories #1, #4, #12 and centromeres of chromosomes #6, #10, #X were labelled by FISH and analysed for chromosome numbers, volumes and spatial distribution during 7 days post irradiation. The numbers of interphase chromosome territories or centromeres, respectively, the positions of the most peripherally and centrally located chromosome territories, and the territory volumes were compared to non-irradiated controls over this time course. Nuclei with three copies of several chromosomes (#1, #6, #10, #12, #X) were found in the irradiated as well as non-irradiated specimens. From day 2 to day 5 post irradiation, chromosome territories (#1, #4, #12) shifted towards the nuclear periphery and their volumes increased 16- to 25-fold. Consequently, chromosome territories returned towards the nuclear centre during day 6 and 7 post irradiation. In comparison to non-irradiated cells (∼500μm(3)), the nuclear volume of irradiated cells was increased 8-fold (to ∼4000μm(3)) at day 7 post irradiation. Additionally, smaller cell nuclei with an average volume of about ∼255μm(3) were detected on day 7. The data suggest a radiation-induced generation of large intra

  7. Sexual Differences in Cell Loss during the Post-Hatch Development of Song Control Nuclei in the Bengalese Finch.

    PubMed

    Chen, XiaoNing; Li, Jia; Zeng, Lei; Zhang, XueBo; Lu, XiaoHua; Zuo, MingXue; Zhang, XinWen; Zeng, ShaoJu

    2015-01-01

    Birdsongs and the regions of their brain that control song exhibit obvious sexual differences. However, the mechanisms underlying these sexual dimorphisms remain unknown. To address this issue, we first examined apoptotic cells labeled with caspase-3 or TUNEL in Bengalese finch song control nuclei - the robust nucleus of the archopallium (RA), the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the high vocal center (HVC) and Area X from post-hatch day (P) 15 to 120. Next, we investigated the expression dynamics of pro-apoptotic (Bid, Bad and Bax) and anti-apoptotic (Bcl-2 and Bcl-xL) genes in the aforementioned nuclei. Our results revealed that the female RA at P45 exhibited marked cell apoptosis, confirmed by low densities of Bcl-xL and Bcl-2. Both the male and female LMAN exhibited apoptotic peaks at P35 and P45, respectively, and the observed cell loss was more extensive in males. A corresponding sharp decrease in the density of Bcl-2 after P35 was observed in both sexes, and a greater density of Bid was noted at P45 in males. In addition, we observed that RA volume and the total number of BDNF-expressing cells decreased significantly after unilateral lesion of the LMAN or HVC (two areas that innervate the RA) and that greater numbers of RA-projecting cells were immunoreactive for BDNF in the LMAN than in the HVC. We reasoned that a decrease in the amount of BDNF transported via HVC afferent fibers might result in an increase in cell apoptosis in the female RA. Our data indicate that cell apoptosis resulting from different pro- and anti-apoptotic agents is involved in generating the differences between male and female song control nuclei.

  8. Sexual Differences in Cell Loss during the Post-Hatch Development of Song Control Nuclei in the Bengalese Finch

    PubMed Central

    Lu, XiaoHua; Zuo, MingXue; Zhang, XinWen; Zeng, ShaoJu

    2015-01-01

    Birdsongs and the regions of their brain that control song exhibit obvious sexual differences. However, the mechanisms underlying these sexual dimorphisms remain unknown. To address this issue, we first examined apoptotic cells labeled with caspase-3 or TUNEL in Bengalese finch song control nuclei - the robust nucleus of the archopallium (RA), the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the high vocal center (HVC) and Area X from post-hatch day (P) 15 to 120. Next, we investigated the expression dynamics of pro-apoptotic (Bid, Bad and Bax) and anti-apoptotic (Bcl-2 and Bcl-xL) genes in the aforementioned nuclei. Our results revealed that the female RA at P45 exhibited marked cell apoptosis, confirmed by low densities of Bcl-xL and Bcl-2. Both the male and female LMAN exhibited apoptotic peaks at P35 and P45, respectively, and the observed cell loss was more extensive in males. A corresponding sharp decrease in the density of Bcl-2 after P35 was observed in both sexes, and a greater density of Bid was noted at P45 in males. In addition, we observed that RA volume and the total number of BDNF-expressing cells decreased significantly after unilateral lesion of the LMAN or HVC (two areas that innervate the RA) and that greater numbers of RA-projecting cells were immunoreactive for BDNF in the LMAN than in the HVC. We reasoned that a decrease in the amount of BDNF transported via HVC afferent fibers might result in an increase in cell apoptosis in the female RA. Our data indicate that cell apoptosis resulting from different pro- and anti-apoptotic agents is involved in generating the differences between male and female song control nuclei. PMID:25938674

  9. Induction of pluripotency by injection of mouse trophectoderm cell nuclei into blastocysts following transplantation into enucleated oocytes.

    PubMed

    Sotomaru, Y; Kato, Y; Tsunoda, Y

    1999-07-15

    Pluripotency of mouse trophectoderm (TE) cells was examined using a nuclear transfer technique. We transferred a TE cell to an enucleated oocyte and cultured the reconstituted oocyte to be blastocyst stage. Then a portion of the inner cell mass (ICM) isolated from the TE-origin blastocyst was injected into the cavity of a fertilized blastocyst to produce a chimeric embryo, which was transferred to a recipient female. Of 319 oocytes reconstituted with TE cells, 263 (82.4%) had a single nucleus (1PN), 3 (0.9%) had 2 nuclei (2PN) and 53 (16.6%) had a nucleus with a polar body (1PN1PB). Although the oocytes with 1PN and 2PN developed to blastocysts (81 of 263, 30.8% and 1 of 3, respectively), only those with 1PN were used to produce chimeric blastocysts. After the transfer of chimeric embryos to recipient females, 7 (28%) of 25 conceptuses analyzed at midgestation showed chimerism. Of those 5 (71%), 6 (86%) and 4 (57%) chimeric conceptuses showed distribution of donor nuclei in the fetus, membrane and placenta, and the distributions were 10 to 65, 10 to 50 and 10 to 15%, respectively. Of the 23 young obtained, 7 (30%; 2 males and 5 females) were coat color chimeras. The contributions of donor nuclei were detected in the brain, lung, heart, liver, kidney, testis, ovary and blood. Each coat-color chimeric mouse was mated with CD-1 male or female mice, but no germ line chimera was obtained. When ICM cells were used as the control nuclear donor, the contribution was equivalent to those of TE cells. In conclusion, pluripotency of mouse TE cells on a somatic line was induced, and chimeric young were obtained using a nuclear transplantation technique.

  10. Sexual Differences in Cell Loss during the Post-Hatch Development of Song Control Nuclei in the Bengalese Finch.

    PubMed

    Chen, XiaoNing; Li, Jia; Zeng, Lei; Zhang, XueBo; Lu, XiaoHua; Zuo, MingXue; Zhang, XinWen; Zeng, ShaoJu

    2015-01-01

    Birdsongs and the regions of their brain that control song exhibit obvious sexual differences. However, the mechanisms underlying these sexual dimorphisms remain unknown. To address this issue, we first examined apoptotic cells labeled with caspase-3 or TUNEL in Bengalese finch song control nuclei - the robust nucleus of the archopallium (RA), the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the high vocal center (HVC) and Area X from post-hatch day (P) 15 to 120. Next, we investigated the expression dynamics of pro-apoptotic (Bid, Bad and Bax) and anti-apoptotic (Bcl-2 and Bcl-xL) genes in the aforementioned nuclei. Our results revealed that the female RA at P45 exhibited marked cell apoptosis, confirmed by low densities of Bcl-xL and Bcl-2. Both the male and female LMAN exhibited apoptotic peaks at P35 and P45, respectively, and the observed cell loss was more extensive in males. A corresponding sharp decrease in the density of Bcl-2 after P35 was observed in both sexes, and a greater density of Bid was noted at P45 in males. In addition, we observed that RA volume and the total number of BDNF-expressing cells decreased significantly after unilateral lesion of the LMAN or HVC (two areas that innervate the RA) and that greater numbers of RA-projecting cells were immunoreactive for BDNF in the LMAN than in the HVC. We reasoned that a decrease in the amount of BDNF transported via HVC afferent fibers might result in an increase in cell apoptosis in the female RA. Our data indicate that cell apoptosis resulting from different pro- and anti-apoptotic agents is involved in generating the differences between male and female song control nuclei. PMID:25938674

  11. Monitoring UVR induced damage in single cells and isolated nuclei using SR-FTIR microspectroscopy and 3D confocal Raman imaging.

    PubMed

    Lipiec, Ewelina; Bambery, Keith R; Heraud, Philip; Kwiatek, Wojciech M; McNaughton, Don; Tobin, Mark J; Vogel, Christian; Wood, Bayden R

    2014-09-01

    SR-FTIR in combination with Principal Component Analysis (PCA) was applied to investigate macromolecular changes in a population of melanocytes and their extracted nuclei induced by environmentally relevant fluxes of UVR (Ultraviolet Radiation). Living cells and isolated cellular nuclei were investigated post-irradiation for three different irradiation dosages (130, 1505, 15,052 Jm(-2) UVR, weighted) after either 24 or 48 hours of incubation. DNA conformational changes were observed in cells exposed to an artificial UVR solar-simulator source as evidenced by a shift in the DNA asymmetric phosphodiester vibration from 1236 cm(-1) to 1242 cm(-1) in the case of the exposed cells and from 1225 cm(-1) to 1242 cm(-1) for irradiated nuclei. PCA Scores plots revealed distinct clustering of spectra from irradiated cells and nuclei from non-irradiated controls in response to the range of applied UVR radiation doses. 3D Raman confocal imaging in combination with k-means cluster analysis was applied to study the effect of the UVR radiation exposure on cellular nuclei. Chemical changes associated with apoptosis were detected and included intra-nuclear lipid deposition along with chromatin condensation. The results reported here demonstrate the utility of SR-FTIR and Raman spectroscopy to probe in situ DNA damage in cell nuclei resulting from UVR exposure. These results are in agreement with the increasing body of evidence that lipid accumulation is a characteristic of aggressive cancer cells, and are involved in the production of membranes for rapid cell proliferation.

  12. Impact of the accuracy of automatic segmentation of cell nuclei clusters on classification of thyroid follicular lesions.

    PubMed

    Jung, Chanho; Kim, Changick

    2014-08-01

    Automatic segmentation of cell nuclei clusters is a key building block in systems for quantitative analysis of microscopy cell images. For that reason, it has received a great attention over the last decade, and diverse automatic approaches to segment clustered nuclei with varying levels of performance under different test conditions have been proposed in literature. To the best of our knowledge, however, so far there is no comparative study on the methods. This study is a first attempt to fill this research gap. More precisely, the purpose of this study is to present an objective performance comparison of existing state-of-the-art segmentation methods. Particularly, the impact of their accuracy on classification of thyroid follicular lesions is also investigated "quantitatively" under the same experimental condition, to evaluate the applicability of the methods. Thirteen different segmentation approaches are compared in terms of not only errors in nuclei segmentation and delineation, but also their impact on the performance of system to classify thyroid follicular lesions using different metrics (e.g., diagnostic accuracy, sensitivity, specificity, etc.). Extensive experiments have been conducted on a total of 204 digitized thyroid biopsy specimens. Our study demonstrates that significant diagnostic errors can be avoided using more advanced segmentation approaches. We believe that this comprehensive comparative study serves as a reference point and guide for developers and practitioners in choosing an appropriate automatic segmentation technique adopted for building automated systems for specifically classifying follicular thyroid lesions.

  13. Fuel cell power plant integrated systems evaluation

    NASA Astrophysics Data System (ADS)

    Bonds, T. L.; Dawes, M. H.; Schnacke, A. W.; Spradlin, L. W.

    1981-01-01

    Power plant configurations for a central station (675 MW) fueled by coal and small dispersed plan generation plants fueled by oil were defined. Capital costs and costs for electricity were evaluated for both plants. Parametric variations and the impact on plants and components are discussed. Alternate oil fueled oil fired cycles as well as several alternate coal gasifiers were examined to show effects on plant performance. The economic attractiveness of the coal fired plant was confirmed and a scenario is established for an oil fired plant with reject heat recovery. Performance for the coal fired plant exceeds the study goal of 6800 Btu/kWh. The oil fired plant performance of 7627 Btu/kWh is very close to the study goal of 7500 Btu/kWh. The development of a finite slice computer model of the carbonate fuel cell is reported and an initial parametric cell and plant performance study was performed using the model. Preliminary subsystem description sheets and plant layout arrangements are presented.

  14. Does flat epithelial atypia have rounder nuclei than columnar cell change/hyperplasia? A morphometric approach to columnar cell lesions of the breast.

    PubMed

    Yamashita, Yoshiko; Ichihara, Shu; Moritani, Suzuko; Yoon, Han-Seung; Yamaguchi, Masahiro

    2016-06-01

    Columnar cell lesions of the breast encompass columnar cell change/hyperplasia (CCC/CCH) and flat epithelial atypia (FEA). These have attracted researchers because emerging data suggest that FEA may represent the earliest histologically detectable non-obligate precursor of breast cancer. However, it is occasionally difficult to distinguish FEA from CCC/CCH because of similar histology. Although the nuclei of FEA are frequently described as relatively round compared with those of CCC/CCH, there are few morphometric studies to support this statement. The aim of this study was to provide objective data as to the nuclear shape in columnar cell lesions. As a shape descriptor, we adopted ellipticity that is defined by the formula 2b/2a, where a is the length of the long axis of the ellipse and b is the length of the short axis. Contrary to circularity, ellipticity reflects the overall configuration of an ellipse irrespective of surface irregularity. Our image analysis included generating whole slide images, extracting glandular cell nuclei, measuring nuclear ellipticity, and superimposing graded colors based on execution of results on the captured images. A total of 7917 nuclei extracted from 22 FEA images and 5010 nuclei extracted from 13 CCC/CCH images were analyzed. There was a significant difference in nuclear roundness between FEA and CCC/CCH with mean ellipticity values of 0.723 and 0.679, respectively (p < 0.001, Welch's t test). Furthermore, FEA with malignancy had significantly rounder nuclei than FEA without malignancy (p < 0.001). Our preliminary results suggest that nuclear ellipticity is a key parameter in reproducibly classifying columnar cell lesions of the breast.

  15. Morphological classification of plant cell deaths

    PubMed Central

    van Doorn, W G; Beers, E P; Dangl, J L; Franklin-Tong, V E; Gallois, P; Hara-Nishimura, I; Jones, A M; Kawai-Yamada, M; Lam, E; Mundy, J; Mur, L A J; Petersen, M; Smertenko, A; Taliansky, M; Van Breusegem, F; Wolpert, T; Woltering, E; Zhivotovsky, B; Bozhkov, P V

    2011-01-01

    Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term ‘apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during tissue and organ formation and elimination, whereas necrosis is typically found under abiotic stress. Some examples of plant PCD cannot be ascribed to either major class and are therefore classified as separate modalities. These are PCD associated with the hypersensitive response to biotrophic pathogens, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined. PMID:21494263

  16. Interplay between cell growth and cell cycle in plants.

    PubMed

    Sablowski, Robert; Carnier Dornelas, Marcelo

    2014-06-01

    The growth of organs and whole plants depends on both cell growth and cell-cycle progression, but the interaction between both processes is poorly understood. In plants, the balance between growth and cell-cycle progression requires coordinated regulation of four different processes: macromolecular synthesis (cytoplasmic growth), turgor-driven cell-wall extension, mitotic cycle, and endocycle. Potential feedbacks between these processes include a cell-size checkpoint operating before DNA synthesis and a link between DNA contents and maximum cell size. In addition, key intercellular signals and growth regulatory genes appear to target at the same time cell-cycle and cell-growth functions. For example, auxin, gibberellin, and brassinosteroid all have parallel links to cell-cycle progression (through S-phase Cyclin D-CDK and the anaphase-promoting complex) and cell-wall functions (through cell-wall extensibility or microtubule dynamics). Another intercellular signal mediated by microtubule dynamics is the mechanical stress caused by growth of interconnected cells. Superimposed on developmental controls, sugar signalling through the TOR pathway has recently emerged as a central control point linking cytoplasmic growth, cell-cycle and cell-wall functions. Recent progress in quantitative imaging and computational modelling will facilitate analysis of the multiple interconnections between plant cell growth and cell cycle and ultimately will be required for the predictive manipulation of plant growth.

  17. Catalysts of plant cell wall loosening

    PubMed Central

    Cosgrove, Daniel J.

    2016-01-01

    The growing cell wall in plants has conflicting requirements to be strong enough to withstand the high tensile forces generated by cell turgor pressure while selectively yielding to those forces to induce wall stress relaxation, leading to water uptake and polymer movements underlying cell wall expansion. In this article, I review emerging concepts of plant primary cell wall structure, the nature of wall extensibility and the action of expansins, family-9 and -12 endoglucanases, family-16 xyloglucan endotransglycosylase/hydrolase (XTH), and pectin methylesterases, and offer a critical assessment of their wall-loosening activity PMID:26918182

  18. Detection of virus-specific antigen in the nuclei or nucleoli of cells infected with Zika or Langat virus.

    PubMed

    Buckley, A; Gould, E A

    1988-08-01

    Two monoclonal antibodies (MAbs) with molecular specificities for either the viral envelope glycoprotein (MAb 541) or the non-structural NS1 glycoprotein (MAb 109) were derived using West Nile and yellow fever (YF) viruses respectively. Their antigenic reactivity with a large number of flaviviruses was tested by indirect immunofluorescence microscopy. Both produced cytoplasmic fluorescent staining patterns with the homologous virus against which they were raised. Additionally, MAb 541 reacted with two substrains of YF virus whereas MAb 109 reacted with Bussuquara, YF and Ntaya viruses. These reactions were exclusively cytoplasmic. Two unexpected patterns of fluorescent labelling were observed when the antibodies were tested with Zika and Langat viruses. MAb 541 produced fluorescent staining of the nuclei, but not the cytoplasm, of cells infected with Zika virus and MAb 109 labelled only the nucleoli of cells infected with Langat virus. Double-labelling experiments showed that the nuclear fluorescent label was confined to virus-infected cells, and antibody absorption experiments with virus-infected cell packs confirmed the virus specificity of the nuclear antigen. The unexpected presence of virus-specific antigen in the nuclei or nucleoli of Zika or Langat virus-infected cells brings into question the role of the nucleus in flavivirus replication.

  19. Improved and robust detection of cell nuclei from four dimensional fluorescence images.

    PubMed

    Bashar, Md Khayrul; Yamagata, Kazuo; Kobayashi, Tetsuya J

    2014-01-01

    Segmentation-free direct methods are quite efficient for automated nuclei extraction from high dimensional images. A few such methods do exist but most of them do not ensure algorithmic robustness to parameter and noise variations. In this research, we propose a method based on multiscale adaptive filtering for efficient and robust detection of nuclei centroids from four dimensional (4D) fluorescence images. A temporal feedback mechanism is employed between the enhancement and the initial detection steps of a typical direct method. We estimate the minimum and maximum nuclei diameters from the previous frame and feed back them as filter lengths for multiscale enhancement of the current frame. A radial intensity-gradient function is optimized at positions of initial centroids to estimate all nuclei diameters. This procedure continues for processing subsequent images in the sequence. Above mechanism thus ensures proper enhancement by automated estimation of major parameters. This brings robustness and safeguards the system against additive noises and effects from wrong parameters. Later, the method and its single-scale variant are simplified for further reduction of parameters. The proposed method is then extended for nuclei volume segmentation. The same optimization technique is applied to final centroid positions of the enhanced image and the estimated diameters are projected onto the binary candidate regions to segment nuclei volumes.Our method is finally integrated with a simple sequential tracking approach to establish nuclear trajectories in the 4D space. Experimental evaluations with five image-sequences (each having 271 3D sequential images) corresponding to five different mouse embryos show promising performances of our methods in terms of nuclear detection, segmentation, and tracking. A detail analysis with a sub-sequence of 101 3D images from an embryo reveals that the proposed method can improve the nuclei detection accuracy by 9% over the previous methods

  20. Improved and Robust Detection of Cell Nuclei from Four Dimensional Fluorescence Images

    PubMed Central

    Bashar, Md. Khayrul; Yamagata, Kazuo; Kobayashi, Tetsuya J.

    2014-01-01

    Segmentation-free direct methods are quite efficient for automated nuclei extraction from high dimensional images. A few such methods do exist but most of them do not ensure algorithmic robustness to parameter and noise variations. In this research, we propose a method based on multiscale adaptive filtering for efficient and robust detection of nuclei centroids from four dimensional (4D) fluorescence images. A temporal feedback mechanism is employed between the enhancement and the initial detection steps of a typical direct method. We estimate the minimum and maximum nuclei diameters from the previous frame and feed back them as filter lengths for multiscale enhancement of the current frame. A radial intensity-gradient function is optimized at positions of initial centroids to estimate all nuclei diameters. This procedure continues for processing subsequent images in the sequence. Above mechanism thus ensures proper enhancement by automated estimation of major parameters. This brings robustness and safeguards the system against additive noises and effects from wrong parameters. Later, the method and its single-scale variant are simplified for further reduction of parameters. The proposed method is then extended for nuclei volume segmentation. The same optimization technique is applied to final centroid positions of the enhanced image and the estimated diameters are projected onto the binary candidate regions to segment nuclei volumes.Our method is finally integrated with a simple sequential tracking approach to establish nuclear trajectories in the 4D space. Experimental evaluations with five image-sequences (each having 271 3D sequential images) corresponding to five different mouse embryos show promising performances of our methods in terms of nuclear detection, segmentation, and tracking. A detail analysis with a sub-sequence of 101 3D images from an embryo reveals that the proposed method can improve the nuclei detection accuracy by 9 over the previous methods

  1. Melanin-concentrating hormone (MCH) immunoreactivity in non-neuronal cells within the raphe nuclei and subventricular region of the brainstem of the cat.

    PubMed

    Torterolo, Pablo; Lagos, Patricia; Sampogna, Sharon; Chase, Michael H

    2008-05-19

    Neurons that utilize melanin-concentrating hormone (MCH) as a neuromodulator are localized within the postero-lateral hypothalamus and zona incerta. These neurons project diffusely throughout the central nervous system and have been implicated in critical physiological processes such as energy homeostasis and sleep. In the present report, we examined the distribution of MCH immunoreactivity in the brainstem of the cat. In addition to MCH+ axons, we found MCH-immunoreactive cells that have not been previously described either in the midbrain raphe nuclei or in the periaqueductal and periventricular areas. These MCH+ cells constituted: 1. ependymal cells that lined the fourth ventricle and aqueduct, 2. ependymal cells with long basal processes that projected deeply into the subventricular (subaqueductal) parenchyma, and, 3. cells in subventricular regions and the midbrain raphe nuclei. The MCH+ cells in the midbrain raphe nuclei were closely related to neuronal processes of serotonergic neurons. Utilizing Neu-N and GFAP immunohistochemistry we determined that the preceding MCH+ cells were neither neurons nor astrocytes. However, we found that vimentin, an intermediate-filament protein that is used as a marker for tanycytes, was specifically co-localized with MCH in these cells. We conclude that MCH is present in tanycytes whose processes innervate the midbrain raphe nuclei and adjacent subependymal regions. Because tanycytes are specialized cells that transport substances from the cerebrospinal fluid (CSF) to neural parenchyma, we suggest that MCH is absorbed from the CSF by tanycytes and subsequently liberate to act upon neurons of brainstem nuclei. PMID:18410908

  2. Regulation of cell division in higher plants

    SciTech Connect

    Jacobs, T.W.

    1992-01-01

    Cell division is arguably the most fundamental of all developmental processes. In higher plants, mitotic activity is largely confined to foci of patterned cell divisions called meristems. From these perpetually embryonic tissues arise the plant's essential organs of light capture, support, protection and reproduction. Once an adequate understanding of plant cell mitotic regulation is attained, unprecedented opportunities will ensue for analyzing and genetically controlling diverse aspects of development, including plant architecture, leaf shape, plant height, and root depth. The mitotic cycle in a variety of model eukaryotic systems in under the control of a regulatory network of striking evolutionary conservation. Homologues of the yeast cdc2 gene, its catalytic product, p34, and the cyclin regulatory subunits of the MPF complex have emerged as ubiquitous mitotic regulators. We have cloned cdc2-like and cyclin genes from pea. As in other eukaryotic model systems, p34 of Pisum sativum is a subunit of a high molecular weight complex which binds the fission yeast p13 protein and displays histone H1 kinase activity in vitro. Our primary objective in this study is to gain baseline information about the regulation of this higher plant cell division control complex in non-dividing, differentiated cells as well as in synchronous and asynchronous mitotic cells. We are investigating cdc2 and cyclin expression at the levels of protein abundance, protein phosphorylation and quaternary associations.

  3. Plant cell nucleolus as a hot spot for iron.

    PubMed

    Roschzttardtz, Hannetz; Grillet, Louis; Isaure, Marie-Pierre; Conéjéro, Geneviève; Ortega, Richard; Curie, Catherine; Mari, Stéphane

    2011-08-12

    Many central metabolic processes require iron as a cofactor and take place in specific subcellular compartments such as the mitochondrion or the chloroplast. Proper iron allocation in the different organelles is thus critical to maintain cell function and integrity. To study the dynamics of iron distribution in plant cells, we have sought to identify the different intracellular iron pools by combining three complementary imaging approaches, histochemistry, micro particle-induced x-ray emission, and synchrotron radiation micro X-ray fluorescence. Pea (Pisum sativum) embryo was used as a model in this study because of its large cell size and high iron content. Histochemical staining with ferrocyanide and diaminobenzidine (Perls/diaminobenzidine) strongly labeled a unique structure in each cell, which co-labeled with the DNA fluorescent stain DAPI, thus corresponding to the nucleus. The unexpected presence of iron in the nucleus was confirmed by elemental imaging using micro particle-induced x-ray emission. X-ray fluorescence on cryo-sectioned embryos further established that, quantitatively, the iron concentration found in the nucleus was higher than in the expected iron-rich organelles such as plastids or vacuoles. Moreover, within the nucleus, iron was particularly accumulated in a subcompartment that was identified as the nucleolus as it was shown to transiently disassemble during cell division. Taken together, our data uncover an as yet unidentified although abundant iron pool in the cell, which is located in the nuclei of healthy, actively dividing plant tissues. This result paves the way for the discovery of a novel cellular function for iron related to nucleus/nucleolus-associated processes.

  4. SYBR Green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content.

    PubMed

    Schoft, Vera K; Chumak, Nina; Bindics, János; Slusarz, Lucyna; Twell, David; Köhler, Claudia; Tamaru, Hisashi

    2015-03-01

    Key message: Purification of pollen nuclei. Germ cell epigenetics is a critical topic in plants and animals. The male gametophyte (pollen) of flowering plants is an attractive model to study genetic and epigenetic reprogramming during sexual reproduction, being composed of only two sperm cells contained within, its companion, vegetative cell. Here, we describe a simple and efficient method to purify SYBR Green-stained sperm and vegetative cell nuclei of Arabidopsis thaliana pollen using fluorescence-activated cell sorting to analyze chromatin and RNA profiles. The method obviates generating transgenic lines expressing cell-type-specific fluorescence reporters and facilitates functional genomic analysis of various mutant lines and accessions. We evaluate the purity and quality of the sorted pollen nuclei and analyze the technique's molecular basis. Our results show that both DNA and RNA contents contribute to SYBR Green-activated nucleus sorting and RNA content differences impact on the separation of sperm and vegetative cell nuclei. We demonstrate the power of the approach by sorting wild-type and polyploid mutant sperm and vegetative cell nuclei from mitotic and meiotic mutants, which is not feasible using cell-type-specific transgenic reporters. Our approach should be applicable to pollen nuclei of crop plants and possibly to cell/nucleus types and cell cycle phases of different species containing substantially different amounts of DNA and/or RNA.

  5. Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture

    NASA Astrophysics Data System (ADS)

    Ducos, Jean-Paul; Terrier, Bénédicte; Courtois, Didier

    Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco.

  6. DIRECT FUEL CELL/TURBINE POWER PLANT

    SciTech Connect

    Hossein Ghezel-Ayagh

    2004-11-01

    This report includes the progress in development of Direct FuelCell/Turbine{reg_sign} (DFC/T{reg_sign}) power plants for generation of clean power at very high efficiencies. The DFC/T power system is based on an indirectly heated gas turbine to supplement fuel cell generated power. The DFC/T power generation concept extends the high efficiency of the fuel cell by utilizing the fuel cell's byproduct heat in a Brayton cycle. Features of the DFC/T system include: electrical efficiencies of up to 75% on natural gas, 60% on coal gas, minimal emissions, simplicity in design, direct reforming internal to the fuel cell, reduced carbon dioxide release to the environment, and potential cost competitiveness with existing combined cycle power plants. The operation of sub-MW hybrid Direct FuelCell/Turbine power plant test facility with a Capstone C60 microturbine was initiated in March 2003. The inclusion of the C60 microturbine extended the range of operation of the hybrid power plant to higher current densities (higher power) than achieved in previous tests using a 30kW microturbine. The design of multi-MW DFC/T hybrid systems, approaching 75% efficiency on natural gas, was initiated. A new concept was developed based on clusters of One-MW fuel cell modules as the building blocks. System analyses were performed, including systems for near-term deployment and power plants with long-term ultra high efficiency objectives. Preliminary assessment of the fuel cell cluster concept, including power plant layout for a 14MW power plant, was performed.

  7. Plant cell shape: modulators and measurements

    PubMed Central

    Ivakov, Alexander; Persson, Staffan

    2013-01-01

    Plant cell shape, seen as an integrative output, is of considerable interest in various fields, such as cell wall research, cytoskeleton dynamics and biomechanics. In this review we summarize the current state of knowledge on cell shape formation in plants focusing on shape of simple cylindrical cells, as well as in complex multipolar cells such as leaf pavement cells and trichomes. We summarize established concepts as well as recent additions to the understanding of how cells construct cell walls of a given shape and the underlying processes. These processes include cell wall synthesis, activity of the actin and microtubule cytoskeletons, in particular their regulation by microtubule associated proteins, actin-related proteins, GTP'ases and their effectors, as well as the recently-elucidated roles of plant hormone signaling and vesicular membrane trafficking. We discuss some of the challenges in cell shape research with a particular emphasis on quantitative imaging and statistical analysis of shape in 2D and 3D, as well as novel developments in this area. Finally, we review recent examples of the use of novel imaging techniques and how they have contributed to our understanding of cell shape formation. PMID:24312104

  8. Plant expansins: diversity and interactions with plant cell walls.

    PubMed

    Cosgrove, Daniel J

    2015-06-01

    Expansins were discovered two decades ago as cell wall proteins that mediate acid-induced growth by catalyzing loosening of plant cell walls without lysis of wall polymers. In the interim our understanding of expansins has gotten more complex through bioinformatic analysis of expansin distribution and evolution, as well as through expression analysis, dissection of the upstream transcription factors regulating expression, and identification of additional classes of expansin by sequence and structural similarities. Molecular analyses of expansins from bacteria have identified residues essential for wall loosening activity and clarified the bifunctional nature of expansin binding to complex cell walls. Transgenic modulation of expansin expression modifies growth and stress physiology of plants, but not always in predictable or even understandable ways.

  9. Plant expansins: diversity and interactions with plant cell walls

    PubMed Central

    Cosgrove, Daniel J.

    2015-01-01

    Expansins were discovered two decades ago as cell wall proteins that mediate acid-induced growth by catalyzing loosening of plant cell walls without lysis of wall polymers. In the interim our understanding of expansins has gotten more complex through bioinformatic analysis of expansin distribution and evolution, as well as through expression analysis, dissection of the upstream transcription factors regulating expression, and identification of additional classes of expansin by sequence and structural similarities. Molecular analyses of expansins from bacteria have identified residues essential for wall loosening activity and clarified the bifunctional nature of expansin binding to complex cell walls. Transgenic modulation of expansin expression modifies growth and stress physiology of plants, but not always in predictable and even understandable ways. PMID:26057089

  10. Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

    SciTech Connect

    Park, Su Hyun; Park, Tae Jun; Lim, In Kyoung

    2011-04-15

    We have previously reported that G-actin accumulation in nuclei is a universal phenomenon of cellular senescence. By employing primary culture of human diploid fibroblast (HDF) and stress-induced premature senescence (SIPS), we explored whether the failure of actin export to cytoplasm is responsible for actin accumulation in nuclei of senescent cells. Expression of exportin 6 (Exp6) and small G-protein, Ran, was significantly reduced in the replicative senescence, but not yet in SIPS, whereas nuclear import of actin by cofilin was already increased in SIPS. After treatment of young HDF cells with H{sub 2}O{sub 2}, rapid reduction of nuclear RanGTP was observed along with cytoplasmic increase of RanGDP. Furthermore, significantly reduced interaction of Exp6 with RanGTP was found by GST-Exp6 pull-down analysis. Failure of RanGTP restoration was accompanied with inhibition of ATP synthesis and NTF2 sequestration in the nuclei along with accordant change of senescence morphology. Indeed, knockdown of Exp6 expression significantly increased actin molecule in the nuclei of young HDF cells. Therefore, actin accumulation in nuclei of senescent cells is most likely due to the failure of RanGTP restoration with ATP deficiency and NTF2 accumulation in nuclei, which result in the decrease of actin export via Exp6 inactivation, in addition to actin import by cofilin activation.

  11. Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells.

    PubMed

    Park, Su Hyun; Park, Tae Jun; Lim, In Kyoung

    2011-04-15

    We have previously reported that G-actin accumulation in nuclei is a universal phenomenon of cellular senescence. By employing primary culture of human diploid fibroblast (HDF) and stress-induced premature senescence (SIPS), we explored whether the failure of actin export to cytoplasm is responsible for actin accumulation in nuclei of senescent cells. Expression of exportin 6 (Exp6) and small G-protein, Ran, was significantly reduced in the replicative senescence, but not yet in SIPS, whereas nuclear import of actin by cofilin was already increased in SIPS. After treatment of young HDF cells with H(2)O(2), rapid reduction of nuclear RanGTP was observed along with cytoplasmic increase of RanGDP. Furthermore, significantly reduced interaction of Exp6 with RanGTP was found by GST-Exp6 pull-down analysis. Failure of RanGTP restoration was accompanied with inhibition of ATP synthesis and NTF2 sequestration in the nuclei along with accordant change of senescence morphology. Indeed, knockdown of Exp6 expression significantly increased actin molecule in the nuclei of young HDF cells. Therefore, actin accumulation in nuclei of senescent cells is most likely due to the failure of RanGTP restoration with ATP deficiency and NTF2 accumulation in nuclei, which result in the decrease of actin export via Exp6 inactivation, in addition to actin import by cofilin activation.

  12. Difference of the Nuclear Green Light Intensity between Papillary Carcinoma Cells Showing Clear Nuclei and Non-neoplastic Follicular Epithelia in Papillary Thyroid Carcinoma

    PubMed Central

    Lee, Hyekyung; Baek, Tae Hwa; Park, Meeja; Lee, Seung Yun; Son, Hyun Jin; Kang, Dong Wook; Kim, Joo Heon; Kim, Soo Young

    2016-01-01

    Background There is subjective disagreement regarding nuclear clearing in papillary thyroid carcinoma. In this study, using digital instruments, we were able to quantify many ambiguous pathologic features and use numeric data to express our findings. Methods We examined 30 papillary thyroid carcinomas. For each case, we selected representative cancer cells showing clear nuclei and surrounding non-neoplastic follicular epithelial cells and evaluated objective values of green light intensity (GLI) for quantitative analysis of nuclear clearing in papillary thyroid carcinoma. Results From 16,274 GLI values from 600 cancer cell nuclei and 13,752 GLI values from 596 non-neoplastic follicular epithelial nuclei, we found a high correlation of 94.9% between GLI and clear nuclei. GLI between the cancer group showing clear nuclei and non-neoplastic follicular epithelia was statistically significant. The overall average level of GLI in the cancer group was over two times higher than the non-neoplastic group despite a wide range of GLI. On a polygonal line graph, there was a fluctuating unique difference between both the cancer and non-neoplastic groups in each patient, which was comparable to the microscopic findings. Conclusions Nuclear GLI could be a useful factor for discriminating between carcinoma cells showing clear nuclei and non-neoplastic follicular epithelia in papillary thyroid carcinoma. PMID:27550048

  13. Distinguishing nuclei-specific benzo[a]pyrene-induced effects from whole-cell alterations in MCF-7 cells using Fourier-transform infrared spectroscopy.

    PubMed

    Obinaju, Blessing E; Fullwood, Nigel J; Martin, Francis L

    2015-09-01

    Exposure to chemicals such as benzo[a]pyrene (B[a]P) can generate intracellular toxic mechanisms. Fourier-transform infrared (FTIR) spectroscopy is a novel approach that allows the non-destructive analysis of underlying chemical bond alterations in patho-physiological processes. This study set out to examine whether B[a]P-induced whole cell alterations could be distinguished from effects on nuclei of exposed cells. Using attenuated total reflection FTIR (ATR-FTIR) spectroscopy, alterations in nuclei isolated from B[a]P-treated MCF-7 cells concentrated either in G0/G1- or S-phase were observed. B[a]P-induced effects in whole-cells included alterations to lipids, DNA and protein spectral regions. Absorbance areas for protein and DNA/RNA regions in B[a]P-treated whole cells differed significantly (P<0.0001) from vehicle controls and these observations correlated with alterations noted in isolated nuclei. Our findings provide evidence that FTIR spectroscopy has the ability to identify specific chemical-induced alterations.

  14. Quantitative Aspects of Cyclosis in Plant Cells.

    ERIC Educational Resources Information Center

    Howells, K. F.; Fell, D. A.

    1979-01-01

    Describes an exercise which is currently used in a course in cell physiology at Oxford Polytechnic in England. This exercise can give students some idea of the molecular events involved in bringing about movement of chloroplasts (and other organelles) in plant cells. (HM)

  15. Identification of c-Yes expression in the nuclei of hepatocellular carcinoma cells: involvement in the early stages of hepatocarcinogenesis.

    PubMed

    Nonomura, Takako; Masaki, Tsutomu; Morishita, Asahiro; Jian, Gong; Uchida, Naohito; Himoto, Takashi; Izuishi, Kunihiko; Iwama, Hisakazu; Yoshiji, Hitoshi; Watanabe, Seishiro; Kurokohchi, Kazutaka; Kuriyama, Shigeki

    2007-01-01

    It is thought that the subcellular distribution of Src-family tyrosine kinases, including c-Yes binding to the cellular membrane, is membranous and/or cytoplasmic. c-Yes protein tyrosine kinase is known to be related to malignant transformation. However, the expression patterns of c-Yes in hepatocellular carcinoma (HCC) remains unknown. In the present study, we report that c-Yes is expressed not only in the membrane and cytoplasm, but also in the nuclei of cancer cells in some human HCC tissues and in a human HCC cell line. We examined the expression and localization of c-Yes in human HCC cell lines (HLE, HLF, PLC/PRF/5 and Hep 3B) by Western blotting and immunohistochemical analyses; we also examined the expression of c-Yes by immunohistochemistry and Western blotting in the tissues of various liver diseases, including 39 samples from HCC patients. We used an antibody array to detect proteins that bind to nuclear c-Yes in PLC/PRF/5 cell line. c-Yes was found to be expressed in the membranes and cytoplasm of HLE, HLF and Hep 3B HCC cells; it was also detected in the nuclei in addition to the membranes and cytoplasm of PLC/PRF/5 HCC cells. HCC with nuclear c-Yes was detected in 5 of 39 cases (13.0%), and nuclear c-Yes expression was not detected in normal, chronic hepatitis or cirrhotic livers. All HCCs with nuclear c-Yes expression were well-differentiated, small tumors at the early stages. In the PLC/PRF/5 cell line, the nuclear localization of c-Yes with cyclin-dependent kinase 1 was confirmed by a protein antibody array. In conclusion, nuclear c-Yes expression was found in cancer cells at the early stages of hepatocarcinogenesis, suggesting that nucleus-located c-Yes may be a useful marker to detect early-stage HCC.

  16. Papillary renal cell carcinoma with oncocytic cells and nonoverlapping low grade nuclei: expanding the morphologic spectrum with emphasis on clinicopathologic, immunohistochemical and molecular features.

    PubMed

    Kunju, Lakshmi P; Wojno, Kirk; Wolf, J Stuart; Cheng, Liang; Shah, Rajal B

    2008-01-01

    Papillary renal cell carcinoma (PRCC), a morphologically and genetically distinct subtype of RCC, is morphologically separated into 2 subtypes, type 1 and 2, for prognostic purposes. Type 1 PRCC (single layer of small cells, scant pale cytoplasm) is more common and has a favorable prognosis compared with type 2 (pseudostratified high-grade nuclei, abundant eosinophilic/oncocytic cytoplasm). We report the clinicopathologic, immunohistochemical, and molecular data of 7 adult papillary tumors with morphological features distinct from type 1 or 2 PRCC. All tumors demonstrated predominant papillary architecture, lined by cells with oncocytic cytoplasm, and nonoverlapping low Fuhrman grade nuclei (1 or 2). Foamy macrophages were noted in 2 of 7 tumors. No case demonstrated necrosis or psammoma bodies. Most tumors (6/7) were small (mean size, 2.0 cm; range, 0.8-5.7 cm) and limited to the kidney. No tumor recurrence or metastasis was identified (median follow-up, 22 months). All tumors demonstrated trisomy for 7 and 17 by fluorescence in situ hybridization analysis and uniform CK 7, CD10, and alpha-methylacyl-coenzyme A racemase expression, characteristic of PRCC. These results suggest that these tumors are distinct from type 1 (owing to oncocytic cells) and type 2 (owing to low-grade nonstratified nuclei, low stage, and good outcome). Awareness of this favorable spectrum of PRCC is important to avoid its potential misinterpretation as an aggressive type 2 PRCC (owing to oncocytic cells) or rarely as an oncocytoma (owing to oncocytic cells and low-grade nuclei). Morphologic spectrum of these PRCCs emphasizes that the future prognostic model of PRCC may need to be based primarily on the nuclear characteristics, irrespective of the cytoplasmic features.

  17. In Vitro plant cell growth in microgravity and on clinostat

    NASA Astrophysics Data System (ADS)

    Laurinavicius, R.; Kenstaviciene, P.; Rupainiene, O.; Necitailo, G.

    1994-08-01

    For the study of gravity's role in the processes of plant cell differentiation in vitro, a model ``seed-seedling-callus'' has been used. Experiments were carried out on board the orbital stations Salyut-7 and Mir as well as on clinostat. They lasted from 18 to 72 days. It was determined that the exclusion of a one-sided action of gravity vector by means of clinostat and spaceflight conditions does not impede the formation and growth of callus tissue; however, at cell and subcellular levels structural and functional changes do take place. No significant changes were observed either on clinostat or in space concerning the accumulation of fresh biomass, while the percentage of dry material in space is lower than in control. Both in microgravity (MG) and in control, even after 72 days of growth, cells with a normally developed ultrastructure are present. In space, however, callus tissue more often contains cells in which the cross-section area of a cells, a nuclei and of mitochondria are smaller and the vacuole area - bigger than in controls. In microgravity a considerable decrease in the number of starch-containing cells and a reduction in the mean area of starch grains in amyloplasts is observed. In space the amount of soluble proteins in callus tissue is 1.5 times greater than in control. However, no differences were observed in fractions when separated by the SDS-PAGE method. In microgravity the changes in cell wall material components was noted. In the space-formed callus changes in the concentration of ions K, Na, Mg, Ca and P were observed. However, the direction of these changes depends on the age of callus. Discussed are the possible reasons for modification of morphological and metabolic parameters of callus cells when grown under changed gravity conditions.

  18. Plant single-cell and single-cell-type metabolomics.

    PubMed

    Misra, Biswapriya B; Assmann, Sarah M; Chen, Sixue

    2014-10-01

    In conjunction with genomics, transcriptomics, and proteomics, plant metabolomics is providing large data sets that are paving the way towards a comprehensive and holistic understanding of plant growth, development, defense, and productivity. However, dilution effects from organ- and tissue-based sampling of metabolomes have limited our understanding of the intricate regulation of metabolic pathways and networks at the cellular level. Recent advances in metabolomics methodologies, along with the post-genomic expansion of bioinformatics knowledge and functional genomics tools, have allowed the gathering of enriched information on individual cells and single cell types. Here we review progress, current status, opportunities, and challenges presented by single cell-based metabolomics research in plants.

  19. Phospholipids of liver cell nuclei during hibernation of Yakutian ground squirrel.

    PubMed

    Lakhina, A A; Markevich, L N; Zakharova, N M; Afanasyev, V N; Kolomiytseva, I K; Fesenko, E E

    2016-07-01

    In hibernating Yakutian ground squirrels S. undulatus, the content of total phospholipids in the nuclei of liver increased by 40% compared to that in animals in summer. In torpid state, the amount of sphingomyelin increased almost 8 times; phosphatidylserine, 7 times; and cardiolipin, 4 times. In active "winter" ground squirrels, the amount of sphingomyelin, phosphatidylserine, and cardiolipin decreased compared to the hibernating individuals but remained high compared to the "summer" ones. The torpor state did not affect the amount of lysophosphatidylcholine and phosphatidylinositol.

  20. Microtubule networks for plant cell division.

    PubMed

    de Keijzer, Jeroen; Mulder, Bela M; Janson, Marcel E

    2014-09-01

    During cytokinesis the cytoplasm of a cell is divided to form two daughter cells. In animal cells, the existing plasma membrane is first constricted and then abscised to generate two individual plasma membranes. Plant cells on the other hand divide by forming an interior dividing wall, the so-called cell plate, which is constructed by localized deposition of membrane and cell wall material. Construction starts in the centre of the cell at the locus of the mitotic spindle and continues radially towards the existing plasma membrane. Finally the membrane of the cell plate and plasma membrane fuse to form two individual plasma membranes. Two microtubule-based cytoskeletal networks, the phragmoplast and the pre-prophase band (PPB), jointly control cytokinesis in plants. The bipolar microtubule array of the phragmoplast regulates cell plate deposition towards a cortical position that is templated by the ring-shaped microtubule array of the PPB. In contrast to most animal cells, plants do not use centrosomes as foci of microtubule growth initiation. Instead, plant microtubule networks are striking examples of self-organizing systems that emerge from physically constrained interactions of dispersed microtubules. Here we will discuss how microtubule-based activities including growth, shrinkage, severing, sliding, nucleation and bundling interrelate to jointly generate the required ordered structures. Evidence mounts that adapter proteins sense the local geometry of microtubules to locally modulate the activity of proteins involved in microtubule growth regulation and severing. Many of the proteins and mechanisms involved have roles in other microtubule assemblies as well, bestowing broader relevance to insights gained from plants. PMID:25136380

  1. Development of interspecies cloned embryos reconstructed with rabbit (Oryctolagus cuniculus) oocytes and cynomolgus monkey (Macaca fascicularis) fibroblast cell nuclei.

    PubMed

    Yamochi, Takayuki; Kida, Yuta; Oh, Noriyoshi; Ohta, Sei; Amano, Tomoko; Anzai, Masayuki; Kato, Hiromi; Kishigami, Satoshi; Mitani, Tasuku; Matsumoto, Kazuya; Saeki, Kazuhiro; Takenoshita, Makoto; Iritani, Akira; Hosoi, Yoshihiko

    2013-11-01

    Interspecies somatic cell nuclear transfer (ISCNT) has been proposed as a technique to produce cloned offspring of endangered species as well as to investigate nucleus-cytoplasm interactions in mammalian embryo. However, it is still not known which embryo culture medium is optimal for ISCNT embryos for the nuclear donor or the oocyte recipient. We assessed the effects of the culture medium on the developmental competence of the ISCNT embryos by introducing cynomolgus monkey (Macaca fascicularis) fibroblast nuclei into enucleated rabbit (Oryctolagus cuniculus) oocytes (monkey-rabbit embryo). The monkey-rabbit ISCNT embryos that were cultured in mCMRL-1066 developed to the blastocyst stage, although all monkey-rabbit ISCNT embryos cultured in M199 were arrested by the 4-cell stage. When monkey-rabbit ISCNT and rabbit-rabbit somatic cell nuclear transfer (SCNT) embryos were cultured in mCMRL-1066, the blastocyst cell numbers of the monkey-rabbit ISCNT embryos corresponded to the cell numbers of the control rabbit-rabbit SCNT embryos, which were produced from a rabbit fibroblast nucleus and an enucleated rabbit oocyte. In addition, the presence of mitochondria, which were introduced with monkey fibroblasts into rabbit recipient cytoplasm, was confirmed up to the blastocyst stage by polymerase chain reaction (PCR). This study demonstrated that: (1) rabbit oocytes can reprogramme cynomolgus monkey somatic cell nuclei, and support preimplantation development; (2) monkey-rabbit ISCNT embryos developed well in monkey culture medium at early embryonic developmental stages; (3) the cell number of monkey-rabbit ISCNT embryos is similar to that of rabbit-rabbit SCNT embryos; and (4) the mitochondrial fate of monkey-rabbit ISCNT embryos is heteroplasmic from the time just after injection to the blastocyst stage that has roots in both rabbit oocytes and monkey fibroblasts.

  2. Monoclonal antibodies against plant cell wall polysaccharides

    SciTech Connect

    Hahn, M.G.; Bucheli, E.; Darvill, A.; Albersheim, P. )

    1989-04-01

    Monoclonal antibodies (McAbs) are useful tools to probe the structure of plant cell wall polysaccharides and to localize these polysaccharides in plant cells and tissues. Murine McAbs were generated against the pectic polysaccharide, rhamnogalacturonan I (RG-I), isolated from suspension-cultured sycamore cells. The McAbs that were obtained were grouped into three classes based upon their reactivities with a variety of plant polysaccharides and membrane glycoproteins. Eleven McAbs (Class I) recognize epitope(s) that appear to be immunodominant and are found in RG-I from sycamore and maize, citrus pectin, polygalacturonic acid, and membrane glycoproteins from suspension-cultured cells of sycamore, maize, tobacco, parsley, and soybean. A second group of five McAbs (Class II) recognize epitope(s) present in sycamore RG-I, but do not bind to any of the other polysaccharides or glycoproteins recognized by Class I. Lastly, one McAb (Class III) reacts with sycamore RG-I, sycamore and tamarind xyloglucan, and sycamore and rice glucuronoarabinoxylan, but does not bind to maize RG-I, polygalacturonic acid or the plant membrane glycoproteins recognized by Class I. McAbs in Classes II and III are likely to be useful in studies of the structure, biosynthesis and localization of plant cell wall polysaccharides.

  3. Laser-mediated perforation of plant cells

    NASA Astrophysics Data System (ADS)

    Wehner, Martin; Jacobs, Philipp; Esser, Dominik; Schinkel, Helga; Schillberg, Stefan

    2007-07-01

    The functional analysis of plant cells at the cellular and subcellular levels requires novel technologies for the directed manipulation of individual cells. Lasers are increasingly exploited for the manipulation of plant cells, enabling the study of biological processes on a subcellular scale including transformation to generate genetically modified plants. In our setup either a picosecond laser operating at 1064 nm wavelength or a continuous wave laser diode emitting at 405 nm are coupled into an inverse microscope. The beams are focused to a spot size of about 1.5 μm and the tobacco cell protoplasts are irradiated. Optoporation is achieved when targeting the laser focal spot at the outermost edge of the plasma membrane. In case of the picosecond laser a single pulse with energy of about 0.4 μJ was sufficient to perforate the plasma membrane enabling the uptake of dye or DNA from the surrounding medium into the cytosol. When the ultraviolet laser diode at a power level of 17 mW is employed an irradiation time of 200 - 500 milliseconds is necessary to enable the uptake of macromolecules. In the presence of an EYFP encoding plasmid with a C-terminal peroxisomal signal sequence in the surrounding medium transient transformation of tobacco protoplasts could be achieved in up to 2% of the optoporated cells. Single cell perforation using this novel optoporation method shows that isolated plant cells can be permeabilized without direct manipulation. This is a valuable procedure for cell-specific applications, particularly where the import of specific molecules into plant cells is required for functional analysis.

  4. Expression of bacterial genes in plant cells.

    PubMed Central

    Fraley, R T; Rogers, S G; Horsch, R B; Sanders, P R; Flick, J S; Adams, S P; Bittner, M L; Brand, L A; Fink, C L; Fry, J S; Galluppi, G R; Goldberg, S B; Hoffmann, N L; Woo, S C

    1983-01-01

    Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed. Images PMID:6308651

  5. DIRECT FUEL CELL/TURBINE POWER PLANT

    SciTech Connect

    Hossein Ghezel-Ayagh

    2003-05-27

    The subMW hybrid DFC/T power plant facility was upgraded with a Capstone C60 microturbine and a state-of-the-art full size fuel cell stack. The integration of the larger microturbine extended the capability of the hybrid power plant to operate at high power ratings with a single gas turbine without the need for supplementary air. The objectives of this phase of subMW hybrid power plant tests are to support the development of process and control and to provide the insight for the design of the packaged subMW hybrid demonstration units. The development of the ultra high efficiency multi-MW power plants was focused on the design of 40 MW power plants with efficiencies approaching 75% (LHV of natural gas). The design efforts included thermodynamic cycle analysis of key gas turbine parameters such as compression ratio.

  6. UV-Induced cell death in plants.

    PubMed

    Nawkar, Ganesh M; Maibam, Punyakishore; Park, Jung Hoon; Sahi, Vaidurya Pratap; Lee, Sang Yeol; Kang, Chang Ho

    2013-01-14

    Plants are photosynthetic organisms that depend on sunlight for energy. Plants respond to light through different photoreceptors and show photomorphogenic development. Apart from Photosynthetically Active Radiation (PAR; 400-700 nm), plants are exposed to UV light, which is comprised of UV-C (below 280 nm), UV-B (280-320 nm) and UV-A (320-390 nm). The atmospheric ozone layer protects UV-C radiation from reaching earth while the UVR8 protein acts as a receptor for UV-B radiation. Low levels of UV-B exposure initiate signaling through UVR8 and induce secondary metabolite genes involved in protection against UV while higher dosages are very detrimental to plants. It has also been reported that genes involved in MAPK cascade help the plant in providing tolerance against UV radiation. The important targets of UV radiation in plant cells are DNA, lipids and proteins and also vital processes such as photosynthesis. Recent studies showed that, in response to UV radiation, mitochondria and chloroplasts produce a reactive oxygen species (ROS). Arabidopsis metacaspase-8 (AtMC8) is induced in response to oxidative stress caused by ROS, which acts downstream of the radical induced cell death (AtRCD1) gene making plants vulnerable to cell death. The studies on salicylic and jasmonic acid signaling mutants revealed that SA and JA regulate the ROS level and antagonize ROS mediated cell death. Recently, molecular studies have revealed genes involved in response to UV exposure, with respect to programmed cell death (PCD).

  7. Osmosis in Poisoned Plant Cells.

    ERIC Educational Resources Information Center

    Tatina, Robert

    1998-01-01

    Describes two simple laboratory exercises that allow students to test hypotheses concerning the requirement of cell energy for osmosis. The first exercise involves osmotically-caused changes in the length of potato tubers and requires detailed quantitative observations. The second exercise involves osmotically-caused changes in turgor of Elodea…

  8. RNA synthesis in isolated nuclei of lactating mammary cells in presence of unmodified and mercury-labeled CTP.

    PubMed Central

    Ganguly, R; Banerjee, M R

    1978-01-01

    Isolated nuclei of lactating mouse mammary gland were capable of supporting DNA-dependent RNA synthesis in vitro in presence of unmodified and mercurated CTP (Hg-CTP) at high ionic condition at 25 degrees C. In presence of unmodified CTP, [3H]UMP incorporation into RNA increased linearly upto 180 min. The kinetic pattern of the reaction and the rate of RNA synthesis were essentially similar when CTP was replaced by Hg-CTP. Both in unmodified and Hg-CTP containing reactions, 70-80% of RNA synthesis was inhibited by alpha-amanitin. Presence of poly(A) in a small portion of the in vitro synthesized messenger-like RNA was detectable by oligo(dT) cellulose chromatography. Both poly(A)+ and poly(A)- RNAs sedimented with a clear peak around 15S region in a formamide-sucrose denaturing gradient. The Hg-RNA after separation from endogenous nuclear RNA by SH-agarose affinity column chromatography also sedimented around 15S region in a formamide-sucrose gradient. The Hg-RNA synthesized in the isolated mammary cell nuclei in vitro should now permit monitoring hormonal regulation of specific gene (casein) transcription in the mammary cells by molecular hybridization of the Hg-RNA with cDNA to casein mRNA. PMID:724523

  9. Calcium signaling in plant cells in microgravity

    NASA Astrophysics Data System (ADS)

    Kordyum, E.

    Changes in the intracellular Ca 2 + concentration in altered gravity (microgravity and clinostating) evidence that Ca2 + signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus - response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in eighties, a review highlighting the performed research and the possible significance of such Ca 2 + changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumably specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca 2 + ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca 2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravis ensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane

  10. Identification and characterization of a haploid germ cell-specific nuclear protein kinase (Haspin) in spermatid nuclei and its effects on somatic cells.

    PubMed

    Tanaka, H; Yoshimura, Y; Nozaki, M; Yomogida, K; Tsuchida, J; Tosaka, Y; Habu, T; Nakanishi, T; Okada, M; Nojima, H; Nishimune, Y

    1999-06-11

    We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I-III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (for haploid germ cell-specific nuclear protein kinase). Western blot analysis showed that specific antibodies recognized a protein of Mr 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G1, resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately one-half of wild-type Haspin was able to bind to a DNA-cellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells. PMID:10358056

  11. Phospholipids of liver cell nuclei during hibernation of Yakutian ground squirrel.

    PubMed

    Lakhina, A A; Markevich, L N; Zakharova, N M; Afanasyev, V N; Kolomiytseva, I K; Fesenko, E E

    2016-07-01

    In hibernating Yakutian ground squirrels S. undulatus, the content of total phospholipids in the nuclei of liver increased by 40% compared to that in animals in summer. In torpid state, the amount of sphingomyelin increased almost 8 times; phosphatidylserine, 7 times; and cardiolipin, 4 times. In active "winter" ground squirrels, the amount of sphingomyelin, phosphatidylserine, and cardiolipin decreased compared to the hibernating individuals but remained high compared to the "summer" ones. The torpor state did not affect the amount of lysophosphatidylcholine and phosphatidylinositol. PMID:27599501

  12. Interactive algorithms for rapid enumeration of hybridization signals in individual whole-cell nuclei inside intact-tissue specimens

    NASA Astrophysics Data System (ADS)

    Lockett, Stephen J.; Thompson, Curtis T.; Mullikin, James C.; Sudar, Damir; Khavari, R.; Hyun, William; Pinkel, Daniel; Gray, Joe W.

    1995-03-01

    Fluorescence in situ hybridization (FISH) is useful for analyzing specific nucleic acid sequences in individual cells. Its application to tissue sections has been limited however because of the difficulties of performing the hybridization and analysis in sections that are thick enough to contain intact nuclei. Recent improvements in FISH permit hybridization with chromosome-specific, centromeric probes throughout 20 micrometers formalin fixed, paraffin- embedded sections, which do contain many intact nuclei. This paper describes software to facilitate analysis of these 3D hybridizations. We have developed two algorithms for analyzing 3D, confocal images of thick sections. One displays 2D, maximum-intensity, projection images through the original 3D image at different angles. When projections are viewed sequentially, the 3D image appears semi-transparent and rotates. The second algorithm allows interactive enumeration of FISH signals. Each signal is marked by the analyst. Then, for each pair of marked signals, a 2D slice image along the line connecting both marked signals and parallel to the z (depth) axis is displayed. From this slice, the analyst decides if the signals are in the same or different nuclei, or if the signals should be rejected because they are in a nucleus truncated by the upper or lower surface of the section. After consideration of all pairs of signals, the algorithm produces a map of the tissue section showing the numbers of signals in each of the intact nucleus. The algorithms enable analysis of small, premalignant and early malignant lesions and infiltrative lesions that cannot be analyzed by other molecular techniques and permit the direct correlation of FISH information with histology/cytology.

  13. Strength and timing of motor responses mediated by rebound firing in the cerebellar nuclei after Purkinje cell activation.

    PubMed

    Witter, Laurens; Canto, Cathrin B; Hoogland, Tycho M; de Gruijl, Jornt R; De Zeeuw, Chris I

    2013-01-01

    The cerebellum refines the accuracy and timing of motor performance. How it encodes information to perform these functions is a major topic of interest. We performed whole cell and extracellular recordings of Purkinje cells (PCs) and cerebellar nuclei neurons (CNs) in vivo, while activating PCs with light in transgenic mice. We show for the first time that graded activation of PCs translates into proportional CN inhibition and induces rebound activity in CNs, which is followed by graded motor contractions timed to the cessation of the stimulus. Moreover, activation of PC ensembles led to disinhibition of climbing fiber activity, which coincided with rebound activity in CNs. Our data indicate that cessation of concerted activity in ensembles of PCs can regulate both timing and strength of movements via control of rebound activity in CNs.

  14. Fixed nuclei as alternative template of BIOMED-2 multiplex polymerase chain reaction for immunoglobulin gene clonality testing in B-cell malignancies.

    PubMed

    Tang, Yuan; Chen, Jie; Wang, Jianchao; Zheng, Ke; Liao, Dianying; Liao, Xiaomei; Liu, Weiping; Wang, Lin

    2015-01-01

    Evaluation of immunoglobulin (Ig) gene rearrangements with BIOMED-2 multiplex PCR has become a standard detection of clonality in mature B cell malignancies. Conventionally, this method is relatively labor-intensive and time-consuming, as it requires DNA isolation from bone marrow aspirates (BM) or peripheral blood (PB) in patients with BM or PB involvement. On the other hand, fluorescence in situ hybridization (FISH) is routinely used as genetic screening in B cell malignancies, but the surplus fixed nuclei initially prepared for FISH usually turn useless afterwards. We sought to use these surplus nuclei after FISH as a template to perform PCR-based Ig gene clonality testing. Templates of 12 patients with mature B cell malignancies, which consisted of both DNA isolated with commercial DNA isolation kit from fresh BM or PB (DNA group) and the fixed nuclei initially prepared for FISH (nuclei group) from the same individuals, were subjected to PCR with BIOMED-2 primer sets for immunoglobulin heavy chain and kappa light chain under recommended conditions. Our result, for the first time, showed a high consistency between the two groups in detecting B cell clonality, which indicates that nuclei for FISH can function as a reliable template comparable to fresh tissue-isolated DNA in PCR based Ig clonality testing. This offers a simple, rapid and more economical alternative to standard Ig testing based on regular DNA.

  15. Direct FuelCell/Turbine Power Plant

    SciTech Connect

    Hossein Ghezel-Ayagh

    2008-09-30

    This report summarizes the progress made in development of Direct FuelCell/Turbine (DFC/T{reg_sign}) power plants for generation of clean power at very high efficiencies. The DFC/T system employs an indirectly heated Turbine Generator to supplement fuel cell generated power. The concept extends the high efficiency of the fuel cell by utilizing the fuel cell's byproduct heat in a Brayton cycle. Features of the DFC/T system include: electrical efficiencies of up to 75% on natural gas, minimal emissions, reduced carbon dioxide release to the environment, simplicity in design, direct reforming internal to the fuel cell, and potential cost competitiveness with existing combined cycle power plants. Proof-of-concept tests using a sub-MW-class DFC/T power plant at FuelCell Energy's (FCE) Danbury facility were conducted to validate the feasibility of the concept and to measure its potential for electric power production. A 400 kW-class power plant test facility was designed and retrofitted to conduct the tests. The initial series of tests involved integration of a full-size (250 kW) Direct FuelCell stack with a 30 kW Capstone microturbine. The operational aspects of the hybrid system in relation to the integration of the microturbine with the fuel cell, process flow and thermal balances, and control strategies for power cycling of the system, were investigated. A subsequent series of tests included operation of the sub-MW Direct FuelCell/Turbine power plant with a Capstone C60 microturbine. The C60 microturbine extended the range of operation of the hybrid power plant to higher current densities (higher power) than achieved in initial tests using the 30kW microturbine. The proof-of-concept test results confirmed the stability and controllability of operating a fullsize (250 kW) fuel cell stack in combination with a microturbine. Thermal management of the system was confirmed and power plant operation, using the microturbine as the only source of fresh air supply to the

  16. Structure of Plant Cell Walls

    PubMed Central

    Weinstein, Larry; Albersheim, Peter

    1979-01-01

    Wild type Bacillus subtilis, when grown on beet araban, secretes into its culture medium an endo-arabanase and two arabinosidases. An alternate procedure to one previously described (Kaji A, T Saheki 1975 Biochim Biophys Acta 410: 354-360) has been developed for the purification of the endo-arabanase. The purified endo-arabanase is shown to be homogeneous by sodium dodecyl sulfate-urea disc gel electrophoresis (molecular weight ≃ 32,000) and by isoelectric focusing (pI = 9.3). The endo-arabanase, acting on a branched araban substrate, has maximal activity at pH 6.0 and preferentially cleaves 5-linked arabinosyl residues. One of the arabinosidases (molecular weight ≃ 65,000, pI = 5.3) has been purified to the point that it contains only one quantitatively minor contaminant, as shown by sodium dodecyl sulfate-urea disc gel electrophoresis and isoelectric focusing. The purified arabinosidase, acting on p-nitrophenyl-α-l-arabinofuranoside, has maximal activity at pH 6.5, and, when acting on a branched araban substrate, preferentially attacks nonreducing terminal arabinosyl residues linked to the 2 or 3 position of other arabinosyl residues. Neither of the two purified enzymes is capable of hydrolyzing a variety of carbohydrate substrates which lack arabinosidic linkages. The purified endo-arabinase is shown to be capable of releasing arabinosyl oligomers from the walls of suspension-cultured sycamore cells, thereby suggesting its usefulness as a probe in studying the structure of the araban component of primary cell walls. PMID:16660741

  17. Cell-to-cell movement of plastids in plants

    PubMed Central

    Thyssen, Gregory; Svab, Zora; Maliga, Pal

    2012-01-01

    Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications. PMID:22308369

  18. Quantification of plant cell coupling with live-cell microscopy.

    PubMed

    Liesche, Johannes; Schulz, Alexander

    2015-01-01

    Movement of nutrients and signaling compounds from cell to cell is an essential process for plant growth and development. To understand processes such as carbon allocation, cell communication, and reaction to pathogen attack it is important to know a specific molecule's capacity to pass a specific cell wall interface. Transport through plasmodesmata, the cell wall channels that directly connect plant cells, is regulated not only by a fixed size exclusion limit, but also by physiological and pathological adaptation. The noninvasive approach described here offers the possibility of precisely determining the plasmodesmata-mediated cell wall permeability for small molecules in living cells.The method is based on photoactivation of the fluorescent tracer caged fluorescein. Non-fluorescent caged fluorescein is applied to a target tissue, where it is taken up passively into all cells. Imaged by confocal microscopy, loaded tracer is activated by UV illumination in a target cell and its spread to neighboring cells monitored. When combined with high-speed acquisition by resonant scanning or spinning disc confocal microscopy, the high signal-to-noise ratio of photoactivation allows collection of three-dimensional (3D) time series. These contain all necessary functional and anatomical data to measure cell coupling in complex tissues noninvasively.

  19. Glycosylation of Fluorophenols by Plant Cell Cultures

    PubMed Central

    Shimoda, Kei; Kubota, Naoji; Kondo, Yoko; Sato, Daisuke; Hamada, Hiroki

    2009-01-01

    Fluoroaromatic compounds are used as agrochemicals and released into environment as pollutants. Glycosylation of 2-, 3-, and 4-fluorophenols using plant cell cultures of Nicotiana tabacum was investigated to elucidate their potential to metabolize these compounds. Cultured N. tabacum cells converted 2-fluorophenol into its β-glucoside (60%) and β-gentiobioside (10%). 4-Fluorophenol was also glycosylated to its β-glucoside (32%) and β-gentiobioside (6%) by N. tabacum cells. On the other hand, N. tabacum glycosylated 3-fluorophenol to β-glucoside (17%). PMID:19564930

  20. Fluorescence activated cell sorting of plant protoplasts.

    PubMed

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-01-01

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  1. Fluorescence activated cell sorting of plant protoplasts.

    PubMed

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-02-18

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  2. Plant cell transformation with Agrobacterium tumefaciens under simulated microgravity

    NASA Astrophysics Data System (ADS)

    Sarnatska, Veresa; Gladun, Hanna; Padalko, Svetlana

    To investigate simulated microgravity (clinorotation) effect on plant cell transformation with Agrobacterium tumefaciens and crown gall formation, the culture of primary explants of potato and Jerusalem artichoke tubers was used. It is found that the efficiency of tumor formation and development in clinorotated explants are considerably reduced. When using the explants isolated from potato tubers clinorotated for 3, 5 and 19 days, drastic reduction of formation and development of crown gall tumors was observed. Conversely, the tumor number and their development increased when potato tubers were clinorotated for one day. As was estimated by us previously, cells of Jerusalem artichoke explants are the most sensitive to agrobacteria on 4-5 h of in vitro culturing and this time corresponds to the certain period of G1-stage of the cell cycle. We have also estimated that this period is characterized by the increase of binding of acridine orange by nuclear chromatin and increase in activity of RNA-polymerase I and II. Inoculation of explants with agrobacteria in this period was the most optimal for transformation and crown gall induction. We estimated that at four - hour clinorotation of explants the intensity of acridine orange binding to nuclei was considerably lower than on 4h in the control. At one-day clinorotation of potato tubers, a considerable increase in template accessibility of chromatin and in activity of RNA-polymerase I and II occurred. These results may serve as an evidence for the ability of plant dormant tissues to respond to microgravity. Another demonstration of dormant tissue response to changed gravity we obtained when investigating pathogenesis-related proteins (PR-proteins). PR-proteins were subjected to nondenaturing PAGE.and we have not found any effect of microgravity on PR-proteins of potato explants with normal or tumorous growth. We may suggest that such response derives from the common effects of two stress factors - wounding and changed

  3. Cell Surfaces in Plant-Microorganism Interactions

    PubMed Central

    Esquerré-Tugayé, Marie-Thérèse; Lamport, Derek T. A.

    1979-01-01

    Infection of muskmelon Cucumis melo seedlings by the fungus Colletotrichum lagenarium causes a 10-fold increase in the amount of cell wall hydroxyproline-rich glycoprotein. Evidence for this increase was provided by studying two specific markers of this glycoprotein, namely hydroxyproline and glycosylated serine. The lability of the O-glycosidic linkage of wall-bound glycosylated serine in the presence of hydrazine, was used to determine the amount of serine which is glycosylated. A large increase in the hydroxyproline content of infected plants is shown, but the ratios of glycosylated serine to hydroxyproline are similar in healthy and infected plants. As far as these markers are concerned, the hydroxyproline-rich glycoproteins secreted into the wall as a result of the disease are similar to those of healthy plants. In addition, the extent of glycosylation of the wall serine, in both healthy and infected plants, decreases as the plant ages. Serine- and hydroxyproline-rich (glyco)peptides were also isolated after trypsinolysis of the wall. These (glyco)peptides include the galactosyl-containing pentapeptide, serine-hydroxyproline4. This pentapeptide is characteristic of cell wall protein. PMID:16660956

  4. Dim-Light Sensitivity of Cells in the Awake Cat's Lateral Geniculate and Medial Interlaminar Nuclei: A Correlation With Behavior

    PubMed Central

    Kang, Incheol; Malpeli, Joseph G.

    2009-01-01

    Contrast thresholds of cells in the dorsal lateral geniculate (LGNd) and medial interlaminar (MIN) nuclei of awake cats were measured for scotopic and mesopic vision with drifting sine gratings (1/8, 2, and 4 cycles/deg [cpd]; 4-Hz temporal frequency). Thresholds for mean firing rate (F0) and temporally modulated responses (F1) were derived with receiver-operating-characteristic analyses and compared with behavioral measures recently reported by Kang and colleagues. Behavioral sensitivity was predicted by the neural responses of the most sensitive combinations of cell class and response mode: Y-cell F1 responses for 1/8 cpd, X-cell F1 responses for 2 cpd, and Y-cell F0 responses for 4 cpd. All previous estimates of neural scotopic increment thresholds in animal models fell between Weber's law (proportional to retinal illuminance) and the deVries–Rose law (proportional to the square root of illuminance). However, psychophysical experiments suggest that under appropriate conditions human scotopic vision follows the deVries–Rose law. If behavioral sensitivity is assumed to be determined by the most sensitive class of cells, this discrepancy is resolved. Under scotopic conditions, off-center Y cells were the most sensitive and these followed the deVries–Rose law fairly closely. MIN Y cells were, on average, 0.25 log units more sensitive than LGNd Y cells under scotopic conditions, supporting a previous proposal that the MIN is a specialization of the carnivore for dim-light vision. We conclude that both physiologically and behaviorally, cat and human scotopic vision are fundamentally similar, including adherence to the deVries–Rose law for detection of Gabor functions. PMID:19458145

  5. Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei.

    PubMed Central

    Brison, O; Kedinger, C; Wilhelm, J

    1977-01-01

    When HeLa cell nuclei, isolated 17 h after infection with adenovirus type 2 (Ad2), were extracted with 200 mM ammonium sulfate, Ad2 nucleoprotein complexes were selectively released. These complexes contained a DNA polymerase activity that corresponded to DNA polymerase molecules actively engaged in Ad2 DNA replication. Under our high-salt (200 mM ammonium sulfate) incubation conditions, where no reinitiation occurred, full-length Ad2 DNA chains were synthesized by elongation of chains that had been initiated in vivo. This conclusion was further supported by density labeling experiments indicating that the in vitro DNA synthesis was semiconservative. Evidence is presented suggesting that at least part of the DNA polymerase molecules engaged in Ad2 DNA replication belong to the gamma class. PMID:916022

  6. [Effect of physical factors on bioelectric properties of cell nuclei of buccal epithelium in students with chronic fatigue syndrome].

    PubMed

    Samoĭlovych, V A; Hutarieva, N V; Tondiĭ, L D

    2005-01-01

    60 students with the syndrome of chronic fatigue have been observed in a sanatorium. 35 patients of the main (first) group underwent hydrokinesotherapy. The patients of the second group attended physical exercises as follows: girls attended shaping classes and contrast douche, boys exercised on different training apparatus and took too contrast douche. An estimation of efficiency of the carried out treatment was conducted with the help of clinical and paraclinical methods of the study. Electrokinetic properties of cellular nuclei of buccalium epithelium were tested to reveal a general nonspecific resistance of the human body. The data of assessment of an electrical potential (Z-potential) showed that clinical improvement coincided with the increase in electrokinetic mobility of cell nucleus of buccalium epithelium (P<0.01). Physical factors proved to have positive influence on homeostas of patients of the main group. This index was not reliable in patients of the second group (P>0.5).

  7. Inhibition of ribonucleic acid efflux from isolated SV40-3T3 cell nuclei by 3'-deoxyadenosine (cordycepin).

    PubMed

    Agutter, P S; McCaldin, B

    1979-05-15

    The effect of 3'-deoxyadenosine (cordycepin) on mRNA efflux from isolated SV40-3T3 cell nuclei has been studied and compared with its effect on the nucleoside triphosphatase activity in the isolated nuclear envelope. Inhibition of mRNA efflux occurs rapidly, but is dependent on the presence of ATP. Half-maximal inhibition occurs with 40 microM-cordycepin. The effect is not simulated by 2'-deoxyadenosine or by actinomycin D, and adenosine provides a substantial degree of protection against it. Cordycepin does not directly inhibit the nucleoside triphosphatase. The stimulation of this enzyme by poly(A) is not affected unless the poly(A) and cordycepin are incubated together with nuclear lysate in the presence of ATP; in this case the stimulation is significantly reduced. Possible interpretations of these results and their relevance for understanding the system in vivo for nucleo-cytoplasmic messenger transport are discussed.

  8. Inhibition of ribonucleic acid efflux from isolated SV40-3T3 cell nuclei by 3'-deoxyadenosine (cordycepin).

    PubMed Central

    Agutter, P S; McCaldin, B

    1979-01-01

    The effect of 3'-deoxyadenosine (cordycepin) on mRNA efflux from isolated SV40-3T3 cell nuclei has been studied and compared with its effect on the nucleoside triphosphatase activity in the isolated nuclear envelope. Inhibition of mRNA efflux occurs rapidly, but is dependent on the presence of ATP. Half-maximal inhibition occurs with 40 microM-cordycepin. The effect is not simulated by 2'-deoxyadenosine or by actinomycin D, and adenosine provides a substantial degree of protection against it. Cordycepin does not directly inhibit the nucleoside triphosphatase. The stimulation of this enzyme by poly(A) is not affected unless the poly(A) and cordycepin are incubated together with nuclear lysate in the presence of ATP; in this case the stimulation is significantly reduced. Possible interpretations of these results and their relevance for understanding the system in vivo for nucleo-cytoplasmic messenger transport are discussed. PMID:226073

  9. Cosmogenic nuclei

    NASA Technical Reports Server (NTRS)

    Raisbeck, G. M.

    1986-01-01

    Cosmogenic nuclei, nuclides formed by nuclear interactions of galactic and solar cosmic rays with extraterrestrial or terrestrial matter are discussed. Long lived radioactive cosmogenic isotopes are focused upon. Their uses in dating, as tracers of the interactions of cosmic rays with matter, and in obtaining information on the variation of primary cosmic ray flux in the past are discussed.

  10. Melatonin protects the integrity of granulosa cells by reducing oxidative stress in nuclei, mitochondria, and plasma membranes in mice

    PubMed Central

    TANABE, Manabu; TAMURA, Hiroshi; TAKETANI, Toshiaki; OKADA, Maki; LEE, Lifa; TAMURA, Isao; MAEKAWA, Ryo; ASADA, Hiromi; YAMAGATA, Yoshiaki; SUGINO, Norihiro

    2014-01-01

    Melatonin protects luteinized granulosa cells (GCs) from oxidative stress in the follicle during ovulation. However, it is unclear in which cellular components (e.g., nuclei, mitochondria, or plasma membranes) melatonin works as an antioxidant. GCs from immature (3 wks) ICR mice were incubated with hydrogen peroxide (H2O2; 0.01, 0.1, 1, 10 mM) in the presence or absence of melatonin (100 μg/ml) for 2 h. DNA damage was assessed by fluorescence-based immunocytochemistry using specific antibodies for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative guanine base damage in DNA, and for histone H2AX phosphorylation (γH2AX), a marker of double-strand breaks of DNA. Mitochondrial function was assessed by the fluorescence intensity of MitoTracker Red probes, which diffuse across the membrane and accumulate in mitochondria with active membrane potentials. Lipid peroxidation of plasma membranes was analyzed by measuring hexanoyl-lysine (HEL), a oxidative stress marker for lipid peroxidation. Apoptosis of GCs was assessed by nuclear fragmentation using DAPI staining, and apoptotic activities were evaluated by caspase-3/7 activities. H2O2 treatment significantly increased the fluorescence intensities of 8-OHdG and γH2AX, reduced the intensity of MitoTracker Red in the mitochondria, increased HEL concentrations in GCs, and enhanced the number of apoptotic cells and caspase-3/7 activities. All these changes were significantly decreased by melatonin treatment. Melatonin reduced oxidative stress-induced DNA damage, mitochondrial dysfunction, lipid peroxidation, and apoptosis in GCs, suggesting that melatonin protects GCs by reducing oxidative stress of cellular components including nuclei, mitochondria, and plasma membranes. Melatonin helps to maintain the integrity of GCs as an antioxidant in the preovulatory follicle. PMID:25366368

  11. Melatonin protects the integrity of granulosa cells by reducing oxidative stress in nuclei, mitochondria, and plasma membranes in mice.

    PubMed

    Tanabe, Manabu; Tamura, Hiroshi; Taketani, Toshiaki; Okada, Maki; Lee, Lifa; Tamura, Isao; Maekawa, Ryo; Asada, Hiromi; Yamagata, Yoshiaki; Sugino, Norihiro

    2015-01-01

    Melatonin protects luteinized granulosa cells (GCs) from oxidative stress in the follicle during ovulation. However, it is unclear in which cellular components (e.g., nuclei, mitochondria, or plasma membranes) melatonin works as an antioxidant. GCs from immature (3 wks) ICR mice were incubated with hydrogen peroxide (H2O2; 0.01, 0.1, 1, 10 mM) in the presence or absence of melatonin (100 μg/ml) for 2 h. DNA damage was assessed by fluorescence-based immunocytochemistry using specific antibodies for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative guanine base damage in DNA, and for histone H2AX phosphorylation (γH2AX), a marker of double-strand breaks of DNA. Mitochondrial function was assessed by the fluorescence intensity of MitoTracker Red probes, which diffuse across the membrane and accumulate in mitochondria with active membrane potentials. Lipid peroxidation of plasma membranes was analyzed by measuring hexanoyl-lysine (HEL), a oxidative stress marker for lipid peroxidation. Apoptosis of GCs was assessed by nuclear fragmentation using DAPI staining, and apoptotic activities were evaluated by caspase-3/7 activities. H2O2 treatment significantly increased the fluorescence intensities of 8-OHdG and γH2AX, reduced the intensity of MitoTracker Red in the mitochondria, increased HEL concentrations in GCs, and enhanced the number of apoptotic cells and caspase-3/7 activities. All these changes were significantly decreased by melatonin treatment. Melatonin reduced oxidative stress-induced DNA damage, mitochondrial dysfunction, lipid peroxidation, and apoptosis in GCs, suggesting that melatonin protects GCs by reducing oxidative stress of cellular components including nuclei, mitochondria, and plasma membranes. Melatonin helps to maintain the integrity of GCs as an antioxidant in the preovulatory follicle.

  12. Postnatal changes in the number of serotonin-immunoreactive cells in midbrain raphe nuclei of male rats.

    PubMed

    Ito, Hiroyuki; Moriizumi, Tetsuji; Shimogawa, Yuji; Yamanouchi, Korehito

    2014-09-01

    To clarify the developmental changes in serotonergic neurons in the subdivisions of the dorsal (DR) and median raphe (MR) nuclei before puberty, the extent of the nuclei and the number of serotonin (5-HT) immunoreactive (ir) cells were measured in 5-, 15-, and 30-day-old rats and 8-week-old (adult) castrated male rats. The brains were fixed and 50 μm frozen sections prepared. After immunostaining for 5-HT, the number of 5-HT-ir cells in a 0.2 × 0.2 mm frame in the dorsal, ventral and lateral subdivisions of the DR (dDR, vDR and lDR, respectively) and MR were counted. Total numbers of 5-HT-ir cells counted in the frame of three sections in each rat were expressed as the number of cells per cubic millimeter (density). The results indicated that the densities of 5-HT-ir cells in the MR were almost the same in all age groups. On the other hand, among the subdivisions of the DR, the mean density of 5-HT-ir cells in 15-day-old rats was higher than that in the 5-day-old group in the lDR only. The area of the three sections of the DR and of the MR was also measured. The area of the DR in 15-day-old rats was found to be twice that in the 5-day-old rats, and differed from the area in 30-day-old rats and adults. There were no differences among the age groups in the areas of the MR. The results indicate that the expression of 5-HT in the lDR and extent of the DR increased to adult levels from days 5 to 15 after birth. In the dDR, vDR and MR, expression of 5-HT at postnatal day 5 was at adult levels already.

  13. 3. Right side of Zinc Plant, from Cell Room midpoint ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. Right side of Zinc Plant, from Cell Room midpoint to Plant Office (foreground) and #5 Roaster and Concentrate Handling (background). View is to the east. - Sullivan Electrolytic Zinc Plant, Government Gulch, Kellogg, Shoshone County, ID

  14. Monitor RNA synthesis in live cell nuclei by using two-photon excited fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Peng, Xiao; Lin, Danying; Wang, Yan; Qi, Jing; Yan, Wei; Qu, Junle

    2015-03-01

    Probing of local molecular environment in cells is of significant value in creating a fundamental understanding of cellular processes and molecular profiles of diseases, as well as studying drug cell interactions. In order to investigate the dynamically changing in subcellular environment during RNA synthesis, we applied two-photon excited fluorescence lifetime imaging microscopy (FLIM) method to monitor the green fluorescent protein (GFP) fused nuclear protein ASF/SF2. The fluorescence lifetime of fluorophore is known to be in inverse correlation with a local refractive index, and thus fluorescence lifetimes of GFP fusions provide real-time information of the molecular environment of ASF/SF2- GFP. The FLIM results showed continuous and significant fluctuations of fluorescence lifetimes of the fluorescent protein fusions in live HeLa cells under physiological conditions. The fluctuations of fluorescence lifetime values indicated the variations of activities of RNA polymerases. Moreover, treatment with pharmacological drugs inhibiting RNA polymerase activities led to irreversible decreases of fluorescence lifetime values. In summary, our study of FLIM imaging of GFP fusion proteins has provided a sensitive and real-time method to investigate RNA synthesis in live cell nuclei.

  15. Characterization of Cellulose Synthesis in Plant Cells

    PubMed Central

    Maleki, Samaneh Sadat; Mohammadi, Kourosh; Ji, Kong-shu

    2016-01-01

    Cellulose is the most significant structural component of plant cell wall. Cellulose, polysaccharide containing repeated unbranched β (1-4) D-glucose units, is synthesized at the plasma membrane by the cellulose synthase complex (CSC) from bacteria to plants. The CSC is involved in biosynthesis of cellulose microfibrils containing 18 cellulose synthase (CesA) proteins. Macrofibrils can be formed with side by side arrangement of microfibrils. In addition, beside CesA, various proteins like the KORRIGAN, sucrose synthase, cytoskeletal components, and COBRA-like proteins have been involved in cellulose biosynthesis. Understanding the mechanisms of cellulose biosynthesis is of great importance not only for improving wood production in economically important forest trees to mankind but also for plant development. This review article covers the current knowledge about the cellulose biosynthesis-related gene family. PMID:27314060

  16. Characterization of Cellulose Synthesis in Plant Cells.

    PubMed

    Maleki, Samaneh Sadat; Mohammadi, Kourosh; Ji, Kong-Shu

    2016-01-01

    Cellulose is the most significant structural component of plant cell wall. Cellulose, polysaccharide containing repeated unbranched β (1-4) D-glucose units, is synthesized at the plasma membrane by the cellulose synthase complex (CSC) from bacteria to plants. The CSC is involved in biosynthesis of cellulose microfibrils containing 18 cellulose synthase (CesA) proteins. Macrofibrils can be formed with side by side arrangement of microfibrils. In addition, beside CesA, various proteins like the KORRIGAN, sucrose synthase, cytoskeletal components, and COBRA-like proteins have been involved in cellulose biosynthesis. Understanding the mechanisms of cellulose biosynthesis is of great importance not only for improving wood production in economically important forest trees to mankind but also for plant development. This review article covers the current knowledge about the cellulose biosynthesis-related gene family. PMID:27314060

  17. Acquisition of multiple nuclei and the activity of DNA polymerase alpha and reinitiation of DNA replication in terminally differentiated adult cardiac muscle cells in culture

    SciTech Connect

    Claycomb, W.C.; Bradshaw, H.D. Jr.

    1983-10-01

    Terminally differentiated ventricular cardiac muscle cells isolated from the adult rat and maintained in cell culture have been observed to acquire multiple nuclei. In one cultured myocyte as many as 10 nuclei have been counted. Apparently, these multiple nuclei are formed by DNA replication followed by karyokinesis; the cells must then fail to complete mitosis and divide. To investigate whether DNA synthesis was occurring, the cells were cultured in the presence of (3H)thymidine and then processed for autoradiography. Mononucleated, binucleated, and multinucleated cells incorporate (3H)thymidine into DNA as evidenced by the high concentration of silver grains over their nuclei. Peak periods of incorporation were observed to occur at 10- to 12-day intervals; at 11, 23, and 33 days after initially placing the cells in culture. When the cells were maintained in the presence of (3H)thymidine continuously from Day 7 to Day 17 of culture, 23% of the cells became labeled. If the cells were cultured continuously for 30 days in the presence of (3H)thymidine, from Day 10 to Day 40, 56% of the cells were labeled. Isopycnic gradient analysis indicates that this thymidine incorporation was into DNA that was being replicated semiconservatively; these experiments did not eliminate the possibility, however, that this incorporation was due to amplification of specific genes, such as those coding for the contractile proteins. The activity of DNA polymerase alpha also returns to these cells. These studies demonstrate that the terminally differentiated mammalian ventricular cardiac muscle cell, previously thought to have permanently lost the capacity to replicate DNA during early development, is able to reinitiate semiconservative DNA replication when grown in culture.

  18. Distinctions in burst spiking between thalamic reticular nucleus cells projecting to the dorsal lateral geniculate and lateral posterior nuclei in the anesthetized rat.

    PubMed

    Kimura, A; Yokoi, I; Imbe, H; Donishi, T; Kaneoke, Y

    2012-12-13

    Thalamic cell activity is under a significant influence of inhibition from the thalamic reticular nucleus (TRN) that is composed of domains connected with first and higher order thalamic nuclei, which are thought to subserve transmission of sensory inputs to the cortex and cortico-thalamo-cortical transmission of cortical outputs, respectively. Provided that TRN cells have distinct activities along with their projections to first and higher order thalamic nuclei, TRN cells could shape cell activities of the two thalamic nuclei in different manners for the distinct functions. In anesthetized rats, visual response and spontaneous activity were recorded from TRN cells projecting to the dorsal lateral geniculate (first order) and lateral posterior (higher order) nuclei (TRN-DLG and TRN-LP cells), using juxta-cellular recording and labeling techniques. TRN-DLG cells had a higher propensity for burst spiking and exhibited bursts of larger numbers of spikes with shorter inter-spike intervals as compared to TRN-LP cells in both visual response and spontaneous activity. Sustained effects of visual input on burst spiking were recognized in recurrent activation of TRN-DLG but not of TRN-LP cells. Further, the features of burst spiking were related with the locations of topographically connected cell bodies and terminal fields. The difference in burst spiking contrasts with the difference between thalamic cells in the DLG and LP, which show low and high levels of burst spiking, respectively. The synergy between thalamic and TRN cell activities with their contrasting features of burst spiking may compose distinctive sensory processing and attentional gating functions of geniculate and extra-geniculate systems.

  19. Fuel cell power plant economic and operational considerations

    NASA Technical Reports Server (NTRS)

    Lance, J. R.

    1984-01-01

    Fuel cell power plants intended for electric utility and cogeneration applications are now in the design and construction stage. This paper describes economic and operational considerations being used in the development and design of plants utilizing air cooled phosphoric acid fuel cells. Fuel cell power plants have some unique characteristics relative to other types of power plants. As a result it was necessary to develop specific definitions of the fuel cell power plant characteristics in order to perform cost of electricity calculations. This paper describes these characteristics and describes the economic analyses used in the Westinghouse fuel cell power plant program.

  20. Plant cell technologies in space: Background, strategies and prospects

    NASA Technical Reports Server (NTRS)

    Kirkorian, A. D.; Scheld, H. W.

    1987-01-01

    An attempt is made to summarize work in plant cell technologies in space. The evolution of concepts and the general principles of plant tissue culture are discussed. The potential for production of high value secondary products by plant cells and differentiated tissue in automated, precisely controlled bioreactors is discussed. The general course of the development of the literature on plant tissue culture is highlighted.

  1. How do plant cell walls extend?

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1993-01-01

    This article briefly summarizes recent work that identifies the biophysical and biochemical processes that give rise to the extension of plant cell walls. I begin with the biophysical notion of stress relaxation of the wall and follow with recent studies of wall enzymes thought to catalyze wall extension and relaxation. Readers should refer to detailed reviews for more comprehensive discussion of earlier literature (Taiz, 1984; Carpita and Gibeaut, 1993; Cosgrove, 1993).

  2. Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

    SciTech Connect

    Fan, Yong; Chen, Xinjie; Luo, Yumei; Chen, Xiaolin; Li, Shaoying; Huang, Yulin; Sun, Xiaofang

    2009-04-24

    The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.

  3. Reproductive and growth performance in Jin Hua pigs cloned from somatic cell nuclei and the meat quality of their offspring.

    PubMed

    Shibata, Masatoshi; Otake, Masayoshi; Tsuchiya, Seiko; Chikyu, Mikio; Horiuchi, Atsushi; Kawarasaki, Tatsuo

    2006-10-01

    Somatic cell cloning is expected to be a valuable method for conserving genetic resources in pigs. In this study, we compared the reproductive and growth performance of Jin Hua cloned pigs with that of naturally bred Jin Hua pigs. In addition, we generated offspring from the cloned sows and examined the productivity and quality of meat in the progeny. The birth weights and growth rates of somatic cell-cloned pigs were similar to those of Jin Hua pigs. The cloned pigs reached puberty very early, and this is typical of the Jin Hua breed. Furthermore, reproductive performance, in terms of traits such as gestation period, litter size, and raising rate in the cloned pigs were similar to Jin Hua pigs. Although the offspring of the cloned (OC) pigs had lower birth weights than the Jin Hua breed, the daily weight gain of the OC pigs was significantly higher, especially at the finishing stage. The carcass quality of the OC pigs had similar characteristics to the Jin Hua breed, namely thick back fat and a small loin area. Furthermore, the meat qualities of the OC pigs were similar to those of Jin Hua pigs in terms of intramuscular fat content and tenderness. These results demonstrate that cloned pigs and their offspring were similar to the Jin Hua breed in most of the growth, reproductive, and meat productive performances. This strongly suggests that pigs cloned from somatic cell nuclei have the potential to be a valuable genetic resource for breeding.

  4. Three-dimensional segmentation of nuclei and mitotic chromosomes for the study of cell divisions in live Drosophila embryos.

    PubMed

    Chinta, Rambabu; Wasser, Martin

    2012-01-01

    Drosophila embryogenesis is an established model to investigate mechanisms and genes related to cell divisions in an intact multicellular organism. Progression through the cell cycle phases can be monitored in vivo using fluorescently labeled fusion proteins and time-lapse microscopy. To measure cellular properties in microscopic images, accurate and fast image segmentation methods are a critical prerequisite. To quantify static and dynamic features of interphase nuclei and mitotic chromosomes, we developed a three-dimensional (3D) segmentation method based on multiple level sets. We tested our method on 3D time-series images of live embryos expressing histone-2Av-green fluorescence protein. Our method is robust to low signal-to-noise ratios inherent to high-speed imaging, fluorescent signals in the cytoplasm, and dynamic changes of shape and texture. Comparisons with manual ground-truth segmentations showed that our method achieves more than 90% accuracy on the object as well as voxel levels and performs consistently throughout all cell cycle phases and developmental stages from syncytial blastoderm to postblastoderm mitotic domains.

  5. Dose to tissue medium or water cavities as surrogate for the dose to cell nuclei at brachytherapy photon energies.

    PubMed

    Enger, Shirin A; Ahnesjö, Anders; Verhaegen, Frank; Beaulieu, Luc

    2012-07-21

    It has been suggested that modern dose calculation algorithms should be able to report absorbed dose both as dose to the local medium, D(m,m,) and as dose to a water cavity embedded in the medium, D(w,m), using conversion factors from cavity theory. Assuming that the cell nucleus with its DNA content is the most important target for biological response, the aim of this study is to investigate, by means of Monte Carlo (MC) simulations, the relationship of the dose to a cell nucleus in a medium, D(n,m,) to D(m,m) and D(w,m), for different combinations of cell nucleus compositions and tissue media for different photon energies used in brachytherapy. As D(n,m) is very impractical to calculate directly for routine treatment planning, while D(m,m) and D(w,m) are much easier to obtain, the questions arise which one of these quantities is the best surrogate for D(n,m) and which cavity theory assumptions should one use for its estimate. The Geant4.9.4 MC code was used to calculate D(m,m,) D(w,m) and D(n,m) for photon energies from 20 (representing the lower energy end of brachytherapy for ¹⁰³Pd or ¹²⁵I) to 300 keV (close to the mean energy of (¹⁹²Ir) and for the tissue media adipose, breast, prostate and muscle. To simulate the cell and its nucleus, concentric spherical cavities were placed inside a cubic phantom (10 × 10 × 10 mm³). The diameter of the simulated nuclei was set to 14 µm. For each tissue medium, three different setups were simulated; (a) D(n,m) was calculated with nuclei embedded in tissues (MC-D(n,m)). Four different published elemental compositions of cell nuclei were used. (b) D(w,m) was calculated with MC (MC-D(w,m)) and compared with large cavity theory calculated D(w,m) (LCT-D(w,m)), and small cavity theory calculated D(w,m) (SCT-D(w,m)). (c) D(m,m) was calculated with MC (MC-D(m,m)). MC-D(w,m) is a good substitute for MC-D(n,m) for all photon energies and for all simulated nucleus compositions and tissue types. SCT-D(w,m) can be used

  6. Plant Cell Adaptive Responses to Microgravity

    NASA Astrophysics Data System (ADS)

    Kordyum, Elizabeth; Kozeko, Liudmyla; Talalaev, Alexandr

    Microgravity is an abnormal environmental condition that plays no role in the functioning of biosphere. Nevertheless, the chronic effect of microgravity in space flight as an unfamiliar factor does not prevent the development of adaptive reactions at the cellular level. In real microgravity in space flight under the more or less optimal conditions for plant growing, namely temperature, humidity, CO2, light intensity and directivity in the hardware angiosperm plants perform an “reproductive imperative”, i.e. they flower, fruit and yield viable seeds. It is known that cells of a multicellular organism not only take part on reactions of the organism but also carry out processes that maintain their integrity. In light of these principles, the problem of the identification of biochemical, physiological and structural patterns that can have adaptive significance at the cellular and subcellular level in real and simulated microgravity is considered. Cytological studies of plants developing in real and simulated microgravity made it possible to establish that the processes of mitosis, cytokinesis, and tissue differentiation of vegetative and generative organs are largely normal. At the same time, under microgravity, essential reconstruction in the structural and functional organization of cell organelles and cytoskeleton, as well as changes in cell metabolism and homeostasis have been described. In addition, new interesting data concerning the influence of altered gravity on lipid peroxidation intensity, the level of reactive oxygen species, and antioxidant system activity, just like on the level of gene expression and synthesis of low-molecular and high-molecular heat shock proteins were recently obtained. So, altered gravity caused time-dependent increasing of the HSP70 and HSP90 levels in cells, that may indicate temporary strengthening of their functional loads that is necessary for re-establish a new cellular homeostasis. Relative qPCR results showed that

  7. Do plant cell walls have a code?

    PubMed

    Tavares, Eveline Q P; Buckeridge, Marcos S

    2015-12-01

    A code is a set of rules that establish correspondence between two worlds, signs (consisting of encrypted information) and meaning (of the decrypted message). A third element, the adaptor, connects both worlds, assigning meaning to a code. We propose that a Glycomic Code exists in plant cell walls where signs are represented by monosaccharides and phenylpropanoids and meaning is cell wall architecture with its highly complex association of polymers. Cell wall biosynthetic mechanisms, structure, architecture and properties are addressed according to Code Biology perspective, focusing on how they oppose to cell wall deconstruction. Cell wall hydrolysis is mainly focused as a mechanism of decryption of the Glycomic Code. Evidence for encoded information in cell wall polymers fine structure is highlighted and the implications of the existence of the Glycomic Code are discussed. Aspects related to fine structure are responsible for polysaccharide packing and polymer-polymer interactions, affecting the final cell wall architecture. The question whether polymers assembly within a wall display similar properties as other biological macromolecules (i.e. proteins, DNA, histones) is addressed, i.e. do they display a code?

  8. The potential of single-cell profiling in plants.

    PubMed

    Efroni, Idan; Birnbaum, Kenneth D

    2016-01-01

    Single-cell transcriptomics has been employed in a growing number of animal studies, but the technique has yet to be widely used in plants. Nonetheless, early studies indicate that single-cell RNA-seq protocols developed for animal cells produce informative datasets in plants. We argue that single-cell transcriptomics has the potential to provide a new perspective on plant problems, such as the nature of the stem cells or initials, the plasticity of plant cells, and the extent of localized cellular responses to environmental inputs. Single-cell experimental outputs require different analytical approaches compared with pooled cell profiles and new tools tailored to single-cell assays are being developed. Here, we highlight promising new single-cell profiling approaches, their limitations as applied to plants, and their potential to address fundamental questions in plant biology. PMID:27048384

  9. 2003 Plant Cell Walls Gordon Conference

    SciTech Connect

    Daniel J. Cosgrove

    2004-09-21

    This conference will address recent progress in many aspects of cell wall biology. Molecular, genetic, and genomic approaches are yielding major advances in our understanding of the composition, synthesis, and architecture of plant cell walls and their dynamics during growth, and are identifying the genes that encode the machinery needed to make their biogenesis possible. This meeting will bring together international scientists from academia, industry and government labs to share the latest breakthroughs and perspectives on polysaccharide biosynthesis, wood formation, wall modification, expansion and interaction with other organisms, and genomic & evolutionary analyses of wall-related genes, as well as to discuss recent ''nanotechnological'' advances that take wall analysis to the level of a single cell.

  10. [Feedback control mechanisms of plant cell expansion

    SciTech Connect

    Cosgrove, D.J.

    1992-01-01

    We have generated considerable evidence for the significance of wall stress relaxation in the control of plant growth and found that several agents (gibberellin, light, genetic loci for dwarf stature) influence growth rate via alteration of wall relaxation. We have refined our methods for measuring wall relaxation and, moreover, have found that wall relaxation properties bear only a distance relationship to wall mechanical properties. We have garnered novel insights into the nature of cell expansion mechanisms by analyzing spontaneous fluctuations of plant growth rate in seedlings. These experiments involved the application of mathematical techniques for analyzing growth rate fluctuations and the development of new instrumentation for measuring and forcing plant growth in a controlled fashion. These studies conclude that growth rate fluctuations generated by the plant as consequence of a feedback control system. This conclusion has important implications for the nature of wall loosening processes and demands a different framework for thinking about growth control. It also implies the existence of a growth rate sensor.

  11. Automated detection of retinal cell nuclei in 3D micro-CT images of zebrafish using support vector machine classification

    NASA Astrophysics Data System (ADS)

    Ding, Yifu; Tavolara, Thomas; Cheng, Keith

    2016-03-01

    Our group is developing a method to examine biological specimens in cellular detail using synchrotron microCT. The method can acquire 3D images of tissue at micrometer-scale resolutions, allowing for individual cell types to be visualized in the context of the entire specimen. For model organism research, this tool will enable the rapid characterization of tissue architecture and cellular morphology from every organ system. This characterization is critical for proposed and ongoing "phenome" projects that aim to phenotype whole-organism mutants and diseased tissues from different organisms including humans. With the envisioned collection of hundreds to thousands of images for a phenome project, it is important to develop quantitative image analysis tools for the automated scoring of organism phenotypes across organ systems. Here we present a first step towards that goal, demonstrating the use of support vector machines (SVM) in detecting retinal cell nuclei in 3D images of wild-type zebrafish. In addition, we apply the SVM classifier on a mutant zebrafish to examine whether SVMs can be used to capture phenotypic differences in these images. The longterm goal of this work is to allow cellular and tissue morphology to be characterized quantitatively for many organ systems, at the level of the whole-organism.

  12. Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy.

    PubMed

    Krause, Marina; Te Riet, Joost; Wolf, Katarina

    2013-12-01

    The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness together with simultaneous visualization of the cantilever-nucleus contact and the fate of the cell. Using cantilevers functionalized with either tips or beads and spring constants ranging from 0.06-10 N m(-1), force-deformation curves were generated from nuclear positions of adherent HT1080 fibrosarcoma cell populations at unchallenged integrity, and a nuclear stiffness range of 0.2 to 2.5 kPa was identified depending on cantilever type and the use of extended fitting models. Chromatin-decondensating agent trichostatin A (TSA) induced nuclear softening of up to 50%, demonstrating the feasibility of our approach. Finally, using a stiff bead-functionalized cantilever pushing at maximal system-intrinsic force, the nucleus was deformed to 20% of its original height which after TSA treatment reduced further to 5% remaining height confirming chromatin organization as an important determinant of nuclear stiffness. Thus, combined AFM-confocal microscopy is a feasible approach to study nuclear compressibility to complement concepts of limiting nuclear deformation in cancer cell invasion and other biological processes.

  13. Head direction cell activity in the anterodorsal thalamus requires intact supragenual nuclei

    PubMed Central

    Clark, Benjamin J.; Brown, Joel E.

    2012-01-01

    Neural activity in several limbic areas varies as a function of the animal's head direction (HD) in the horizontal plane. Lesions of the vestibular periphery abolish this HD cell signal, suggesting an essential role for vestibular afference in HD signal generation. The organization of brain stem pathways conveying vestibular information to the HD circuit is poorly understood; however, recent anatomical work has identified the supragenual nucleus (SGN) as a putative relay. To test this hypothesis, we made lesions of the SGN in rats and screened for HD cells in the anterodorsal thalamus. In animals with complete bilateral lesions, the overall number of HD cells was significantly reduced relative to control animals. In animals with unilateral lesions of the SGN, directional activity was present, but the preferred firing directions of these cells were unstable and less influenced by the rotation of an environmental landmark. In addition, we found that preferred directions displayed large directional shifts when animals foraged for food in a darkened environment and when they were navigating from a familiar environment to a novel one, suggesting that the SGN plays a critical role in projecting essential self-motion (idiothetic) information to the HD cell circuit. PMID:22875899

  14. Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy

    NASA Astrophysics Data System (ADS)

    Krause, Marina; te Riet, Joost; Wolf, Katarina

    2013-12-01

    The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness together with simultaneous visualization of the cantilever-nucleus contact and the fate of the cell. Using cantilevers functionalized with either tips or beads and spring constants ranging from 0.06-10 N m-1, force-deformation curves were generated from nuclear positions of adherent HT1080 fibrosarcoma cell populations at unchallenged integrity, and a nuclear stiffness range of 0.2 to 2.5 kPa was identified depending on cantilever type and the use of extended fitting models. Chromatin-decondensating agent trichostatin A (TSA) induced nuclear softening of up to 50%, demonstrating the feasibility of our approach. Finally, using a stiff bead-functionalized cantilever pushing at maximal system-intrinsic force, the nucleus was deformed to 20% of its original height which after TSA treatment reduced further to 5% remaining height confirming chromatin organization as an important determinant of nuclear stiffness. Thus, combined AFM-confocal microscopy is a feasible approach to study nuclear compressibility to complement concepts of limiting nuclear deformation in cancer cell invasion and other biological processes.

  15. Molecular regulation of plant cell wall extensibility

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1998-01-01

    Gravity responses in plants often involve spatial and temporal changes in cell growth, which is regulated primarily by controlling the ability of the cell wall to extend. The wall is thought to be a cellulose-hemicellulose network embedded in a hydrated matrix of complex polysaccharides and a small amount of structural protein. The wall extends by a form of polymer creep, which is mediated by expansins, a novel group of wall-loosening proteins. Expansins were discovered during a molecular dissection of the "acid growth" behavior of cell walls. Expansin alters the rheology of plant walls in profound ways, yet its molecular mechanism of action is still uncertain. It lacks detectable hydrolytic activity against the major components of the wall, but it is able to disrupt noncovalent adhesion between wall polysaccharides. The discovery of a second family of expansins (beta-expansins) sheds light on the biological role of a major group of pollen allergens and implies that expansins have evolved for diverse developmental functions. Finally, the contribution of other processes to wall extensibility is briefly summarized.

  16. Molecular regulation of plant cell wall extensibility.

    PubMed

    Cosgrove, D J

    1998-05-01

    Gravity responses in plants often involve spatial and temporal changes in cell growth, which is regulated primarily by controlling the ability of the cell wall to extend. The wall is thought to be a cellulose-hemicellulose network embedded in a hydrated matrix of complex polysaccharides and a small amount of structural protein. The wall extends by a form of polymer creep, which is mediated by expansins, a novel group of wall-loosening proteins. Expansins were discovered during a molecular dissection of the "acid growth" behavior of cell walls. Expansin alters the rheology of plant walls in profound ways, yet its molecular mechanism of action is still uncertain. It lacks detectable hydrolytic activity against the major components of the wall, but it is able to disrupt noncovalent adhesion between wall polysaccharides. The discovery of a second family of expansins (beta-expansins) sheds light on the biological role of a major group of pollen allergens and implies that expansins have evolved for diverse developmental functions. Finally, the contribution of other processes to wall extensibility is briefly summarized. PMID:11540640

  17. DNA double-strand breaks alter the spatial arrangement of homologous loci in plant cells.

    PubMed

    Hirakawa, Takeshi; Katagiri, Yohei; Ando, Tadashi; Matsunaga, Sachihiro

    2015-01-01

    Chromatin dynamics and arrangement are involved in many biological processes in nuclei of eukaryotes including plants. Plants have to respond rapidly to various environmental stimuli to achieve growth and development because they cannot move. It is assumed that the alteration of chromatin dynamics and arrangement support the response to these stimuli; however, there is little information in plants. In this study, we investigated the chromatin dynamics and arrangement with DNA damage in Arabidopsis thaliana by live-cell imaging with the lacO/LacI-EGFP system and simulation analysis. It was revealed that homologous loci kept a constant distance in nuclei of A. thaliana roots in general growth. We also found that DNA double-strand breaks (DSBs) induce the approach of the homologous loci with γ-irradiation. Furthermore, AtRAD54, which performs an important role in the homologous recombination repair pathway, was involved in the pairing of homologous loci with γ-irradiation. These results suggest that homologous loci approach each other to repair DSBs, and AtRAD54 mediates these phenomena.

  18. DNA double-strand breaks alter the spatial arrangement of homologous loci in plant cells

    PubMed Central

    Hirakawa, Takeshi; Katagiri, Yohei; Ando, Tadashi; Matsunaga, Sachihiro

    2015-01-01

    Chromatin dynamics and arrangement are involved in many biological processes in nuclei of eukaryotes including plants. Plants have to respond rapidly to various environmental stimuli to achieve growth and development because they cannot move. It is assumed that the alteration of chromatin dynamics and arrangement support the response to these stimuli; however, there is little information in plants. In this study, we investigated the chromatin dynamics and arrangement with DNA damage in Arabidopsis thaliana by live-cell imaging with the lacO/LacI-EGFP system and simulation analysis. It was revealed that homologous loci kept a constant distance in nuclei of A. thaliana roots in general growth. We also found that DNA double-strand breaks (DSBs) induce the approach of the homologous loci with γ-irradiation. Furthermore, AtRAD54, which performs an important role in the homologous recombination repair pathway, was involved in the pairing of homologous loci with γ-irradiation. These results suggest that homologous loci approach each other to repair DSBs, and AtRAD54 mediates these phenomena. PMID:26046331

  19. Measuring the elasticity of plant cells with atomic force microscopy.

    PubMed

    Braybrook, Siobhan A

    2015-01-01

    The physical properties of biological materials impact their functions. This is most evident in plants where the cell wall contains each cell's contents and connects each cell to its neighbors irreversibly. Examining the physical properties of the plant cell wall is key to understanding how plant cells, tissues, and organs grow and gain the shapes important for their respective functions. Here, we present an atomic force microscopy-based nanoindentation method for examining the elasticity of plant cells at the subcellular, cellular, and tissue level. We describe the important areas of experimental design to be considered when planning and executing these types of experiments and provide example data as illustration.

  20. Become a Laboratory Investigator: Detect the Presence of Nuclei in Red Blood Cells.

    ERIC Educational Resources Information Center

    Puckering, Amanda L.; Synenki, Lauren R.; Moore, Kristin; Steapleton, Melissa; Hammond, Paul; Pomart, Katrina; Sisken, Dorothy

    2003-01-01

    Presents lab exercises in which students use the microscope to study cells. Describes these activities as allowing students to work independently and become more proficient at microscope use. Students also gain a deeper understanding of the more invisible world of science. (Author/KHR)

  1. Bioinformatic and mass spectrometry identification of Anaplasma phagocytophilum proteins translocated into host cell nuclei

    PubMed Central

    Sinclair, Sara H. G.; Garcia-Garcia, Jose C.; Dumler, J. Stephen

    2015-01-01

    Obligate intracellular bacteria have an arsenal of proteins that alter host cells to establish and maintain a hospitable environment for replication. Anaplasma phagocytophilum secrets Ankyrin A (AnkA), via a type IV secretion system, which translocates to the nucleus of its host cell, human neutrophils. A. phagocytophilum-infected neutrophils have dramatically altered phenotypes in part explained by AnkA-induced transcriptional alterations. However, it is unlikely that AnkA is the sole effector to account for infection-induced transcriptional changes. We developed a simple method combining bioinformatics and iTRAQ protein profiling to identify potential bacterial-derived nuclear-translocated proteins that could impact transcriptional programming in host cells. This approach identified 50 A. phagocytophilum candidate genes or proteins. The encoding genes were cloned to create GFP fusion protein-expressing clones that were transfected into HEK-293T cells. We confirmed nuclear translocation of six proteins: APH_0062, RplE, Hup, APH_0382, APH_0385, and APH_0455. Of the six, APH_0455 was identified as a type IV secretion substrate and is now under investigation as a potential nucleomodulin. Additionally, application of this approach to other intracellular bacteria such as Mycobacterium tuberculosis, Chlamydia trachomatis and other intracellular bacteria identified multiple candidate genes to be investigated. PMID:25705208

  2. Isolation of nuclei from yeast.

    PubMed

    Bhargava, M M; Halvorson, H O

    1971-05-01

    A method for isolation of nuclei from Saccharomyces cervisiae in high yield is described. The DNA/protein ratio of the isolated nuclei is 10 times higher than that of whole cells. Examination of these nuclei in phase and electron microscopes has shown them to be round bodies having a double membrane, microtubules, and a dark crescent at one end. The optimum conditions for extraction and resolution of histones of these nuclei on acrylamide gels have been investigated. The nuclei have an active RNA polymerase (E.C. 2.7.7.6) and are able to synthesize RNA in vitro. They are also readily stainable with Giemsa's, Feulgen's, and acridine orange methods. PMID:19866769

  3. [EFFECTS OF DIFFERENT CLASSES OF PLANT HORMONES ON MAMMALIAN CELLS].

    PubMed

    Vildanova, M S; Smirnova, E A

    2016-01-01

    Plant hormones are signal molecules of different chemical structure, secreted by plant cells and acting at low concentrations as regulators of plant growth and differentiation. Certain plant hormones are similar to animal hormones or can be produced by animal cells. A number of studies show that the effect of biologically active components of plant origin including plant/phytohormones is much wider than was previously thought, but so far there are no objective criteria for assessing the influence of phytohormones on the physiological state of animal cells. Presented in the survey data show that plant hormones, which have different effects on plant growth and development (jasmonic, abscisic and gibberellic acids), are not neutral to the cells of animal origin, and animal cells response to them may be either positive or negative. PMID:27220246

  4. High-level expression of ice nuclei in Erwinia herbicola is induced by phosphate starvation and low temperature.

    PubMed

    Fall, A L; Fall, R

    1998-06-01

    In laboratory cultures of ice nucleation-active (Ice+) Erwinia herbicola isolates, it has been difficult to achieve high-level expression of ice nuclei, especially nuclei active at temperatures warmer than -5 degrees C (i.e., type 1 ice nuclei). Here we demonstrate that starvation for phosphate and exposure to low temperature triggers expression of ice nuclei in E. herbicola cultures. Starvation for nitrogen, sulfur, or iron was less effective. Under optimal conditions with two different strains, essentially all cells produced ice nuclei active at -10 degrees C or warmer, with an average of 22% containing type 1 ice nuclei within 1 h of a low-temperature shift. These conditions did not greatly enhance the shedding of ice nucleation-active membrane vesicles that are known to be produced by Ice+ E. herbicola isolates. These results support the theory that the Ice+ phenotype may allow nutrient-limited epiphytes to trigger freezing damage, releasing nutrients from host plants. PMID:9608750

  5. Quantitative phosphoproteomics in nuclei of vasopressin-sensitive renal collecting duct cells

    PubMed Central

    Bolger, Steven J.; Hurtado, Patricia A. Gonzales; Hoffert, Jason D.; Saeed, Fahad; Pisitkun, Trairak

    2012-01-01

    Vasopressin regulates transport across the collecting duct epithelium in part via effects on gene transcription. Transcriptional regulation occurs partially via changes in phosphorylation of transcription factors, transcriptional coactivators, and protein kinases in the nucleus. To test whether vasopressin alters the nuclear phosphoproteome of vasopressin-sensitive cultured mouse mpkCCD cells, we used stable isotope labeling and mass spectrometry to quantify thousands of phosphorylation sites in nuclear extracts and nuclear pellet fractions. Measurements were made in the presence and absence of the vasopressin analog dDAVP. Of the 1,251 sites quantified, 39 changed significantly in response to dDAVP. Network analysis of the regulated proteins revealed two major clusters (“cell-cell adhesion” and “transcriptional regulation”) that were connected to known elements of the vasopressin signaling pathway. The hub proteins for these two clusters were the transcriptional coactivator β-catenin and the transcription factor c-Jun. Phosphorylation of β-catenin at Ser552 was increased by dDAVP [log2(dDAVP/vehicle) = 1.79], and phosphorylation of c-Jun at Ser73 was decreased [log2(dDAVP/vehicle) = −0.53]. The β-catenin site is known to be targeted by either protein kinase A or Akt, both of which are activated in response to vasopressin. The c-Jun site is a canonical target for the MAP kinase Jnk2, which is downregulated in response to vasopressin in the collecting duct. The data support the idea that vasopressin-mediated control of transcription in collecting duct cells involves selective changes in the nuclear phosphoproteome. All data are available to users at http://helixweb.nih.gov/ESBL/Database/mNPPD/. PMID:22992673

  6. Dynamic simulation of a direct carbonate fuel cell power plant

    SciTech Connect

    Ernest, J.B.; Ghezel-Ayagh, H.; Kush, A.K.

    1996-12-31

    Fuel Cell Engineering Corporation (FCE) is commercializing a 2.85 MW Direct carbonate Fuel Cell (DFC) power plant. The commercialization sequence has already progressed through construction and operation of the first commercial-scale DFC power plant on a U.S. electric utility, the 2 MW Santa Clara Demonstration Project (SCDP), and the completion of the early phases of a Commercial Plant design. A 400 kW fuel cell stack Test Facility is being built at Energy Research Corporation (ERC), FCE`s parent company, which will be capable of testing commercial-sized fuel cell stacks in an integrated plant configuration. Fluor Daniel, Inc. provided engineering, procurement, and construction services for SCDP and has jointly developed the Commercial Plant design with FCE, focusing on the balance-of-plant (BOP) equipment outside of the fuel cell modules. This paper provides a brief orientation to the dynamic simulation of a fuel cell power plant and the benefits offered.

  7. Effects of hypergravity on the development of cell number and asymmetry in fish brain nuclei

    NASA Astrophysics Data System (ADS)

    Anken, R. H.; Werner, K.; Rahmann, H.

    Larval cichlid fish ( Oreochromis mossambicus) siblings were subjected to 3g hypergravity (hg) and total darkness for 21 days during development and subsequently processed for conventional histology. Further siblings reared at 1g and alternating light/dark (12h:12h) conditions served as contros. Cell number counts of the visual Nucleus isthmi (Ni) versus the vestibular Nucleus magnocellularis (Nm) revealed that in experimental animals total cell number was decreased in the Ni, possibly due to retarded growth as a result of the lack of visual input whereas no effect was observed in the Nm. Calculating the percentual asymmetry in cell number (i.e., right vs. the left side of the brain), no effects of hg/darkness were seen in the Ni, whereas asymmetry was slightly increased in the Nm. Since the asymmetry of inner ear otoliths is decreased under hg, this finding may indicate efferent vestibular action of the CNS on the level of the Nm by means of a feedback mechanism.

  8. Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells.

    PubMed

    Trinkle-Mulcahy, L; Sleeman, J E; Lamond, A I

    2001-12-01

    Protein phosphatase 1 (PP1) is expressed in mammalian cells as three closely related isoforms, alpha, beta/delta and gamma1, which are encoded by separate genes. It has yet to be determined whether the separate isoforms behave in a similar fashion or play distinct roles in vivo. We report here on analyses by fluorescence microscopy of functional and fluorescently tagged PP1 isoforms in live cells. PP1alpha and PP1gamma fluorescent protein fusions show largely complimentary localization patterns, particularly within the nucleus where tagged PP1gamma accumulates in the nucleolus, whereas tagged PP1alpha is primarily found in the nucleoplasm. Overexpression of NIPP1 (nuclear inhibitor of PP1), a PP1 targeting subunit that accumulates at interchromatin granule clusters in the nucleoplasm, results in a retargeting of both isoforms to these structures, indicating that steady-state localization is based, at least in part, on relative affinities for various targeting subunits. Photobleaching analyses show that PP1gamma is rapidly exchanging between the nucleolar, nucleoplasmic and cytoplasmic compartments. Fluorescence resonance energy transfer (FRET) analyses indicate that the direct interaction of the two proteins predominantly occurs at or near interchromatin granule clusters. These data indicate that PP1 isoforms are highly mobile in cells and can be dynamically (re)localized through direct interaction with targeting subunits. PMID:11739654

  9. Reorganization of Synaptic Connections and Perineuronal Nets in the Deep Cerebellar Nuclei of Purkinje Cell Degeneration Mutant Mice.

    PubMed

    Blosa, M; Bursch, C; Weigel, S; Holzer, M; Jäger, C; Janke, C; Matthews, R T; Arendt, T; Morawski, M

    2016-01-01

    The perineuronal net (PN) is a subtype of extracellular matrix appearing as a net-like structure around distinct neurons throughout the whole CNS. PNs surround the soma, proximal dendrites, and the axonal initial segment embedding synaptic terminals on the neuronal surface. Different functions of the PNs are suggested which include support of synaptic stabilization, inhibition of axonal sprouting, and control of neuronal plasticity. A number of studies provide evidence that removing PNs or PN-components results in renewed neurite growth and synaptogenesis. In a mouse model for Purkinje cell degeneration, we examined the effect of deafferentation on synaptic remodeling and modulation of PNs in the deep cerebellar nuclei. We found reduced GABAergic, enhanced glutamatergic innervations at PN-associated neurons, and altered expression of the PN-components brevican and hapln4. These data refer to a direct interaction between ECM and synapses. The altered brevican expression induced by activated astrocytes could be required for an adequate regeneration by promoting neurite growth and synaptogenesis.

  10. Reorganization of Synaptic Connections and Perineuronal Nets in the Deep Cerebellar Nuclei of Purkinje Cell Degeneration Mutant Mice

    PubMed Central

    Blosa, M.; Bursch, C.; Weigel, S.; Holzer, M.; Jäger, C.; Janke, C.; Matthews, R. T.; Arendt, T.; Morawski, M.

    2016-01-01

    The perineuronal net (PN) is a subtype of extracellular matrix appearing as a net-like structure around distinct neurons throughout the whole CNS. PNs surround the soma, proximal dendrites, and the axonal initial segment embedding synaptic terminals on the neuronal surface. Different functions of the PNs are suggested which include support of synaptic stabilization, inhibition of axonal sprouting, and control of neuronal plasticity. A number of studies provide evidence that removing PNs or PN-components results in renewed neurite growth and synaptogenesis. In a mouse model for Purkinje cell degeneration, we examined the effect of deafferentation on synaptic remodeling and modulation of PNs in the deep cerebellar nuclei. We found reduced GABAergic, enhanced glutamatergic innervations at PN-associated neurons, and altered expression of the PN-components brevican and hapln4. These data refer to a direct interaction between ECM and synapses. The altered brevican expression induced by activated astrocytes could be required for an adequate regeneration by promoting neurite growth and synaptogenesis. PMID:26819763

  11. Reorganization of Synaptic Connections and Perineuronal Nets in the Deep Cerebellar Nuclei of Purkinje Cell Degeneration Mutant Mice.

    PubMed

    Blosa, M; Bursch, C; Weigel, S; Holzer, M; Jäger, C; Janke, C; Matthews, R T; Arendt, T; Morawski, M

    2016-01-01

    The perineuronal net (PN) is a subtype of extracellular matrix appearing as a net-like structure around distinct neurons throughout the whole CNS. PNs surround the soma, proximal dendrites, and the axonal initial segment embedding synaptic terminals on the neuronal surface. Different functions of the PNs are suggested which include support of synaptic stabilization, inhibition of axonal sprouting, and control of neuronal plasticity. A number of studies provide evidence that removing PNs or PN-components results in renewed neurite growth and synaptogenesis. In a mouse model for Purkinje cell degeneration, we examined the effect of deafferentation on synaptic remodeling and modulation of PNs in the deep cerebellar nuclei. We found reduced GABAergic, enhanced glutamatergic innervations at PN-associated neurons, and altered expression of the PN-components brevican and hapln4. These data refer to a direct interaction between ECM and synapses. The altered brevican expression induced by activated astrocytes could be required for an adequate regeneration by promoting neurite growth and synaptogenesis. PMID:26819763

  12. BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.

    PubMed

    Huang, Jiaojiao; Zhang, Hongyong; Yao, Jing; Qin, Guosong; Wang, Feng; Wang, Xianlong; Luo, Ailing; Zheng, Qiantao; Cao, Chunwei; Zhao, Jianguo

    2016-01-01

    Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.

  13. Characterization of ionic currents of cells of the subfornical organ that project to the supraoptic nuclei

    NASA Technical Reports Server (NTRS)

    Johnson, R. F.; Beltz, T. G.; Jurzak, M.; Wachtel, R. E.; Johnson, A. K.

    1999-01-01

    The subfornical organ (SFO) is a forebrain structure that converts peripheral blood-borne signals reflecting the hydrational state of the body to neural signals and then through efferent fibers conveys this information to several central nervous system structures. One of the forebrain areas receiving input from the SFO is the supraoptic nucleus (SON), a source of vasopressin synthesis and control of release from the posterior pituitary. Little is known of the transduction and transmission processes by which this conversion of systemic information to brain input occurs. As a step in elucidating these mechanisms, the present study characterized the ionic currents of dissociated cells of the SFO that were identified as neurons that send efferents to the SON. A retrograde tracer was injected into the SON area in eleven-day-old rats. After three days for retrograde transport of the label, the SFOs of these animals were dissociated and plated for tissue culture. The retrograde tracer was used to identify the soma of SFO cells projecting to the SON so that voltage-dependent ionic currents using whole-cell voltage clamp methods could be studied. The three types of currents in labeled SFO neurons were characterized as a 1) rapid, transient inward current that can be blocked by tetrodotoxin (TTX) characteristic of a sodium current; 2) slow-onset sustained outward current that can be blocked by tetraethylammonium (TEA) characteristic of a delayed rectifier potassium current; and 3) remaining outward current that has a rapid-onset and transient characteristic of a potassium A-type current. Copyright 1999 Elsevier Science B.V.

  14. Historical review of research on plant cell dedifferentiation.

    PubMed

    Sugiyama, Munetaka

    2015-05-01

    Plant cell dedifferentiation has long attracted interest as a key process for understanding the plasticity of plant development. In early studies, typical examples of plant cell dedifferentiation were described as physiological and cytological changes associated with wound healing or regenerative development. Subsequently, plant tissue and cell culture techniques, in which exciting progress was achieved after discovery of the hormonal control of cell proliferation and organogenesis in vitro in the 1950s, have been used extensively to study dedifferentiation. The pioneer studies of plant tissue/cell culture led to the hypothesis that many mature plant cells retain totipotency and related dedifferentiation to the initial step of the expression of totipotency. Plant tissue/cell cultures have provided experimental systems not only for physiological analysis, but also for genetic and molecular biological analysis, of dedifferentiation. More recently, proteomic, transcriptomic, and epigenetic analyses have been applied to the study of plant cell dedifferentiation. All of these works have expanded our knowledge of plant cell dedifferentiation, and current research is contributing to unraveling the molecular mechanisms. The present article provides a brief overview of the history of research on plant cell dedifferentiation. PMID:25725626

  15. Historical review of research on plant cell dedifferentiation.

    PubMed

    Sugiyama, Munetaka

    2015-05-01

    Plant cell dedifferentiation has long attracted interest as a key process for understanding the plasticity of plant development. In early studies, typical examples of plant cell dedifferentiation were described as physiological and cytological changes associated with wound healing or regenerative development. Subsequently, plant tissue and cell culture techniques, in which exciting progress was achieved after discovery of the hormonal control of cell proliferation and organogenesis in vitro in the 1950s, have been used extensively to study dedifferentiation. The pioneer studies of plant tissue/cell culture led to the hypothesis that many mature plant cells retain totipotency and related dedifferentiation to the initial step of the expression of totipotency. Plant tissue/cell cultures have provided experimental systems not only for physiological analysis, but also for genetic and molecular biological analysis, of dedifferentiation. More recently, proteomic, transcriptomic, and epigenetic analyses have been applied to the study of plant cell dedifferentiation. All of these works have expanded our knowledge of plant cell dedifferentiation, and current research is contributing to unraveling the molecular mechanisms. The present article provides a brief overview of the history of research on plant cell dedifferentiation.

  16. Evolution and diversity of green plant cell walls.

    PubMed

    Popper, Zoë A

    2008-06-01

    Plant cells are surrounded by a dynamic cell wall that performs many essential biological roles, including regulation of cell expansion, the control of tissue cohesion, ion-exchange and defence against microbes. Recent evidence shows that the suite of polysaccharides and wall proteins from which the plant cell wall is composed shows variation between monophyletic plant taxa. This is likely to have been generated during the evolution of plant groups in response to environmental stress. Understanding the natural variation and diversity that exists between cell walls from different taxa is key to facilitating their future exploitation and manipulation, for example by increasing lignocellulosic content or reducing its recalcitrance for use in biofuel generation.

  17. Role of Calcium and Calmodulin in Plant Cell Regulation

    NASA Technical Reports Server (NTRS)

    Cormier, M. J.

    1983-01-01

    The role of calcium and calmodulin in plant cell regulation is discussed. Experiments are done to discover the level of calcium in plants and animals. The effect of intracellular calcium on photosynthesis is discussed.

  18. Roles of membrane trafficking in plant cell wall dynamics

    PubMed Central

    Ebine, Kazuo; Ueda, Takashi

    2015-01-01

    The cell wall is one of the characteristic components of plant cells. The cell wall composition differs among cell types and is modified in response to various environmental conditions. To properly generate and modify the cell wall, many proteins are transported to the plasma membrane or extracellular space through membrane trafficking, which is one of the key protein transport mechanisms in eukaryotic cells. Given the diverse composition and functions of the cell wall in plants, the transport of the cell wall components and proteins that are involved in cell wall-related events could be specialized for each cell type, i.e., the machinery for cell wall biogenesis, modification, and maintenance could be transported via different trafficking pathways. In this review, we summarize the recent progress in the current understanding of the roles and mechanisms of membrane trafficking in plant cells and focus on the biogenesis and regulation of the cell wall. PMID:26539200

  19. Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei.

    PubMed

    Rauch, Joachim; Knoch, Tobias A; Solovei, Irina; Teller, Kathrin; Stein, Stefan; Buiting, Karin; Horsthemke, Bernhard; Langowski, Jörg; Cremer, Thomas; Hausmann, Michael; Cremer, Christoph

    2008-01-01

    Despite the major advancements during the last decade with respect to both knowledge of higher order chromatin organization in the cell nucleus and the elucidation of epigenetic mechanisms of gene control, the true three-dimensional (3D) chromatin structure of endogenous active and inactive gene loci is not known. The present study was initiated as an attempt to close this gap. As a model case, we compared the chromatin architecture between the genetically active and inactive domains of the imprinted Prader-Willi syndrome (PWS) locus in human fibroblast and lymphoblastoid cell nuclei by 3D fluorescence in situ hybridization and quantitative confocal laser scanning microscopy. The volumes and 3D compactions of identified maternal and paternal PWS domains were determined in stacks of light optical serial sections using a novel threshold-independent approach. Our failure to detect volume and compaction differences indicates that possible differences are below the limits of light optical resolution. To overcome this limitation, spectral precision distance microscopy, a method of localization microscopy at the nanometer scale, was used to measure 3D distances between differentially labeled probes located both within the PWS region and in its neighborhood. This approach allows the detection of intranuclear differences between 3D distances down to about 70-90 nm, but again did not reveal clearly detectable differences between active and inactive PWS domains. Despite this failure, a comparison of the experimental 3D distance measurements with computer simulations of chromatin folding strongly supports a non-random higher order chromatin configuration of the PWS locus and argues against 3D configurations based on giant chromatin loops. Our results indicate that the search for differences between endogenous active and inactive PWS domains must be continued at still smaller scales than hitherto possible with conventional light microscopic procedures. The possibilities to

  20. Efficient Delivery of DOX to Nuclei of Hepatic Carcinoma Cells in the Subcutaneous Tumor Model Using pH-Sensitive Pullulan-DOX Conjugates.

    PubMed

    Li, Huanan; Cui, Yani; Sui, Junhui; Bian, Shaoquan; Sun, Yong; Liang, Jie; Fan, Yujiang; Zhang, Xingdong

    2015-07-29

    A series of pullulan-doxorubicin conjugates (Pu-DOXs) were investigated for effectively delivering DOX to nuclei of hepatic carcinoma cells in subcutaneous tumor model. These Pu-DOXs were prepared by conjugating DOX onto pullulan molecule via pH-responsive hydrazone bond using spacers with different alkane chain length. The highest drug loading content of Pu-DOXs went up to nearly 50%, and the diameter of Pu-DOX nanoparticles ranged from 50 to 170 nm, as measured by DLS and TEM. These Pu-DOX nanoparticles could rapidly release DOX in the acidic environment at pH = 5.0 while being kept relatively stable in neural conditions. The in vitro cell coculture experiments revealed that these Pu-DOX nanoparticles were selectively internalized by hepatic carcinoma cells through receptor-mediated endocytosis via asialoglycoprotein receptor on the hepatic carcinoma cell surface. DOX was rapidly released from Pu-DOX nanoparticles in acidic endosome/lysosome, diffused into cell nuclei due to its strong affinity to nucleic acid, inhibited the cell proliferation, and accelerated the cell apoptosis. In the nude mice subcutaneous hepatic carcinoma model, Pu-DOX nanoparticles efficiently accumulated in the tumor site through the enhanced permeation and retention effect. Then DOX was specifically internalized by hepatic carcinoma cells and rapidly diffused into the nuclei of cells. Compared with the control group in in vivo experiments, these Pu-DOX nanoparticles effectively inhibited solid tumor growth, prolonging the lifetime of the experimental animal. These pH sensitive nanoparticles might provide an important clinical implication for targeted hepatic carcinoma therapy with high efficiency and low systematic toxicity.

  1. Segmentation of cell nuclei in heterogeneous microscopy images: a reshapable templates approach.

    PubMed

    Alilou, Mehdi; Kovalev, Vassili; Taimouri, Vahid

    2013-01-01

    Histological tissue images typically exhibit very sophisticated spatial color patterns. It is of great clinical importance to extract qualitative and quantitative information from these images. As an ad hoc solution, various unsupervised approaches address the object detection and segmentation problem which are suitable for limited classes of histology images. In this paper, we propose a general purpose localization and segmentation method which utilizes reshapable templates. The method combines both pixel- and object-level features for detecting regions of interest. Segmentation is carried out in two levels including both the coarse and fine ones. A set of simple-shaped templates is used for coarse segmentation. A content based template reshaping algorithm is proposed for fine segmentation of target objects. Experimentation was done using a publicly available image data set which contains 7931 manually labeled cells of heterogeneous histology images. The experiments have demonstrated acceptable level of detection and segmentation results for the proposed approach (precision=0.904, recall=0.870 and Zijdenbos similarity index=73%). Thus, the prototype software developed based on proposed method can be considered as a potential tool for pathologists in clinical process.

  2. mRNA export from mammalian cell nuclei is dependent on GANP.

    PubMed

    Wickramasinghe, Vihandha O; McMurtrie, Paul I A; Mills, Anthony D; Takei, Yoshinori; Penrhyn-Lowe, Sue; Amagase, Yoko; Main, Sarah; Marr, Jackie; Stewart, Murray; Laskey, Ronald A

    2010-01-12

    Bulk nuclear export of messenger ribonucleoproteins (mRNPs) through nuclear pore complexes (NPCs) is mediated by NXF1. It binds mRNPs through adaptor proteins such as ALY and SR splicing factors and mediates translocation through the central NPC transport channel via transient interactions with FG nucleoporins. Here, we show that mammalian cells require GANP (germinal center-associated nuclear protein) for efficient mRNP nuclear export and for efficient recruitment of NXF1 to NPCs. Separate regions of GANP show local homology to FG nucleoporins, the yeast mRNA export factor Sac3p, and the mammalian MCM3 acetyltransferase. GANP interacts with both NXF1 and NPCs and partitions between NPCs and the nuclear interior. GANP depletion inhibits mRNA export, with retention of mRNPs and NXF1 in punctate foci within the nucleus. The GANP N-terminal region that contains FG motifs interacts with the NXF1 FG-binding domain. Overexpression of this GANP fragment leads to nuclear accumulation of both poly(A)(+)RNA and NXF1. Treatment with transcription inhibitors redistributes GANP from NPCs into foci throughout the nucleus. These results establish GANP as an integral component of the mammalian mRNA export machinery and suggest a model whereby GANP facilitates the transfer of NXF1-containing mRNPs to NPCs. PMID:20005110

  3. Transcribed DNA is preferentially located in the perichromatin region of mammalian cell nuclei

    SciTech Connect

    Niedojadlo, Janusz; Perret-Vivancos, Cecile; Kalland, Karl-Henning; Cmarko, Dusan; Cremer, Thomas; Driel, Roel van; Fakan, Stanislav

    2011-02-15

    The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.

  4. Auxin regulation of cell polarity in plants.

    PubMed

    Pan, Xue; Chen, Jisheng; Yang, Zhenbiao

    2015-12-01

    Auxin is well known to control pattern formation and directional growth at the organ/tissue levels via the nuclear TIR1/AFB receptor-mediated transcriptional responses. Recent studies have expanded the arena of auxin actions as a trigger or key regulator of cell polarization and morphogenesis. These actions require non-transcriptional responses such as changes in the cytoskeleton and vesicular trafficking, which are commonly regulated by ROP/Rac GTPase-dependent pathways. These findings beg for the question about the nature of auxin receptors that regulate these responses and renew the interest in ABP1 as a cell surface auxin receptor, including the work showing auxin-binding protein 1 (ABP1) interacts with the extracellular domain of the transmembrane kinase (TMK) receptor-like kinases in an auxin-dependent manner, as well as the debate on this auxin binding protein discovered about 40 years ago. This review highlights recent work on the non-transcriptional auxin signaling mechanisms underscoring cell polarity and shape formation in plants.

  5. Auxin regulation of cell polarity in plants.

    PubMed

    Pan, Xue; Chen, Jisheng; Yang, Zhenbiao

    2015-12-01

    Auxin is well known to control pattern formation and directional growth at the organ/tissue levels via the nuclear TIR1/AFB receptor-mediated transcriptional responses. Recent studies have expanded the arena of auxin actions as a trigger or key regulator of cell polarization and morphogenesis. These actions require non-transcriptional responses such as changes in the cytoskeleton and vesicular trafficking, which are commonly regulated by ROP/Rac GTPase-dependent pathways. These findings beg for the question about the nature of auxin receptors that regulate these responses and renew the interest in ABP1 as a cell surface auxin receptor, including the work showing auxin-binding protein 1 (ABP1) interacts with the extracellular domain of the transmembrane kinase (TMK) receptor-like kinases in an auxin-dependent manner, as well as the debate on this auxin binding protein discovered about 40 years ago. This review highlights recent work on the non-transcriptional auxin signaling mechanisms underscoring cell polarity and shape formation in plants. PMID:26599954

  6. Over-expression of GFP-FEZ1 causes generation of multi-lobulated nuclei mediated by microtubules in HEK293 cells

    SciTech Connect

    Lanza, Daniel C.F.; Trindade, Daniel M.; Assmann, Eliana M.; Kobarg, Joerg

    2008-06-10

    FEZ1 (Fasciculation and elongation protein zeta 1) is an ortholog of the Caenorhabditis elegans protein UNC-76, involved in neuronal development and axon outgrowth, in that worm. Mammalian FEZ1 has already been reported to cooperate with PKC-zeta in the differentiation and polarization of PC12 neuronal cells. Furthermore, FEZ1 is associated with kinesin 1 and JIP1 to form a cargo-complex responsible for microtubule based transport of mitochondria along axons. FEZ1 can also be classified as a hub protein, since it was reported to interact with over 40 different proteins in yeast two-hybrid screens, including at least nine nuclear proteins. Here, we transiently over-expressed GFP-FEZ1full in human HEK293 and HeLa cells in order to study the sub-cellular localization of GFP-FEZ1. We observed that over 40% of transiently transfected cells at 3 days post-transfection develop multi-lobulated nuclei, which are also called flower-like nuclei. We further demonstrated that GFP-FEZ1 localizes either to the cytoplasm or the nuclear fraction, and that the appearance of the flower-like nuclei depends on intact microtubule function. Finally, we show that FEZ1 co-localizes with both, {alpha}- and especially with {gamma}-tubulin, which localizes as a centrosome like structure at the center of the multiple lobules. In summary, our data suggest that FEZ1 has an important centrosomal function and supply new mechanistic insights to the formation of flower-like nuclei, which are a phenotypical hallmark of human leukemia cells.

  7. In situ methods to localize transgenes and transcripts in interphase nuclei: a tool for transgenic plant research

    PubMed Central

    Santos, Ana Paula; Wegel, Eva; Allen, George C; Thompson, William F; Stoger, Eva; Shaw, Peter; Abranches, Rita

    2006-01-01

    Genetic engineering of commercially important crops has become routine in many laboratories. However, the inability to predict where a transgene will integrate and to efficiently select plants with stable levels of transgenic expression remains a limitation of this technology. Fluorescence in situ hybridization (FISH) is a powerful technique that can be used to visualize transgene integration sites and provide a better understanding of transgene behavior. Studies using FISH to characterize transgene integration have focused primarily on metaphase chromosomes, because the number and position of integration sites on the chromosomes are more easily determined at this stage. However gene (and transgene) expression occurs mainly during interphase. In order to accurately predict the activity of a transgene, it is critical to understand its location and dynamics in the three-dimensional interphase nucleus. We and others have developed in situ methods to visualize transgenes (including single copy genes) and their transcripts during interphase from different tissues and plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transgene activity. Furthermore, this approach is useful for studying nuclear organization and the dynamics of genes and chromatin. PMID:17081287

  8. Cell biology of plant gravity sensing

    NASA Astrophysics Data System (ADS)

    Sack, F. D.

    1994-08-01

    The debate about whether gravity sensing relies upon statoliths (amyloplasts that sediment) has intensified with recent findings of gravitropism in starchless mutants and of claims of hydrostatic gravity sensing. Starch and significant plastid sedimentation are not necessary for reduced sensing in mutant roots, but plastids might function here if there were a specialized receptor for plastid mass e.g. in the ER. Alternatively, components in addition to amyloplasts might provide mass for sensing. The nucleus is dense and its position is regulated, but no direct data exist for its role in sensing. If the weight of the protoplast functioned in sensing, why would there be specific cytological specializations favoring sedimentation rather than cell mass? Gravity has multiple effects on plants in addition to gravitropism. There may be more than one mechanism of gravity sensing.

  9. Programmed cell death in the plant immune system

    PubMed Central

    Coll, N S; Epple, P; Dangl, J L

    2011-01-01

    Cell death has a central role in innate immune responses in both plants and animals. Besides sharing striking convergences and similarities in the overall evolutionary organization of their innate immune systems, both plants and animals can respond to infection and pathogen recognition with programmed cell death. The fact that plant and animal pathogens have evolved strategies to subvert specific cell death modalities emphasizes the essential role of cell death during immune responses. The hypersensitive response (HR) cell death in plants displays morphological features, molecular architectures and mechanisms reminiscent of different inflammatory cell death types in animals (pyroptosis and necroptosis). In this review, we describe the molecular pathways leading to cell death during innate immune responses. Additionally, we present recently discovered caspase and caspase-like networks regulating cell death that have revealed fascinating analogies between cell death control across both kingdoms. PMID:21475301

  10. Plant and animal stem cells: similar yet different.

    PubMed

    Heidstra, Renze; Sabatini, Sabrina

    2014-05-01

    The astonishingly long lives of plants and their regeneration capacity depend on the activity of plant stem cells. As in animals, stem cells reside in stem cell niches, which produce signals that regulate the balance between self-renewal and the generation of daughter cells that differentiate into new tissues. Plant stem cell niches are located within the meristems, which are organized structures that are responsible for most post-embryonic development. The continuous organ production that is characteristic of plant growth requires a robust regulatory network to keep the balance between pluripotent stem cells and differentiating progeny. Components of this network have now been elucidated and provide a unique opportunity for comparing strategies that were developed in the animal and plant kingdoms, which underlie the logic of stem cell behaviour.

  11. A unique perylene-based DNA intercalator: localization in cell nuclei and inhibition of cancer cells and tumors.

    PubMed

    Xu, Zejun; Guo, Kunru; Yu, Jieshi; Sun, Haili; Tang, Jun; Shen, Jie; Müllen, Klaus; Yang, Wantai; Yin, Meizhen

    2014-10-29

    To date, perylene derivatives have not been explored as DNA intercalator to inhibit cancer cells by intercalating into the base pairs of DNA. Herein, a water-soluble perylene bisimide (PBDI) that efficiently intercalates into the base pairs of DNA is synthesized. Excitingly, PBDI is superior to the commercial DNA intercalator, amonafide, for specific nuclear accumulation and effective suppression of cancer cells and tumors.

  12. Proteomic profiling of nuclei from native renal inner medullary collecting duct cells using LC-MS/MS

    PubMed Central

    Tchapyjnikov, Dmitry; Li, Yuedan; Pisitkun, Trairak; Hoffert, Jason D.; Yu, Ming-Jiun

    2010-01-01

    Vasopressin is a peptide hormone that regulates renal water excretion in part through its actions on the collecting duct. The regulation occurs in part via control of transcription of genes coding for the water channels aquaporin-2 (Aqp2) and aquaporin-3 (Aqp3). To identify transcription factors expressed in collecting duct cells, we have carried out LC-MS/MS-based proteomic profiling of nuclei isolated from native rat inner medullary collecting ducts (IMCDs). To maximize the number of proteins identified, we matched spectra to rat amino acid sequences using three different search algorithms (SEQUEST, InsPecT, and OMSSA). All searches were coupled to target-decoy methodology to limit false-discovery identifications to 2% of the total for single-peptide identifications. In addition, we developed a computational tool (ProMatch) to identify and eliminate ambiguous identifications. With this approach, we identified >3,500 proteins, including 154 proteins classified as “transcription factor” proteins (Panther Classification System). Among these, are members of CREB, ETS, RXR, NFAT, HOX, GATA, EBOX, EGR, MYT1, KLF, and CP2 families, which were found to have evolutionarily conserved putative binding sites in the 5′-flanking region or first intron of the Aqp2 gene, as well as members of EBOX, NR2, GRE, MAZ, KLF, and SP1 families corresponding to conserved sites in the 5′-flanking region of the Aqp3 gene. In addition, several novel phosphorylation sites in nuclear proteins were identified using the neutral loss-scanning LC-MS3 technique. The newly identified proteins have been incorporated into the IMCD Proteome Database (http://dir.nhlbi.nih.gov/papers/lkem/imcd/). PMID:19996160

  13. 76 FR 63702 - In the Matter of the Designation of Conspiracy of Fire Nuclei, aka Conspiracy of the Nuclei of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-13

    ... Matter of the Designation of Conspiracy of Fire Nuclei, aka Conspiracy of the Nuclei of Fire, aka Conspiracy of Cells of Fire, aka Synomosia of Pyrinon Tis Fotias, aka Thessaloniki-Athens Fire Nuclei... January 23, 2003, I hereby determine that the organization known as Conspiracy of Fire Nuclei, also...

  14. Projections to the anterodorsal thalamus and lateral mammillary nuclei arise from different cell populations within the postsubiculum: implications for the control of head direction cells.

    PubMed

    Yoder, Ryan M; Taube, Jeffrey S

    2011-10-01

    The neural representation of directional heading is encoded by a population of cells located in a circuit that includes the postsubiculum (PoS), anterodorsal thalamus (ADN), and lateral mammillary nuclei (LMN). Throughout this circuit, many cells rely on both movement- and landmark-related information to discharge as a function of the animal's directional heading. The PoS projects to both the ADN and LMN, and these connections may convey critical spatial information about landmarks, because lesions of the PoS disrupt landmark control in head direction (HD) cells and hippocampal place cells [Goodridge and Taube (1997) J Neurosci 17:9315-9330; Calton et al. (2003) J Neurosci 23:9719-9731]. The PoS → ADN projection originates in the deep layers of PoS, but no studies have determined whether the PoS → LMN projection originates from the same cells that project to ADN. To address this issue, two distinct cholera toxin-subunit B (CTB) fluorophore conjugates (Alexa Fluor 488 and Alexa Fluor 594) were injected into the LMN and ADN of the same rats, and PoS sections were examined for cell bodies containing either or both CTB conjugates. Results indicated that the PoS → LMN projection originates exclusively from a thin layer of cells located superficial to the layer(s) of PoS → ADN projection cells, with no overlap. To verify the laminar distribution and morphological characteristics of PoS → LMN and PoS → ADN cells, biotinylated dextran amine was injected into LMN or ADN of different rats, and tissue sections were counterstained with thionin. Results indicated that the PoS → LMN projection arises from large pyramidal cells in layer IV, whereas the PoS → ADN projection arises from a heterogeneous cell population in layers V/VI. This study provides the first evidence that the PoS → ADN and PoS → LMN projections arise from distinct, nonoverlapping cell layers in PoS. Functionally, the PoS may provide landmark information to HD cells in LMN.

  15. Tanshinone IIA enhances bystander cell killing of cancer cells expressing Drosophila melanogaster deoxyribonucleoside kinase in nuclei and mitochondria.

    PubMed

    Jiang, Haiyang; Zhao, Lei; Dong, Xiaoshen; He, Anning; Zheng, Caiwei; Johansson, Magnus; Karlsson, Anna; Zheng, Xinyu

    2015-09-01

    Heterologous expression of the Drosophila melanogaster multi-substrate deoxyribonucleoside kinase (Dm-dNK) increases the sensitivity of cancer cells to several cytotoxic nucleoside analogs. Thus, it may be used as a suicide gene in combined gene/chemotherapy treatment of cancer. To further characterize this potential suicide gene, we constructed two retroviral vectors that enabled the expression of Dm-dNK in cancer cells. One vector harbored the wild‑type enzyme that localized to the nucleus. The other vector harbored a mitochondrial localized mutant enzyme that was constructed by deleting the nuclear localization signal and fusing it to a mitochondrial import signal of cytochrome c oxidase. A thymidine kinase-deficient osteosarcoma cell line was transduced with the recombinant viruses. The sensitivity and bystander cell killing in the presence of pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)‑2'‑deoxyuridine and 1-β-D-arabinofuranosylthymine were investigated. Tanshinone IIA is a constituent of Danshen; a traditional Chinese medicine used in the treatment of cardiovascular diseases. This study also looked at the influence of Tanshinone IIA on the bystander effect and the underlying mechanisms. We showed that sensitivity of the osteosarcoma cell line to the nucleoside analogs and the efficiency of bystander cell killing were independent of the subcellular localization of Dm-dNK. The enhanced effect of tanshinone IIA on the bystander effect was related to the increased expression of Cx43 and Cx26.

  16. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    ERIC Educational Resources Information Center

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  17. Formative cell divisions: principal determinants of plant morphogenesis.

    PubMed

    Smolarkiewicz, Michalina; Dhonukshe, Pankaj

    2013-03-01

    Formative cell divisions utilizing precise rotations of cell division planes generate and spatially place asymmetric daughters to produce different cell layers. Therefore, by shaping tissues and organs, formative cell divisions dictate multicellular morphogenesis. In animal formative cell divisions, the orientation of the mitotic spindle and cell division planes relies on intrinsic and extrinsic cortical polarity cues. Plants lack known key players from animals, and cell division planes are determined prior to the mitotic spindle stage. Therefore, it appears that plants have evolved specialized mechanisms to execute formative cell divisions. Despite their profound influence on plant architecture, molecular players and cellular mechanisms regulating formative divisions in plants are not well understood. This is because formative cell divisions in plants have been difficult to track owing to their submerged positions and imprecise timings of occurrence. However, by identifying a spatiotemporally inducible cell division plane switch system applicable for advanced microscopy techniques, recent studies have begun to uncover molecular modules and mechanisms for formative cell divisions. The identified molecular modules comprise developmentally triggered transcriptional cascades feeding onto microtubule regulators that now allow dissection of the hierarchy of the events at better spatiotemporal resolutions. Here, we survey the current advances in understanding of formative cell divisions in plants in the context of embryogenesis, stem cell functionality and post-embryonic organ formation.

  18. Formative cell divisions: principal determinants of plant morphogenesis.

    PubMed

    Smolarkiewicz, Michalina; Dhonukshe, Pankaj

    2013-03-01

    Formative cell divisions utilizing precise rotations of cell division planes generate and spatially place asymmetric daughters to produce different cell layers. Therefore, by shaping tissues and organs, formative cell divisions dictate multicellular morphogenesis. In animal formative cell divisions, the orientation of the mitotic spindle and cell division planes relies on intrinsic and extrinsic cortical polarity cues. Plants lack known key players from animals, and cell division planes are determined prior to the mitotic spindle stage. Therefore, it appears that plants have evolved specialized mechanisms to execute formative cell divisions. Despite their profound influence on plant architecture, molecular players and cellular mechanisms regulating formative divisions in plants are not well understood. This is because formative cell divisions in plants have been difficult to track owing to their submerged positions and imprecise timings of occurrence. However, by identifying a spatiotemporally inducible cell division plane switch system applicable for advanced microscopy techniques, recent studies have begun to uncover molecular modules and mechanisms for formative cell divisions. The identified molecular modules comprise developmentally triggered transcriptional cascades feeding onto microtubule regulators that now allow dissection of the hierarchy of the events at better spatiotemporal resolutions. Here, we survey the current advances in understanding of formative cell divisions in plants in the context of embryogenesis, stem cell functionality and post-embryonic organ formation. PMID:23248201

  19. Nuclear export in plants. Use of geminivirus movement proteins for a cell-based export assay.

    PubMed Central

    Ward, B M; Lazarowitz, S G

    1999-01-01

    The nuclear export of proteins and RNAs has been studied in heterokaryons or by microinjecting test substrates into nuclei of HeLa cells or Xenopus oocytes. We have previously shown that the two movement proteins BR1 and BL1 encoded by the plant pathogenic squash leaf curl virus act in a coordinated manner to facilitate virus cell-to-cell movement and that one of these (BR1) is a nuclear shuttle protein. By using a novel in vivo cell-based assay for nuclear export in which nuclear-localized BR1 is trapped by BL1 and redirected to the cortical cytoplasm, we demonstrate that residues 177 to 198 of BR1 contain a leucine-rich nuclear export signal (NES) of the type found in the Rev protein encoded by the human immunodeficiency virus and in Xenopus TFIIIA. We further show that the TFIIIA NES can functionally replace the NES of BR1 in both nuclear export and viral infectivity. These findings suggest that this basic pathway for nuclear export is highly conserved among plant and animal cells and in yeast. PMID:10402428

  20. Low concentrations of the toxin ophiobolin A lead to an arrest of the cell cycle and alter the intracellular partitioning of glutathione between the nuclei and cytoplasm.

    PubMed

    Locato, Vittoria; Uzal, Esther Novo; Cimini, Sara; Zonno, Maria Chiara; Evidente, Antonio; Micera, Alessandra; Foyer, Christine H; De Gara, Laura

    2015-05-01

    Ophiobolin A, a tetracyclic sesterpenoid produced by phytopathogenic fungi, is responsible for catastrophic losses in crop yield but its mechanism of action is not understood. The effects of ophiobolin A were therefore investigated on the growth and redox metabolism of Tobacco Bright Yellow-2 (TBY-2) cell cultures by applying concentrations of the toxin that did not promote cell death. At concentrations between 2 and 5 μM, ophiobolin A inhibited growth and proliferation of the TBY-2 cells, which remained viable. Microscopic and cytofluorimetric analyses showed that ophiobolin A treatment caused a rapid decrease in mitotic index, with a lower percentage of the cells at G1 and increased numbers of cells at the S/G2 phases. Cell size was not changed following treatment suggesting that the arrest of cell cycle progression was not the result of a block on cell growth. The characteristic glutathione redox state and the localization of glutathione in the nucleus during cell proliferation were not changed by ophiobolin A. However, subsequent decreases in glutathione and the re-distribution of glutathione between the cytoplasm and nuclei after mitosis occurring in control cells, as well as the profile of glutathionylated proteins, were changed in the presence of the toxin. The profile of poly ADP-ribosylated proteins were also modified by ophiobolin A. Taken together, these data provide evidence of the mechanism of ophiobolin A action as a cell cycle inhibitor and further demonstrate the link between nuclear glutathione and the cell cycle regulation, suggesting that glutathione-dependent redox controls in the nuclei prior to cell division are of pivotal importance.

  1. Loss of Ptf1a Leads to a Widespread Cell-Fate Misspecification in the Brainstem, Affecting the Development of Somatosensory and Viscerosensory Nuclei

    PubMed Central

    Iskusnykh, Igor Y.; Steshina, Ekaterina Y.

    2016-01-01

    The brainstem contains diverse neuronal populations that regulate a wide range of processes vital to the organism. Proper cell-fate specification decisions are critical to achieve neuronal diversity in the CNS, but the mechanisms regulating cell-fate specification in the developing brainstem are poorly understood. Previously, it has been shown that basic helix-loop-helix transcription factor Ptf1a is required for the differentiation and survival of neurons of the inferior olivary and cochlear brainstem nuclei, which contribute to motor coordination and sound processing, respectively. In this study, we show that the loss of Ptf1a compromises the development of the nucleus of the solitary tract, which processes viscerosensory information, and the spinal and principal trigeminal nuclei, which integrate somatosensory information of the face. Combining genetic fate-mapping, birth-dating, and gene expression studies, we found that at least a subset of brainstem abnormalities in Ptf1a−/− mice are mediated by a dramatic cell-fate misspecification in rhombomeres 2–7, which results in the production of supernumerary viscerosensory and somatosensory neurons of the Lmx1b lineage at the expense of Pax2+ GABAergic viscerosensory and somatosensory neurons, and inferior olivary neurons. Our data identify Ptf1a as a major regulator of cell-fate specification decisions in the developing brainstem, and as a previously unrecognized developmental regulator of both viscerosensory and somatosensory brainstem nuclei. SIGNIFICANCE STATEMENT Cell-fate specification decisions are critical for normal CNS development. Although extensively studied in the cerebellum and spinal cord, the mechanisms mediating cell-fate decisions in the brainstem, which regulates a wide range of processes vital to the organism, remain largely unknown. Here we identified mouse Ptf1a as a novel regulator of cell-fate decisions during both early and late brainstem neurogenesis, which are critical for proper

  2. Experimental approaches to study plant cell walls during plant-microbe interactions

    PubMed Central

    Xia, Ye; Petti, Carloalberto; Williams, Mark A.; DeBolt, Seth

    2014-01-01

    Plant cell walls provide physical strength, regulate the passage of bio-molecules, and act as the first barrier of defense against biotic and abiotic stress. In addition to providing structural integrity, plant cell walls serve an important function in connecting cells to their extracellular environment by sensing and transducing signals to activate cellular responses, such as those that occur during pathogen infection. This mini review will summarize current experimental approaches used to study cell wall functions during plant-pathogen interactions. Focus will be paid to cell imaging, spectroscopic analyses, and metabolic profiling techniques. PMID:25352855

  3. Experimental approaches to study plant cell walls during plant-microbe interactions.

    PubMed

    Xia, Ye; Petti, Carloalberto; Williams, Mark A; DeBolt, Seth

    2014-01-01

    Plant cell walls provide physical strength, regulate the passage of bio-molecules, and act as the first barrier of defense against biotic and abiotic stress. In addition to providing structural integrity, plant cell walls serve an important function in connecting cells to their extracellular environment by sensing and transducing signals to activate cellular responses, such as those that occur during pathogen infection. This mini review will summarize current experimental approaches used to study cell wall functions during plant-pathogen interactions. Focus will be paid to cell imaging, spectroscopic analyses, and metabolic profiling techniques.

  4. Electrophoretic mobility of cell nuclei (EMN index) as a biomarker of the biological aging process: Considering the association between EMN index and age.

    PubMed

    Czapla, Z; McPhail, S M

    2015-12-01

    The present study examined whether a specific property of cell microstructures may be useful as a biomarker of aging. Specifically, the association between age and changes of cellular structures reflected in electrophoretic mobility of cell nuclei index (EMN index) values across the adult lifespan was examined. This report considers findings from cross sections of females (n=1273) aged 18-98 years, and males (n=506) aged 19-93 years. A Biotest apparatus was used to perform intracellular microelectrophoresis on buccal epithelial cells collected from each individual. EMN index was calculated on the basis of the number of epithelial cells with mobile nuclei in reference to the cells with immobile nuclei per 100cells. Regression analyses indicated a significant negative association between EMN index value and age for men (r=-0.71, p<0.001) and women (r=-0.60, p<0.001); demonstrating a key requirement that must be met by a biomarker of aging. The strength of association observed between EMN index and age for both men and women was encouraging and supports the potential use of EMN index for determining a biological age of an individual (or a group). In this study, a new attempt of complex explanation of cellular mechanisms contributing to age related changes of the EMN index was made. In this study, a new attempt of complex explanation of cellular mechanisms contributing to age related changes of the EMN index was made. EMN index has demonstrated potential to meet criteria proposed for biomarkers of aging and further investigations are necessary.

  5. ENERGY PRODUCTION AND POLLUTION PREVENTION AT SEWAGE TREATMENT PLANTS USING FUEL CELL POWER PLANTS

    EPA Science Inventory

    The paper discusses energy production and pollution prevention at sewage treatment plants using fuel cell power plants. Anaerobic digester gas (ADG) is produced at waste water treatment plants during the anaerobic treatment of sewage to reduce solids. The major constituents are...

  6. Progress and prospects for phosphoric acid fuel cell power plants

    SciTech Connect

    Bonville, L.J.; Scheffler, G.W.; Smith, M.J.

    1996-12-31

    International Fuel Cells (IFC) has developed the fuel cell power plant as a new, on-site power generation source. IFC`s commercial fuel cell product is the 200-kW PC25{trademark} power plant. To date over 100 PC25 units have been manufactured. Fleet operating time is in excess of one million hours. Individual units of the initial power plant model, the PC25 A, have operated for more than 30,000 hours. The first model {open_quotes}C{close_quotes} power plant has over 10,000 hours of operation. The manufacturing, application and operation of this power plant fleet has established a firm base for design and technology development in terms of a clear understanding of the requirements for power plant reliability and durability. This fleet provides the benchmark against which power plant improvements must be measured.

  7. Cell biology of the plant-powdery mildew interaction.

    PubMed

    Hückelhoven, Ralph; Panstruga, Ralph

    2011-12-01

    Powdery mildew fungi represent a paradigm for obligate biotrophic parasites, which only propagate in long-lasting intimate interactions with living host cells. These highly specialized phytopathogens induce re-organization of host cell architecture and physiology for their own demands. This probably includes the corruption of basal host cellular functions for successful fungal pathogenesis. Recent studies revealed secretory processes by both interaction partners as key incidents of the combat at the plant-fungus interface. The analysis of cellular events during plant-powdery mildew interactions may not only lead to a better understanding of plant pathological features, but may also foster novel discoveries in the area of plant cell biology.

  8. The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear lamins

    NASA Technical Reports Server (NTRS)

    Tong, C. G.; Dauwalder, M.; Clawson, G. A.; Hatem, C. L.; Roux, S. J.

    1993-01-01

    The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.

  9. Methods for degrading or converting plant cell wall polysaccharides

    DOEpatents

    Berka, Randy; Cherry, Joel

    2008-08-19

    The present invention relates to methods for converting plant cell wall polysaccharides into one or more products, comprising: treating the plant cell wall polysaccharides with an effective amount of a spent whole fermentation broth of a recombinant microorganism, wherein the recombinant microorganism expresses one or more heterologous genes encoding enzymes which degrade or convert the plant cell wall polysaccharides into the one or more products. The present invention also relates to methods for producing an organic substance, comprising: (a) saccharifying plant cell wall polysaccharides with an effective amount of a spent whole fermentation broth of a recombinant microorganism, wherein the recombinant microorganism expresses one or more heterologous genes encoding enzymes which degrade or convert the plant cell wall polysaccharides into saccharified material; (b) fermenting the saccharified material of step (a) with one or more fermenting microoganisms; and (c) recovering the organic substance from the fermentation.

  10. The role of root border cells in plant defense.

    PubMed

    Hawes, M C; Gunawardena, U; Miyasaka, S; Zhao, X

    2000-03-01

    The survival of a plant depends upon the capacity of root tips to sense and move towards water and other nutrients in the soil. Perhaps because of the root tip's vital role in plant health, it is ensheathed by large populations of detached somatic cells - root 'border' cells - which have the ability to engineer the chemical and physical properties of the external environment. Of particular significance, is the production by border cells of specific chemicals that can dramatically alter the behavior of populations of soilborne microflora. Molecular approaches are being used to identify and manipulate the expression of plant genes that control the production and the specialized properties of border cells in transgenic plants. Such plants can be used to test the hypothesis that these unusual cells act as a phalanx of biological 'goalies', which neutralize dangers to newly generated root tissue as the root tip makes its way through soil.

  11. Cell polarity in plants: a PARspective on PINs.

    PubMed

    Geldner, Niko

    2009-02-01

    Plants have acquired the ability for organized multicellular development independent from animals. Because of this, they represent an independent example in nature for the development of coordinated, complex cell polarity from the simple polarity found in unicellular eukaryotes. Plants display a striking array of polarized cell types, with different axes of polarity being defined in one cell. The most investigated and best understood aspect of plant polarity is the apical-basal polarity of the PIN family of auxin efflux facilitators, which are of crucial importance for the organization of the entire plant body. Striking differences exist between the PAR-polarity modules known in animals and the ways PINs polarize plant cells. Nonetheless, a common regulatory logic probably applies to all polarizing eukaryotic cells, which includes self-reinforcing, positive feedback loops, intricate interactions between membrane-attached proteins, lipid signatures, and the targeting of transmembrane proteins to the correct domains of the plasma membrane. PMID:18993110

  12. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

    PubMed

    Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-03-10

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

  13. Plant cell piercing by a predatory mite: evidence and implications.

    PubMed

    Adar, E; Inbar, M; Gal, S; Issman, L; Palevsky, E

    2015-02-01

    Omnivorous arthropods can play an important role as beneficial natural enemies because they can sustain their populations on plants when prey is scarce, thereby providing prophylactic protection against an array of herbivores. Although some omnivorous mite species of the family Phytoseiidae consume plant cell-sap, the feeding mechanism and its influence on the plant are not known. Using scanning electron microscopy we demonstrated that the omnivorous predatory mite Euseius scutalis penetrates epidermal cells of pepper foliage and wax membranes. Penetration holes were teardrop shape to oval, of 2-5 µm diameter. The similarities between penetration holes in pollen grains and in epidermal cells implied that the same penetration mechanism is used for pollen feeding and plant cell-sap uptake. Variation in shape and size of penetration holes in leaves and a wax membrane were attributed to different mite life stages, depth of penetration or the number of chelicerae puncturing (one or both). Punctured stomata, epidermal and vein cells appeared flat and lacking turgor. When the mite penetrated and damaged a single cell, neighboring cells were most often intact. In a growth chamber experiment very large numbers of E. scutalis negatively affected the growth of young pepper plants. Consequently caution should be taken when applying cell-piercing predators to young plants. Further studies are needed to take advantage of the potential sustainability of plant cell-sap feeding predators.

  14. Plant cell wall proteomics: the leadership of Arabidopsis thaliana

    PubMed Central

    Albenne, Cécile; Canut, Hervé; Jamet, Elisabeth

    2013-01-01

    Plant cell wall proteins (CWPs) progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses, and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose, and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cell walls need to be modified and customized during plant development and in response to environmental cues, thus contributing to plant adaptation. CWPs play essential roles in all these physiological processes and particularly in the dynamics of cell walls, which requires organization and rearrangements of polysaccharides as well as cell-to-cell communication. In the last 10 years, plant cell wall proteomics has greatly contributed to a wider knowledge of CWPs. This update will deal with (i) a survey of plant cell wall proteomics studies with a focus on Arabidopsis thaliana; (ii) the main protein families identified and the still missing peptides; (iii) the persistent issue of the non-canonical CWPs; (iv) the present challenges to overcome technological bottlenecks; and (v) the perspectives beyond cell wall proteomics to understand CWP functions. PMID:23641247

  15. Exposure to acoustic stimuli promotes the development and differentiation of neural stem cells from the cochlear nuclei through the clusterin pathway.

    PubMed

    Xue, Tao; Wei, Li; Zha, Ding-Jun; Qiao, Li; Lu, Lian-Jun; Chen, Fu-Quan; Qiu, Jian-Hua

    2015-03-01

    Stem cell therapy has attracted widespread attention for a number of diseases. Recently, neural stem cells (NSCs) from the cochlear nuclei have been identified, indicating a potential direction for the treatment of sensorineural hearing loss. Acoustic stimuli play an important role in the development of the auditory system. In this study, we aimed to determine whether acoustic stimuli induce NSC development and differentiation through the upregulation of clusterin (CLU) in NSCs isolated from the cochlear nuclei. To further clarify the underlying mechanisms involved in the development and differentiation of NSCs exposed to acoustic stimuli, we successfully constructed animal models in which was CLU silenced by an intraperitoneal injection of shRNA targeting CLI. As expected, the NSCs from rats treated with LV-CLU shRNA exhibited a lower proliferation ratio when exposed to an augmented acoustic environment (AAE). Furthermore, the inhibition of cell apoptosis induced by exposure to AAE was abrogated after silencing the expression of the CLU gene. During the differentiation of acoustic stimuli-exposed stem cells into neurons, the number of astrocytes was significantly reduced, as evidenced by the expression of the cell markers, microtubule associated protein‑2 (MAP-2) and glial fibrillary acidic protein (GFAP), which was markedly inhibited when the CLU gene was silenced. Our results indicate that acoustic stimuli may induce the development and differentiation of NSCs from the cochlear nucleus mainly through the CLU pathway. Our study suggests that CLU may be a novel target for the treatment of sensorineural hearing loss.

  16. Cell biology of molybdenum in plants.

    PubMed

    Mendel, Ralf R

    2011-10-01

    The transition element molybdenum (Mo) is of essential importance for (nearly) all biological systems as it is required by enzymes catalyzing important reactions within the cell. The metal itself is biologically inactive unless it is complexed by a special cofactor. With the exception of bacterial nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor (Moco) which is the active compound at the catalytic site of all other Mo-enzymes. In plants, the most prominent Mo-enzymes are nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and the mitochondrial amidoxime reductase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also includes iron as well as copper in an indispensable way. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco-carrier/binding proteins that also participate in Moco-insertion into the cognate apoproteins. Xanthine dehydrogenase and aldehyde oxidase, but not the other Mo-enzymes, require a final step of posttranslational activation of their catalytic Mo-center for becoming active.

  17. (Hydroxyproline-rich glycoproteins of the plant cell wall)

    SciTech Connect

    Varner, J.E.

    1990-01-01

    We are studying the chemistry and architecture of plant cells walls, the extracellular matrices that taken together shape the plant and provide mechanical support for the plant. Cell walls are dynamic structures that regulate, or are the site of, many physiological processes, in addition to being the cells' first line of defense against invading pathogens. In the past year we have examined the role of the cell wall enzyme ascorbic acid oxidase as related to the structure of the wall and its possible interactions with hydroxyproline-rich glycoproteins of the wall.

  18. Penium margaritaceum as a model organism for cell wall analysis of expanding plant cells.

    PubMed

    Rydahl, Maja G; Fangel, Jonatan U; Mikkelsen, Maria Dalgaard; Johansen, I Elisabeth; Andreas, Amanda; Harholt, Jesper; Ulvskov, Peter; Jørgensen, Bodil; Domozych, David S; Willats, William G T

    2015-01-01

    The growth of a plant cell encompasses a complex set of subcellular components interacting in a highly coordinated fashion. Ultimately, these activities create specific cell wall structural domains that regulate the prime force of expansion, internally generated turgor pressure. The precise organization of the polymeric networks of the cell wall around the protoplast also contributes to the direction of growth, the shape of the cell, and the proper positioning of the cell in a tissue. In essence, plant cell expansion represents the foundation of development. Most studies of plant cell expansion have focused primarily upon late divergent multicellular land plants and specialized cell types (e.g., pollen tubes, root hairs). Here, we describe a unicellular green alga, Penium margaritaceum (Penium), which can serve as a valuable model organism for understanding cell expansion and the underlying mechanics of the cell wall in a single plant cell.

  19. Multidimensional solid-state NMR spectroscopy of plant cell walls.

    PubMed

    Wang, Tuo; Phyo, Pyae; Hong, Mei

    2016-09-01

    Plant biomass has become an important source of bio-renewable energy in modern society. The molecular structure of plant cell walls is difficult to characterize by most atomic-resolution techniques due to the insoluble and disordered nature of the cell wall. Solid-state NMR (SSNMR) spectroscopy is uniquely suited for studying native hydrated plant cell walls at the molecular level with chemical resolution. Significant progress has been made in the last five years to elucidate the molecular structures and interactions of cellulose and matrix polysaccharides in plant cell walls. These studies have focused on primary cell walls of growing plants in both the dicotyledonous and grass families, as represented by the model plants Arabidopsis thaliana, Brachypodium distachyon, and Zea mays. To date, these SSNMR results have shown that 1) cellulose, hemicellulose, and pectins form a single network in the primary cell wall; 2) in dicot cell walls, the protein expansin targets the hemicellulose-enriched region of the cellulose microfibril for its wall-loosening function; and 3) primary wall cellulose has polymorphic structures that are distinct from the microbial cellulose structures. This article summarizes these key findings, and points out future directions of investigation to advance our fundamental understanding of plant cell wall structure and function.

  20. Multidimensional solid-state NMR spectroscopy of plant cell walls.

    PubMed

    Wang, Tuo; Phyo, Pyae; Hong, Mei

    2016-09-01

    Plant biomass has become an important source of bio-renewable energy in modern society. The molecular structure of plant cell walls is difficult to characterize by most atomic-resolution techniques due to the insoluble and disordered nature of the cell wall. Solid-state NMR (SSNMR) spectroscopy is uniquely suited for studying native hydrated plant cell walls at the molecular level with chemical resolution. Significant progress has been made in the last five years to elucidate the molecular structures and interactions of cellulose and matrix polysaccharides in plant cell walls. These studies have focused on primary cell walls of growing plants in both the dicotyledonous and grass families, as represented by the model plants Arabidopsis thaliana, Brachypodium distachyon, and Zea mays. To date, these SSNMR results have shown that 1) cellulose, hemicellulose, and pectins form a single network in the primary cell wall; 2) in dicot cell walls, the protein expansin targets the hemicellulose-enriched region of the cellulose microfibril for its wall-loosening function; and 3) primary wall cellulose has polymorphic structures that are distinct from the microbial cellulose structures. This article summarizes these key findings, and points out future directions of investigation to advance our fundamental understanding of plant cell wall structure and function. PMID:27552739

  1. Plant lipid bodies and cell-cell signaling

    PubMed Central

    van der Schoot, Christiaan; Paul, Laju K.; Paul, Sheetal Babu; Rinne, Päivi L.H.

    2011-01-01

    Plant lipid droplets are found in seeds and in post-embryonic tissues. Lipid droplets in seeds have been intensively studied, but those in post-embryonic tissues are less well characterised. Although known by a variety of names, here we will refer to all of them as lipid bodies (LBs). LBs are unique spherical organelles which bud off from the endoplasmic reticulum, and are composed of a single phospholipid (PL) layer enclosing a core of triacylglycerides. The PL monolayer is coated with oleosin, a structural protein that stabilizes the LB, restricts its size, and prevents fusion with adjacent LBs. Oleosin is uniquely present at LBs and is regarded as a LB marker. Although initially viewed as simple stores for energy and carbon, the emerging view is that LBs also function in cytoplasmic signalling, with the minor LB proteins caleosin and steroleosin in a prominent role. Apart from seeds, a variety of vegetative and floral structures contain LBs. Recently, it was found that numerous LBs emerge in the shoot apex of perennial plants during seasonal growth arrest and bud formation. They appear to function in dormancy release by reconstituting cell-cell signalling paths in the apex. As apices and orthodox seeds proceed through comparable cycles of dormancy and dehydration, the question arises to what degree LBs in apices share functions with those in seeds. We here review what is known about LBs, particularly in seeds, and speculate about possible unique functions of LBs in post-embryonic tissues in general and in apices in particular. PMID:22057325

  2. Plant cell tissue culture: A potential source of chemicals

    SciTech Connect

    Scott, C.D.; Dougall, D.K.

    1987-08-01

    Higher plants produce many industrially important products. Among these are drugs and medicinal chemicals, essential oils and flavors, vegetable oils and fats, fine and specialty chemicals, and even some commodity chemicals. Although, currently, whole-plant extraction is the primary means of harvesting these materials, the advent of plant cell tissue culture could be a much more effective method of producing many types of phytochemicals. The use of immobilized plant cells in an advanced bioreactor configuration with excretion of the product into the reactor medium may represent the most straightforward way of commercializing such techniques for lower-value chemicals. Important research and development opportunities in this area include screening for plant cultures for nonmedical, lower-value chemicals; understanding and controlling plant cell physiology and biochemistry; optimizing effective immobilization methods; developing more efficient bioreactor concepts; and perfecting product extraction and purification techniques. 62 refs., 2 figs.

  3. The stem cell concept in plants: a matter of debate.

    PubMed

    Laux, Thomas

    2003-05-01

    Throughout their life, which can last for over a thousand years, plants have the fascinating ability to give rise to new organs from founder cells in their apical meristems. Whether these founder cells are equivalent to the pluripotent stem cells in animals has been a long-standing controversy amongst plant scientists. Here, this controversy will be addressed in light of classical observations and recent findings. PMID:12732137

  4. The connection between chromatin motion on the 100 nm length scale and core histone dynamics in live XTC-2 cells and isolated nuclei.

    PubMed

    Davis, Sara K; Bardeen, Christopher J

    2004-01-01

    The diffusive motion of DNA-containing chromatin in live cells and isolated nuclei is investigated using a two-photon standing wave fluorescence photobleaching experiment with 100 nm spatial resolution. The chromatin is labeled using the minor groove binding dye Hoechst 33342. In live cells, the mean diffusion rate is 5 x 10(-4) micro m2/s, with considerable cell-to-cell variation. This diffusion is highly constrained and cannot be observed in a standard, single beam fluorescence recovery after photobleaching experiment. To determine the chemical origin of the diffusion, we study motion in isolated nuclei and vary the strength of the histone-DNA interactions by changing the ionic strength and using chemical and photocross-linking experiments. At higher NaCl concentrations, we see increased chromatin diffusion as the histone-DNA interaction is weakened due to ionic screening, whereas photocross-linking the core histones to the DNA results in a complete absence of diffusive motion. These trends are consistent with the 100 nm scale motion being correlated with the interactions of histone proteins with the DNA. If chromatin diffusion is connected to the nucleosomal dynamics on much smaller length scales, this may provide a way to assay biochemical activity in vivo based on larger scale macromolecular dynamics observed via fluorescence microscopy.

  5. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope

    PubMed Central

    Lamm, Christian E.; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-01-01

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell’s nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein. PMID:26978388

  6. Plant cell wall characterization using scanning probe microscopy techniques

    PubMed Central

    Yarbrough, John M; Himmel, Michael E; Ding, Shi-You

    2009-01-01

    Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy. PMID:19703302

  7. [Transfer of T-DNA from agrobacteria into plant cells through cell walls and membranes].

    PubMed

    Chumakov, M I

    2001-01-01

    Discusses probable routes of agrobacterial penetration through the plant integumental tissues, cell wall, and plant cell plasmodesma. Analyzes the contribution of extracellular structures of agrobacteria in penetration through barriers of a plant cell, primary contact (adhesion), and during DNA transfer from bacterial (E. coli, A. tumefaciens) to recipient (bacterial or plant) cells. Discusses the relationship between donor cell adhesion to recipient cell surface and the infectious and conjugation processes. Considers the probable role of piles in conjugative transfer of agrobacterial DNA through membranes of donor and recipient (bacterial and plant) cells. Analyzes the contribution of the plant cell cytoskeleton to T-DNA transfer. Suggests a model of transport of T-DNA-VirD2 complex and VirE2 proteins through independent channels consisting of vir-coded proteins. PMID:11236737

  8. Identification of proteins associated with ligand-activated estrogen receptor α in human breast cancer cell nuclei by tandem affinity purification and nano LC-MS/MS.

    PubMed

    Tarallo, Roberta; Bamundo, Angela; Nassa, Giovanni; Nola, Ernesto; Paris, Ornella; Ambrosino, Concetta; Facchiano, Angelo; Baumann, Marc; Nyman, Tuula A; Weisz, Alessandro

    2011-01-01

    Estrogen receptor α (ER-α) is a key mediator of estrogen actions in breast cancer (BC) cells. Understanding the effects of ligand-activated ER-α in target cells requires identification of the molecular partners acting in concert with this nuclear receptor to transduce the hormonal signal. We applied tandem affinity purification (TAP), glycerol gradient centrifugation and MS analysis to isolate and identify proteins interacting with ligand-activated ER-α in MCF-7 cell nuclei. This led to the identification of 264 ER-associated proteins, whose functions highlight the hinge role of ER-α in the coordination of multiple hormone-regulated nuclear processes in BC cells. PMID:21182205

  9. Effect of 17β-estradiol and flavonoids on the regulation of expression of newly identified oestrogen responsive genes in a rat raphe nuclei-derived cell line.

    PubMed

    Amer, Dena A M; Jähne, Maria; Weigt, Carmen; Kretzschmar, Georg; Vollmer, Günter

    2012-10-01

    Due to the health risks attributed to perimenopausal hormone therapy, phytoestrogens such as flavonoids are receiving widespread attention to help alleviate menopausal symptoms, including hormone-driven mood disorders. Based on our previous reporter gene study regarding their transactivational activity in raphe nuclei cells from a brain region involved in regulation of mood disturbances, we herein study their effects on the regulation of expression of 17β-estradiol (E2)-regulated genes. DNA microarray was used to globally assess E2-induced gene expression in RNDA cells, a rat raphe nuclei-derived cellular model expressing oestrogen receptor β. Out of 212 regulated genes, six were selected for verification and as endpoints for the effect of flavonoids on the regulation of mRNA expression in proliferating as well as differentiating RNDA cells. Under proliferative conditions, E2 up-regulated mRNA expression of Cml-5, Sox-18 and Krt-19. Similar effects were observed in response to 8-prenylnaringenin (8-PN), genistein (GEN), daidzein (DAI) and equol (EQ). In line with E2, mRNA expression of Nefm and Zdhhc-2 was down-regulated following 8-PN, GEN, DAI, EQ and naringenin treatment. No regulation was observed on Slc6a4 mRNA expression in response to E2 or the flavonoids in proliferating RNDA cells. When cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards E2 and the tested flavonoids were noticed. These expression studies additionally highlighted some of the genes as markers for RNDA cellular differentiation. RNDA cells should prove useful to elucidate molecular and cellular mechanisms of exogenous oestrogen receptor ligands with neural cell populations. PMID:22213181

  10. Root Border Cells and Their Role in Plant Defense.

    PubMed

    Hawes, Martha; Allen, Caitilyn; Turgeon, B Gillian; Curlango-Rivera, Gilberto; Minh Tran, Tuan; Huskey, David A; Xiong, Zhongguo

    2016-08-01

    Root border cells separate from plant root tips and disperse into the soil environment. In most species, each root tip can produce thousands of metabolically active cells daily, with specialized patterns of gene expression. Their function has been an enduring mystery. Recent studies suggest that border cells operate in a manner similar to mammalian neutrophils: Both cell types export a complex of extracellular DNA (exDNA) and antimicrobial proteins that neutralize threats by trapping pathogens and thereby preventing invasion of host tissues. Extracellular DNases (exDNases) of pathogens promote virulence and systemic spread of the microbes. In plants, adding DNase I to root tips eliminates border cell extracellular traps and abolishes root tip resistance to infection. Mutation of genes encoding exDNase activity in plant-pathogenic bacteria (Ralstonia solanacearum) and fungi (Cochliobolus heterostrophus) results in reduced virulence. The study of exDNase activities in plant pathogens may yield new targets for disease control. PMID:27215971

  11. Root Border Cells and Their Role in Plant Defense.

    PubMed

    Hawes, Martha; Allen, Caitilyn; Turgeon, B Gillian; Curlango-Rivera, Gilberto; Minh Tran, Tuan; Huskey, David A; Xiong, Zhongguo

    2016-08-01

    Root border cells separate from plant root tips and disperse into the soil environment. In most species, each root tip can produce thousands of metabolically active cells daily, with specialized patterns of gene expression. Their function has been an enduring mystery. Recent studies suggest that border cells operate in a manner similar to mammalian neutrophils: Both cell types export a complex of extracellular DNA (exDNA) and antimicrobial proteins that neutralize threats by trapping pathogens and thereby preventing invasion of host tissues. Extracellular DNases (exDNases) of pathogens promote virulence and systemic spread of the microbes. In plants, adding DNase I to root tips eliminates border cell extracellular traps and abolishes root tip resistance to infection. Mutation of genes encoding exDNase activity in plant-pathogenic bacteria (Ralstonia solanacearum) and fungi (Cochliobolus heterostrophus) results in reduced virulence. The study of exDNase activities in plant pathogens may yield new targets for disease control.

  12. Rare earth elements activate endocytosis in plant cells

    PubMed Central

    Wang, Lihong; Li, Jigang; Zhou, Qing; Yang, Guangmei; Ding, Xiao Lan; Li, Xiaodong; Cai, Chen Xin; Zhang, Zhao; Wei, Hai Yan; Lu, Tian Hong; Deng, Xing Wang; Huang, Xiao Hua

    2014-01-01

    It has long been observed that rare earth elements (REEs) regulate multiple facets of plant growth and development. However, the underlying mechanisms remain largely unclear. Here, using electron microscopic autoradiography, we show the life cycle of a light REE (lanthanum) and a heavy REE (terbium) in horseradish leaf cells. Our data indicate that REEs were first anchored on the plasma membrane in the form of nanoscale particles, and then entered the cells by endocytosis. Consistently, REEs activated endocytosis in plant cells, which may be the cellular basis of REE actions in plants. Moreover, we discovered that a portion of REEs was successively released into the cytoplasm, self-assembled to form nanoscale clusters, and finally deposited in horseradish leaf cells. Taken together, our data reveal the life cycle of REEs and their cellular behaviors in plant cells, which shed light on the cellular mechanisms of REE actions in living organisms. PMID:25114214

  13. Rare earth elements activate endocytosis in plant cells.

    PubMed

    Wang, Lihong; Li, Jigang; Zhou, Qing; Yang, Guangmei; Ding, Xiao Lan; Li, Xiaodong; Cai, Chen Xin; Zhang, Zhao; Wei, Hai Yan; Lu, Tian Hong; Deng, Xing Wang; Huang, Xiao Hua

    2014-09-01

    It has long been observed that rare earth elements (REEs) regulate multiple facets of plant growth and development. However, the underlying mechanisms remain largely unclear. Here, using electron microscopic autoradiography, we show the life cycle of a light REE (lanthanum) and a heavy REE (terbium) in horseradish leaf cells. Our data indicate that REEs were first anchored on the plasma membrane in the form of nanoscale particles, and then entered the cells by endocytosis. Consistently, REEs activated endocytosis in plant cells, which may be the cellular basis of REE actions in plants. Moreover, we discovered that a portion of REEs was successively released into the cytoplasm, self-assembled to form nanoscale clusters, and finally deposited in horseradish leaf cells. Taken together, our data reveal the life cycle of REEs and their cellular behaviors in plant cells, which shed light on the cellular mechanisms of REE actions in living organisms.

  14. Guard cell protoplasts: isolation, culture, and regeneration of plants.

    PubMed

    Tallman, Gary

    2006-01-01

    Guard cell protoplasts have been used extensively in short-term experiments designed to elucidate the signal transduction mechanisms that regulate stomatal movements. The utility of uard cell protoplasts for other types of longer-term signal transduction experiments is just now being realized. Because highly purified, primary isolates of guard cell protoplasts are synchronous initially, they are uniform in their responses to changes in culture conditions. Such isolates have demonstrated potential to reveal mechanisms that underlie hormonal signalling for plant cell survival, cell cycle re-entry, reprogramming of genes during dedifferentiation to an embryogenic state, and plant cell thermotolerance. Plants have been regenerated from cultured guard cell protoplasts of two species: Nicotiana glauca (Graham), tree tobacco, and Beta vulgaris, sugar beet. Plants genetically engineered for herbicide tolerance have been regenerated from cultured guard cell protoplasts of B. vulgaris. The method for isolating, culturing, and regenerating plants from guard cell protoplasts of N. glauca is described here. A recently developed procedure for large-scale isolation of these cells from as many as nine leaves per experiment is described. Using this protocol, yields of 1.5-2 x 10(7) per isolate may be obtained. Such yields are sufficient for standard methods of molecular, biochemical, and proteomic analysis.

  15. Cytoplasmic calcium stimulates exocytosis in a plant secretory cell

    PubMed Central

    Tester, Mark; Zorec, Robert

    1992-01-01

    Although exocytosis is likely to occur in plant cells, the control of this process is the subject of speculation, as no direct measurements of vesicle fusion to the plasma membrane have been made. We used the patch clamp technique to monitor the secretory activity of single aleurone protoplasts by measuring membrane capacitance (Cm), while dialyzing the cytosol with different Ca2+ containing solutions. Secretory activity increased with [Ca2+]i ∼ 1 μM. This demonstrates directly the existence of exocytosis in plant cells, and suggests that both plant and animal cells share common mechanisms (cytosolic Ca2+) for the control of exocytotic secretion. PMID:19431846

  16. Live cell imaging of the cytoskeleton and cell wall enzymes in plant cells.

    PubMed

    Sampathkumar, Arun; Wightman, Raymond

    2015-01-01

    The use of live imaging techniques to visualize the dynamic changes and interactions within plant cells has given us detailed information on the function and organization of the cytoskeleton and cell wall associated proteins. This information has grown with the constant improvement in imaging hardware and molecular tools. In this chapter, we describe the procedure for the preparation and live visualization of fluorescent protein fusions associated with the cytoskeleton and the cell wall in Arabidopsis. PMID:25408450

  17. Regulation of cell division in higher plants. Progress report

    SciTech Connect

    Jacobs, T.W.

    1992-07-01

    Cell division is arguably the most fundamental of all developmental processes. In higher plants, mitotic activity is largely confined to foci of patterned cell divisions called meristems. From these perpetually embryonic tissues arise the plant`s essential organs of light capture, support, protection and reproduction. Once an adequate understanding of plant cell mitotic regulation is attained, unprecedented opportunities will ensue for analyzing and genetically controlling diverse aspects of development, including plant architecture, leaf shape, plant height, and root depth. The mitotic cycle in a variety of model eukaryotic systems in under the control of a regulatory network of striking evolutionary conservation. Homologues of the yeast cdc2 gene, its catalytic product, p34, and the cyclin regulatory subunits of the MPF complex have emerged as ubiquitous mitotic regulators. We have cloned cdc2-like and cyclin genes from pea. As in other eukaryotic model systems, p34 of Pisum sativum is a subunit of a high molecular weight complex which binds the fission yeast p13 protein and displays histone H1 kinase activity in vitro. Our primary objective in this study is to gain baseline information about the regulation of this higher plant cell division control complex in non-dividing, differentiated cells as well as in synchronous and asynchronous mitotic cells. We are investigating cdc2 and cyclin expression at the levels of protein abundance, protein phosphorylation and quaternary associations.

  18. Plant cell organelle proteomics in response to abiotic stress.

    PubMed

    Hossain, Zahed; Nouri, Mohammad-Zaman; Komatsu, Setsuko

    2012-01-01

    Proteomics is one of the finest molecular techniques extensively being used for the study of protein profiling of a given plant species experiencing stressed conditions. Plants respond to a stress by alteration in the pattern of protein expression, either by up-regulating of the existing protein pool or by the synthesizing novel proteins primarily associated with plants antioxidative defense mechanism. Improved protein extraction protocols and advance techniques for identification of novel proteins have been standardized in different plant species at both cellular and whole plant level for better understanding of abiotic stress sensing and intracellular stress signal transduction mechanisms. In contrast, an in-depth proteome study of subcellular organelles could generate much detail information about the intrinsic mechanism of stress response as it correlates the possible relationship between the protein abundance and plant stress tolerance. Although a wealth of reviews devoted to plant proteomics are available, review articles dedicated to plant cell organelle proteins response under abiotic stress are very scanty. In the present review, an attempt has been made to summarize all significant contributions related to abiotic stresses and their impacts on organelle proteomes for better understanding of plants abiotic stress tolerance mechanism at protein level. This review will not only provide new insights into the plants stress response mechanisms, which are necessary for future development of genetically engineered stress tolerant crop plants for the benefit of humankind, but will also highlight the importance of studying changes in protein abundance within the cell organelles in response to abiotic stress.

  19. Plant and algal cell walls: diversity and functionality

    PubMed Central

    Popper, Zoë A.; Ralet, Marie-Christine; Domozych, David S.

    2014-01-01

    Background Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore, wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes (plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. Scope The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every

  20. Genes and plant cell walls: a difficult relationship.

    PubMed

    Wojtaszek, P

    2000-08-01

    Chemical information, carried by genes, is one of several types of information important for the functioning of cells and organisms. While genes govern the two-dimensional flow of information, the cell walls are at the basis of a structural, three-dimensional framework of plant form and growth. Recent data show the walls to be a cellular 'organelle' undergoing dynamic changes in response to a plethora of stimuli. In this review, an integrated approach, rooted in the organismal perspective, is taken to consider the role of cell walls in the biology of plants. First, the complexity of molecular and biochemical events leading to the biosynthesis of wall components is described within the framework of its spatial cellular organisation, and the major regulatory check-points are characterised. Second, cell walls form a structural and functional continuum within the whole plant and thus could be defined in relation to the protoplasts that produce them and in relation to the plant itself. Model systems of suspension-cultured cells are used to reveal the existence of a bidirectional exchange of information between the protoplast and its walls. The 'plasticity' of plant cell reactions, seen in defence responses or in changes in wall composition, to e.g. stress, plant growth regulators or chemical agents as well as the role of cell walls and/or wall components in somatic embryogenesis are also discussed. Third, being a continuum within the plant body, the walls fulfil vital functions in plant growth and development. The examples characterised include the determination of cellular polarity and the plane of cell division, cytokinesis, and the role of plasmodesmata in cell-to-cell communication and the formation of functional symplastic domains. Fourth, the exocellular control of morphogenetic processes is described and the potential of cell walls as determinants or reservoirs of positional information is indicated. Particular emphasis is put on the (bio)chemical signals coming

  1. Specification of epidermal cell fate in plant shoots.

    PubMed

    Takada, Shinobu; Iida, Hiroyuki

    2014-01-01

    Land plants have evolved a single layer of epidermal cells, which are characterized by mostly anticlinal cell division patterns, formation of a waterproof coat called cuticle, and unique cell types such as stomatal guard cells and trichomes. The shoot epidermis plays important roles not only to protect plants from dehydration and pathogens but also to ensure their proper organogenesis and growth control. Extensive molecular genetic studies in Arabidopsis and maize have identified a number of genes that are required for epidermal cell differentiation. However, the mechanism that specifies shoot epidermal cell fate during plant organogenesis remains largely unknown. Particularly, little is known regarding positional information that should restrict epidermal cell fate to the outermost cell layer of the developing organs. Recent studies suggested that certain members of the HD-ZIP class IV homeobox genes are possible master regulators of shoot epidermal cell fate. Here, we summarize the roles of the regulatory genes that are involved in epidermal cell fate specification and discuss the possible mechanisms that limit the expression and/or activity of the master transcriptional regulators to the outermost cell layer in plant shoots. PMID:24616724

  2. Plant cell wall dynamics and wall-related susceptibility in plant-pathogen interactions.

    PubMed

    Bellincampi, Daniela; Cervone, Felice; Lionetti, Vincenzo

    2014-01-01

    The cell wall is a dynamic structure that often determines the outcome of the interactions between plants and pathogens. It is a barrier that pathogens need to breach to colonize the plant tissue. While fungal necrotrophs extensively destroy the integrity of the cell wall through the combined action of degrading enzymes, biotrophic fungi require a more localized and controlled degradation of the cell wall in order to keep the host cells alive and utilize their feeding structures. Also bacteria and nematodes need to degrade the plant cell wall at a certain stage of their infection process, to obtain nutrients for their growth. Plants have developed a system for sensing pathogens and monitoring the cell wall integrity, upon which they activate defense responses that lead to a dynamic cell wall remodeling required to prevent the disease. Pathogens, on the other hand, may exploit the host cell wall metabolism to support the infection. We review here the strategies utilized by both plants and pathogens to prevail in the cell wall battleground.

  3. Role of proline in cell wall synthesis and plant development and its implications in plant ontogeny.

    PubMed

    Kavi Kishor, Polavarapu B; Hima Kumari, P; Sunita, M S L; Sreenivasulu, Nese

    2015-01-01

    Proline is a proteogenic amino acid and accumulates both under stress and non-stress conditions as a beneficial solute in plants. Recent discoveries point out that proline plays an important role in plant growth and differentiation across life cycle. It is a key determinant of many cell wall proteins that plays important roles in plant development. The role of extensins, arabinogalactan proteins and hydroxyproline- and proline-rich proteins as important components of cell wall proteins that play pivotal roles in cell wall signal transduction cascades, plant development and stress tolerance is discussed in this review. Molecular insights are also provided here into the plausible roles of proline transporters modulating key events in plant development. In addition, the roles of proline during seed developmental transitions including storage protein synthesis are discussed. PMID:26257754

  4. Role of proline in cell wall synthesis and plant development and its implications in plant ontogeny

    PubMed Central

    Kavi Kishor, Polavarapu B.; Hima Kumari, P.; Sunita, M. S. L.; Sreenivasulu, Nese

    2015-01-01

    Proline is a proteogenic amino acid and accumulates both under stress and non-stress conditions as a beneficial solute in plants. Recent discoveries point out that proline plays an important role in plant growth and differentiation across life cycle. It is a key determinant of many cell wall proteins that plays important roles in plant development. The role of extensins, arabinogalactan proteins and hydroxyproline- and proline-rich proteins as important components of cell wall proteins that play pivotal roles in cell wall signal transduction cascades, plant development and stress tolerance is discussed in this review. Molecular insights are also provided here into the plausible roles of proline transporters modulating key events in plant development. In addition, the roles of proline during seed developmental transitions including storage protein synthesis are discussed. PMID:26257754

  5. Modulation of programmed cell death by medicinal plants.

    PubMed

    Thatte, U; Bagadey, S; Dahanukar, S

    2000-02-01

    Programmed cell death (apoptosis), a form of cell death, described by Kerr and Wyllie some 20 years ago, has generated considerable interest in recent years. The mechanisms by which this mode of cell death (seen both in animal and plant cells), takes place have been examined in detail. Extracellular signals and intracellular events have been elaborated. Of interest to the clinician, is the concentrated effort to study pharmacological modulation of programmed cell death. The attempt to influence the natural phenomenon of programmed cell death stems from the fact that it is reduced (like in cancer) or increased (like in neurodegenerative diseases) in several clinical situations. Thus, chemicals that can modify programmed cell death are likely to be potentially useful drugs. From foxglove, which gave digitalis to the Pacific Yew from which came taxol, plants have been a source of research material for useful drugs. Recently, a variety of plant extracts have been investigated for their ability to influence the apoptotic process. This article discusses some of the interesting data. The ability of plants to influence programmed cell death in cancerous cells in an attempt to arrest their proliferation has been the topic of much research. Various cell-lines like HL60, human hepatocellular carcinoma cell line (KIM-1), a cholangiocarcinoma cell-line (KMC-1), B-cell hybridomas, U937 a monocytic cell-line, HeLa cells, human lymphoid leukemia (MOLT-4B) cells and K562 cells have been studied. The agents found to induce programmed cell death (measured either morphologically or flow cytometrically) included extracts of plants like mistletoe and Semicarpus anacardium. Isolated compounds like bryonolic acid (from Trichosanthes kirilowii var. Japonica, crocin (from saffron) and allicin (from Allium sativum) have also been found to induce programmed cell death and therefore arrest proliferation. Even Chinese herbal medicine "Sho-saiko-to" induces programmed cell death in selected

  6. Evolution and diversity of plant cell walls: from algae to flowering plants.

    PubMed

    Popper, Zoë A; Michel, Gurvan; Hervé, Cécile; Domozych, David S; Willats, William G T; Tuohy, Maria G; Kloareg, Bernard; Stengel, Dagmar B

    2011-01-01

    All photosynthetic multicellular Eukaryotes, including land plants and algae, have cells that are surrounded by a dynamic, complex, carbohydrate-rich cell wall. The cell wall exerts considerable biological and biomechanical control over individual cells and organisms, thus playing a key role in their environmental interactions. This has resulted in compositional variation that is dependent on developmental stage, cell type, and season. Further variation is evident that has a phylogenetic basis. Plants and algae have a complex phylogenetic history, including acquisition of genes responsible for carbohydrate synthesis and modification through a series of primary (leading to red algae, green algae, and land plants) and secondary (generating brown algae, diatoms, and dinoflagellates) endosymbiotic events. Therefore, organisms that have the shared features of photosynthesis and possession of a cell wall do not form a monophyletic group. Yet they contain some common wall components that can be explained increasingly by genetic and biochemical evidence.

  7. The Transport of Ions Across Plant Cell Membranes.

    ERIC Educational Resources Information Center

    Baker, D. A.

    1981-01-01

    Presented is one of a series of articles designed to help science teachers keep current on ideas in specific areas of biology. This article provides information about ion transport in plant cells. (PB)

  8. Development of molten carbonate fuel cell power plant, volume 1

    NASA Astrophysics Data System (ADS)

    1985-03-01

    The technical results of a molten carbonate fuel cell power plant evelopment program are presented which establish the necessary technology base and demonstrate readiness to proceed with the fabrication and test of full size prototype stacks for coal fueled molten carbonate fuel cell power plants. The effort covered power plant systems studies, fuel cell component technology development, fuel cell stack design and analysis, manufacturing process definition, and an extensive experimental program. The reported results include: the definition and projected costs for a reference coal fueled power plant system based on user requirements, state-of-the-art advances in anode and electrolyte matrix technology, the detailed description of an internally manifolded stack design concept offering a number of attractive advantages, and the specification of the fabrication processes and methods necessary to produce and assemble this design. Results from the experimental program are documented.

  9. Investigating Wound Healing in Plant Cells: This Spud's for You!

    ERIC Educational Resources Information Center

    Thomson, Norm

    2000-01-01

    Presents classroom inquiry-based investigations to investigate wound healing in plant tissues and cells. Students create their own research problems and the investigations can be related to the National Science Standards. (SAH)

  10. 31. SOUTH PLANT NORTHERN EDGE, SHOWING CELL BUILDING (BUILDING 242) ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    31. SOUTH PLANT NORTHERN EDGE, SHOWING CELL BUILDING (BUILDING 242) AT LEFT, LABORATORY (BUILDING 241) AT CENTER AND CAUSTIC FUSION PLANT (BUILDING 254) AT RIGHT. VIEW TO SOUTHWEST. - Rocky Mountain Arsenal, Bounded by Ninety-sixth Avenue & Fifty-sixth Avenue, Buckley Road, Quebec Street & Colorado Highway 2, Commerce City, Adams County, CO

  11. Effector biology during biotrophic invasion of plant cells

    PubMed Central

    Chaudhari, Prateek; Ahmed, Bulbul; Joly, David L; Germain, Hugo

    2014-01-01

    Several obligate biotrophic phytopathogens, namely oomycetes and fungi, invade and feed on living plant cells through specialized structures known as haustoria. Deploying an arsenal of secreted proteins called effectors, these pathogens balance their parasitic propagation by subverting plant immunity without sacrificing host cells. Such secreted proteins, which are thought to be delivered by haustoria, conceivably reprogram host cells and instigate structural modifications, in addition to the modulation of various cellular processes. As effectors represent tools to assist disease resistance breeding, this short review provides a bird’s eye view on the relationship between the virulence function of effectors and their subcellular localization in host cells. PMID:25513771

  12. Hydrogen peroxide as a signal controlling plant programmed cell death

    PubMed Central

    Gechev, Tsanko S.; Hille, Jacques

    2005-01-01

    Hydrogen peroxide (H2O2) has established itself as a key player in stress and programmed cell death responses, but little is known about the signaling pathways leading from H2O2 to programmed cell death in plants. Recently, identification of key regulatory mutants and near-full genome coverage microarray analysis of H2O2-induced cell death have begun to unravel the complexity of the H2O2 network. This review also describes a novel link between H2O2 and sphingolipids, two signals that can interplay and regulate plant cell death. PMID:15631987

  13. BUB1 and SURVIVIN proteins are not degraded after a prolonged mitosis and accumulate in the nuclei of HCT116 cells

    PubMed Central

    Andonegui-Elguera, Marco A; Cáceres-Gutiérrez, Rodrigo E; Luna-Maldonado, Fernando; López-Saavedra, Alejandro; Díaz-Chávez, José; Cisneros-Soberanis, Fernanda; Prada, Diddier; Mendoza-Pérez, Julia; Herrera, Luis A

    2016-01-01

    Spindle poisons activate the spindle assembly checkpoint and prevent mitotic exit until cells die or override the arrest. Several studies have focused on spindle poison-mediated cell death, but less is known about consequences in cells that survive a mitotic arrest. During mitosis, proteins such as CYCLIN B, SECURIN, BUB1 and SURVIVIN are degraded in order to allow mitotic exit, and these proteins are maintained at low levels in the next interphase. In contrast, exit from a prolonged mitosis depends only on degradation of CYCLIN B; it is not known whether the levels of other proteins decrease or remain high. Here, we analyzed the levels and localization of the BUB1 and SURVIVIN proteins in cells that escaped from a paclitaxel-mediated prolonged mitosis. We compared cells with a short arrest (HCT116 cells) with cells that spent more time in mitosis (HT29 cells) after paclitaxel treatment. BUB1 and SURVIVIN were not degraded and remained localized to the nuclei of HCT116 cells after a mitotic arrest. Moreover, BUB1 nuclear foci were observed; BUB1 did not colocalize with centromere proteins. In HT29 cells, the levels of BUB1 and SURVIVIN decreased during the arrest, and these proteins were not present in cells that reached the next interphase. Using time-lapse imaging, we observed morphological heterogeneity in HCT116 cells that escaped from the arrest; this heterogeneity was due to the cytokinesis-like mechanism by which the cells exited mitosis. Thus, our results show that high levels of BUB1 and SURVIVIN can be maintained after a mitotic arrest, which may promote resistance to cell death.

  14. Roles of Atox1 and p53 in the trafficking of copper-64 to tumor cell nuclei: implications for cancer therapy.

    PubMed

    Beaino, Wissam; Guo, Yunjun; Chang, Albert J; Anderson, Carolyn J

    2014-03-01

    Owing to its cytotoxicity, free copper is chelated by protein side chains and does not exist in vivo. Several chaperones transport copper to various cell compartments, but none have been identified that traffic copper to the nucleus. Copper-64 decays by β (+) and β (-) emission, allowing positron emission tomography and targeted radionuclide therapy for cancer. Because the delivery of (64)Cu to the cell nucleus may enhance the therapeutic effect of copper radiopharmaceuticals, elucidation of the pathway(s) involved in transporting copper to the tumor cell nucleus is important for optimizing treatment. We identified Atox1 as one of the proteins that binds copper in the nucleus. Mouse embryonic fibroblast cells, positive and negative for Atox1, were used to determine the role of Atox1 in (64)Cu transport to the nucleus. Mouse embryonic fibroblast Atox1(+/+) cells accumulated more (64)Cu in the nucleus than did Atox1(-/-) cells. HCT 116 colorectal cancer cells expressing p53 (+/+) and not expressing p53 (-/-) were used to evaluate the role of this tumor suppressor protein in (64)Cu transport. In cells treated with cisplatin, the uptake of (64)Cu in the nucleus of HCT 116 p53(+/+) cells was greater than that in HCT 116 p53(-/-) cells. Atox1 expression increased in HCT 116 p53(+/+) and p53(-/-) cells treated with cisplatin; however, Atox1 localized to the nuclei of p53(+/+) cells more than in the p53(-/-) cells. The data presented here indicate that Atox1 is involved in copper transport to the nucleus, and cisplatin affects nuclear transport of (64)Cu in HCT 116 cells by upregulating the expression and the nuclear localization of Atox1.

  15. Structural Studies of Complex Carbohydrates of Plant Cell Walls

    SciTech Connect

    Darvill, Alan; Hahn, Michael G.; O'Neill, Malcolm A.; York, William S.

    2015-02-17

    Most of the solar energy captured by land plants is converted into the polysaccharides (cellulose, hemicellulose, and pectin) that are the predominant components of the cell wall. These walls, which account for the bulk of plant biomass, have numerous roles in the growth and development of plants. Moreover, these walls have a major impact on human life as they are a renewable source of biomass, a source of diverse commercially useful polymers, a major component of wood, and a source of nutrition for humans and livestock. Thus, understanding the molecular mechanisms that lead to wall assembly and how cell walls and their component polysaccharides contribute to plant growth and development is essential to improve and extend the productivity and value of plant materials. The proposed research will develop and apply advanced analytical and immunological techniques to study specific changes in the structures and interactions of the hemicellulosic and pectic polysaccharides that occur during differentiation and in response to genetic modification and chemical treatments that affect wall biosynthesis. These new techniques will make it possible to accurately characterize minute amounts of cell wall polysaccharides so that subtle changes in structure that occur in individual cell types can be identified and correlated to the physiological or developmental state of the plant. Successful implementation of this research will reveal fundamental relationships between polysaccharide structure, cell wall architecture, and cell wall functions.

  16. The biphasic interphase-mitotic polarity of cell nuclei induced under DNA replication stress seems to be correlated with Pin2 localization in root meristems of Allium cepa.

    PubMed

    Żabka, Aneta; Trzaskoma, Paweł; Winnicki, Konrad; Polit, Justyna Teresa; Chmielnicka, Agnieszka; Maszewski, Janusz

    2015-02-01

    Long-term treatment of Allium cepa seedlings with low concentration of hydroxyurea (HU) results in a disruption of cell cycle checkpoints, leading root apex meristem (RAM) cells to an abnormal organization of nuclear structures forming interphase (I) and mitotic (M) domains of chromatin at opposite poles of the nucleus. Thus far, both critical cell length and an uneven distribution of cyclin B-like proteins along the nuclear axis have been recognized as essential factors needed to facilitate the formation of biphasic interphase-mitotic (IM) cells. Two new aspects with respect to their emergence are investigated in this study. The first concerns a relationship between the polarity of increasing chromatin condensation (IM orientation) and the acropetal (base→apex) alignment of RAM cell files. The second problem involves the effects of auxin (IAA), on the frequency of IM cells. We provide evidence that there is an association between the advanced M-poles of the IM cell nuclei and the polarized accumulation sites of auxin efflux carriers (PIN2 proteins) and IAA. Furthermore, our observations reveal exclusion regions for PIN2 proteins in the microtubule-rich structures, such as preprophase bands (PPBs) and phragmoplast. The current and previous studies have prompted us to formulate a hypothetical mechanism linking PIN2-mediated unilateral localization of IAA and the induction of bipolar IM cells in HU-treated RAMs of A. cepa.

  17. The biphasic interphase-mitotic polarity of cell nuclei induced under DNA replication stress seems to be correlated with Pin2 localization in root meristems of Allium cepa.

    PubMed

    Żabka, Aneta; Trzaskoma, Paweł; Winnicki, Konrad; Polit, Justyna Teresa; Chmielnicka, Agnieszka; Maszewski, Janusz

    2015-02-01

    Long-term treatment of Allium cepa seedlings with low concentration of hydroxyurea (HU) results in a disruption of cell cycle checkpoints, leading root apex meristem (RAM) cells to an abnormal organization of nuclear structures forming interphase (I) and mitotic (M) domains of chromatin at opposite poles of the nucleus. Thus far, both critical cell length and an uneven distribution of cyclin B-like proteins along the nuclear axis have been recognized as essential factors needed to facilitate the formation of biphasic interphase-mitotic (IM) cells. Two new aspects with respect to their emergence are investigated in this study. The first concerns a relationship between the polarity of increasing chromatin condensation (IM orientation) and the acropetal (base→apex) alignment of RAM cell files. The second problem involves the effects of auxin (IAA), on the frequency of IM cells. We provide evidence that there is an association between the advanced M-poles of the IM cell nuclei and the polarized accumulation sites of auxin efflux carriers (PIN2 proteins) and IAA. Furthermore, our observations reveal exclusion regions for PIN2 proteins in the microtubule-rich structures, such as preprophase bands (PPBs) and phragmoplast. The current and previous studies have prompted us to formulate a hypothetical mechanism linking PIN2-mediated unilateral localization of IAA and the induction of bipolar IM cells in HU-treated RAMs of A. cepa. PMID:25462968

  18. 3D Plant cell architecture of Arabidopsis thaliana (Brassicaceae) using focused ion beam–scanning electron microscopy1

    PubMed Central

    Bhawana; Miller, Joyce L.; Cahoon, A. Bruce

    2014-01-01

    • Premise of the study: Focused ion beam–scanning electron microscopy (FIB-SEM) combines the ability to sequentially mill the sample surface and obtain SEM images that can be used to create 3D renderings with micron-level resolution. We have applied FIB-SEM to study Arabidopsis cell architecture. The goal was to determine the efficacy of this technique in plant tissue and cellular studies and to demonstrate its usefulness in studying cell and organelle architecture and distribution. • Methods: Seed aleurone, leaf mesophyll, stem cortex, root cortex, and petal lamina from Arabidopsis were fixed and embedded for electron microscopy using protocols developed for animal tissues and modified for use with plant cells. Each sample was sectioned using the FIB and imaged with SEM. These serial images were assembled to produce 3D renderings of each cell type. • Results: Organelles such as nuclei and chloroplasts were easily identifiable, and other structures such as endoplasmic reticula, lipid bodies, and starch grains were distinguishable in each tissue. • Discussion: The application of FIB-SEM produced 3D renderings of five plant cell types and offered unique views of their shapes and internal content. These results demonstrate the usefulness of FIB-SEM for organelle distribution and cell architecture studies. PMID:25202629

  19. MOLTEN CARBONATE FUEL CELL POWER PLANT LOCATED AT TERMINAL ISLAND WASTEWATER TREATMENT PLANT

    SciTech Connect

    William W. Glauz

    2004-09-01

    The Los Angeles Department of Water and Power (LADWP) has developed one of the most recognized fuel cell demonstration programs in the United States. In addition to their high efficiencies and superior environmental performance, fuel cells and other generating technologies that can be located at or near the load, offers several electric utility benefits. Fuel cells can help further reduce costs by reducing peak electricity demand, thereby deferring or avoiding expenses for additional electric utility infrastructure. By locating generators near the load, higher reliability of service is possible and the losses that occur during delivery of electricity from remote generators are avoided. The potential to use renewable and locally available fuels, such as landfill or sewage treatment waste gases, provides another attractive outlook. In Los Angeles, there are also many oil producing areas where the gas by-product can be utilized. In June 2000, the LADWP contracted with FCE to install and commission the precommercial 250kW MCFC power plant. The plant was delivered, installed, and began power production at the JFB in August 2001. The plant underwent manufacturer's field trials up for 18 months and was replace with a commercial plant in January 2003. In January 2001, the LADWP contracted with FCE to provide two additional 250kW MCFC power plants. These commercial plants began operations during mid-2003. The locations of these plants are at the Terminal Island Sewage Treatment Plant at the Los Angeles Harbor (for eventual operation on digester gas) and at the LADWP Main Street Service Center east of downtown Los Angeles. All three carbonate fuel cell plants received partial funding through the Department of Defense's Climate Change Fuel Cell Buydown Program. This report covers the technical evaluation and benefit-cost evaluation of the Terminal Island 250kW MCFC power plant during its first year of operation from June 2003 to July 2004.

  20. Effects of ultraviolet radiation on plant cells.

    PubMed

    Hollósy, F

    2002-01-01

    Recent measurements of ozone levels have led to concern that the stratospheric ozone layer is being depleted as a result of contamination with man-made chlorofluorocarbons. Concomitantly, the amounts of solar UV-B radiation reaching the Earth's surface is increasing. UV-B radiation has been shown to be harmful to living organisms, damaging DNA, proteins, lipids and membranes. Plants, which use sunlight for photosynthesis and are unable to avoid exposure to enhanced levels of UV-B radiation, are at risk. Thus, mechanisms by which plants may protect themselves from UV radiation are of particular interest. This review will summarizes the main aspects of ultraviolet radiation on plants at physiological and biochemical level, with particular emphasis on protective structures and mechanisms.

  1. Centrality of host cell death in plant-microbe interactions.

    PubMed

    Dickman, Martin B; Fluhr, Robert

    2013-01-01

    Programmed cell death (PCD) is essential for proper growth, development, and cellular homeostasis in all eukaryotes. The regulation of PCD is of central importance in plant-microbe interactions; notably, PCD and features associated with PCD are observed in many host resistance responses. Conversely, pathogen induction of inappropriate cell death in the host results in a susceptible phenotype and disease. Thus, the party in control of PCD has a distinct advantage in these battles. PCD processes appear to be of ancient origin, as indicated by the fact that many features of cell death strategy are conserved between animals and plants; however, some of the details of death execution differ. Mammalian core PCD genes, such as caspases, are not present in plant genomes. Similarly, pro- and antiapoptotic mammalian regulatory elements are absent in plants, but, remarkably, when expressed in plants, successfully impact plant PCD. Thus, subtle structural similarities independent of sequence homology appear to sustain operational equivalence. The vacuole is emerging as a key organelle in the modulation of plant PCD. Under different signals for cell death, the vacuole either fuses with the plasmalemma membrane or disintegrates. Moreover, the vacuole appears to play a key role in autophagy; evidence suggests a prosurvival function for autophagy, but other studies propose a prodeath phenotype. Here, we describe and discuss what we know and what we do not know about various PCD pathways and how the host integrates signals to activate salicylic acid and reactive oxygen pathways that orchestrate cell death. We suggest that it is not cell death as such but rather the processes leading to cell death that contribute to the outcome of a given plant-pathogen interaction. PMID:23915134

  2. Dissecting calcium oscillators in plant cells.

    PubMed

    Harper, J F

    2001-09-01

    To understand Ca2+ signaling, we need to identify all the Ca2+ transporters and their regulatory components. The first Ca2+ transporters to be cloned from plants and shown to have regulated activity were calmodulin-dependent Ca2+ -pumps. The regulation of these pumps suggests that being able to change the rate of Ca2+ efflux is important for Ca2+ signaling. The identification of pumps and antiporters in different subcellular locations is helping to dissect the complexities of Ca2+ signaling in plants.

  3. Polarity establishment, morphogenesis, and cultured plant cells in space

    NASA Technical Reports Server (NTRS)

    Krikorian, Abraham D.

    1989-01-01

    Plant development entails an orderly progression of cellular events both in terms of time and geometry. There is only circumstantial evidence that, in the controlled environment of the higher plant embryo sac, gravity may play a role in embryo development. It is still not known whether or not normal embryo development and differentiation in higher plants can be expected to take place reliably and efficiently in the micro g space environment. It seems essential that more attention be given to studying aspects of reproductive biology in order to be confident that plants will survive seed to seed to seed in a space environment. Until the time arrives when successive generations of plants can be grown, the best that can be done is utilize the most appropriate systems and begin, piece meal, to accumulate information on important aspects of plant reproduction. Cultured plant cells can play an important role in these activities since they can be grown so as to be morphogenetically competent, and thus can simulate those embryogenic events more usually identified with fertilized eggs in the embryo sac of the ovule in the ovary. Also, they can be manipulated with relative ease. The extreme plasticity of such demonstrably totipotent cell systems provides a means to test environmental effects such as micro g on a potentially free-running entity. The successful manipulation and management of plant cells and propagules in space also has significance for exploitation of biotechnologies in space since such systems, perforce, are an important vehicle whereby many genetic engineering manipulations are achieved.

  4. Plant cell culture strategies for the production of natural products

    PubMed Central

    Ochoa-Villarreal, Marisol; Howat, Susan; Hong, SunMi; Jang, Mi Ok; Jin, Young-Woo; Lee, Eun-Kyong; Loake, Gary J.

    2016-01-01

    Plants have evolved a vast chemical cornucopia to support their sessile lifestyles. Man has exploited this natural resource since Neolithic times and currently plant-derived chemicals are exploited for a myriad of applications. However, plant sources of most high-value natural products (NPs) are not domesticated and therefore their production cannot be undertaken on an agricultural scale. Further, these plant species are often slow growing, their populations limiting, the concentration of the target molecule highly variable and routinely present at extremely low concentrations. Plant cell and organ culture constitutes a sustainable, controllable and environmentally friendly tool for the industrial production of plant NPs. Further, advances in cell line selection, biotransformation, product secretion, cell permeabilisation, extraction and scale-up, among others, are driving increases in plant NP yields. However, there remain significant obstacles to the commercial synthesis of high-value chemicals from these sources. The relatively recent isolation, culturing and characterisation of cambial meristematic cells (CMCs), provides an emerging platform to circumvent many of these potential difficulties. [BMB Reports 2016; 49(3): 149-158] PMID:26698871

  5. Endocytosis of cell surface material mediates cell plate formation during plant cytokinesis.

    PubMed

    Dhonukshe, Pankaj; Baluska, Frantisek; Schlicht, Markus; Hlavacka, Andrej; Samaj, Jozef; Friml, Jirí; Gadella, Theodorus W J

    2006-01-01

    Dividing plant cells perform a remarkable task of building a new cell wall within the cytoplasm in a few minutes. A long-standing paradigm claims that this primordial cell wall, known as the cell plate, is generated by delivery of newly synthesized material from Golgi apparatus-originated secretory vesicles. Here, we show that, in diverse plant species, cell surface material, including plasma membrane proteins, cell wall components, and exogenously applied endocytic tracers, is rapidly delivered to the forming cell plate. Importantly, this occurs even when de novo protein synthesis is blocked. In addition, cytokinesis-specific syntaxin KNOLLE as well as plasma membrane (PM) resident proteins localize to endosomes that fuse to initiate the cell plate. The rate of endocytosis is strongly enhanced during cell plate formation, and its genetic or pharmacological inhibition leads to cytokinesis defects. Our results reveal that endocytic delivery of cell surface material significantly contributes to cell plate formation during plant cytokinesis.

  6. Superresolution live imaging of plant cells using structured illumination microscopy.

    PubMed

    Komis, George; Mistrik, Martin; Šamajová, Olga; Ovečka, Miroslav; Bartek, Jiri; Šamaj, Jozef

    2015-08-01

    Although superresolution (SR) approaches have been routinely used for fixed or living material from other organisms, the use of time-lapse structured illumination microscopy (SIM) imaging in plant cells still remains under-developed. Here we describe a validated method for time-lapse SIM that focuses on cortical microtubules of different plant cell types. By using one of the existing commercially available SIM platforms, we provide a user-friendly and easy-to-follow protocol that may be widely applied to the imaging of plant cells. This protocol includes steps describing calibration of the microscope and channel alignment, generation of an experimental point spread function (PSF), preparation of appropriate observation chambers with available plant material, image acquisition, reconstruction and validation. This protocol can be carried out within two to three working days.

  7. Chromosomes and plant cell division in space

    NASA Technical Reports Server (NTRS)

    Krikorian, A. D.

    1988-01-01

    The objectives were: examination of chromosomal aberrations; development of an experimental system; and engineering design units (EDUs) evaluation. Evaluation criteria are presented. Procedures were developed for shuttle-based investigations which result in the procurement of plant root tips for subsequent cytological examination.

  8. Programmed cell death in C. elegans, mammals and plants.

    PubMed

    Lord, Christina E N; Gunawardena, Arunika H L A N

    2012-08-01

    Programmed cell death (PCD) is the regulated removal of cells within an organism and plays a fundamental role in growth and development in nearly all eukaryotes. In animals, the model organism Caenorhabditis elegans (C. elegans) has aided in elucidating many of the pathways involved in the cell death process. Various analogous PCD processes can also be found within mammalian PCD systems, including vertebrate limb development. Plants and animals also appear to share hallmarks of PCD, both on the cellular and molecular level. Cellular events visualized during plant PCD resemble those seen in animals including: nuclear condensation, DNA fragmentation, cytoplasmic condensation, and plasma membrane shrinkage. Recently the molecular mechanisms involved in plant PCD have begun to be elucidated. Although few regulatory proteins have been identified as conserved across all eukaryotes, molecular features such as the participation of caspase-like proteases, Bcl-2-like family members and mitochondrial proteins appear to be conserved between plant and animal systems. Transgenic expression of mammalian and C. elegans pro- and anti-apoptotic genes in plants has been observed to dramatically influence the regulatory pathways of plant PCD. Although these genes often show little to no sequence similarity they can frequently act as functional substitutes for one another, thus suggesting that action may be more important than sequence resemblance. Here we present a summary of these findings, focusing on the similarities, between mammals, C. elegans, and plants. An emphasis will be placed on the mitochondria and its role in the cell death pathway within each organism. Through the comparison of these systems on both a cellular and molecular level we can begin to better understand PCD in plant systems, and perhaps shed light on the pathways, which are controlling the process. This manuscript adds to the field of PCD in plant systems by profiling apoptotic factors, to scale on a protein

  9. From plants to animals; the role of plant cell death in ruminant herbivores.

    PubMed

    Kingston-Smith, Alison H; Davies, Teri E; Edwards, Joan E; Theodorou, Michael K

    2008-01-01

    Plant cell death occurring as a result of adverse environmental conditions is known to limit crop production. It is less well recognized that plant cell death processes can also contribute to the poor environmental footprint of ruminant livestock production. Although the forage cells ingested by grazing ruminant herbivores will ultimately die, the lack of oxygen, elevated temperature, and challenge by microflora experienced in the rumen induce regulated plant stress responses resulting in DNA fragmentation and autolytic protein breakdown during the cell death process. Excessive ruminal proteolysis contributes to the inefficient conversion of plant to microbial and animal protein which results in up to 70% of the ingested nitrogen being returned to the land as the nitrogenous pollutants ammonia and urea. This constitutes a significant challenge for sustainable livestock production. As it is estimated that 25% of cultivated land worldwide is assigned to livestock production, it is clear that understanding the fundamental biology underlying cell death in ingested forage will have a highly significant role in minimizing the impact of human activities. This review examines our current understanding of plant metabolism in the rumen and explores opportunities for exploitation of plant genetics to advance sustainable land use.

  10. The plant cell wall: a dynamic barrier against pathogen invasion.

    PubMed

    Underwood, William

    2012-01-01

    Prospective plant pathogens must overcome the physical barrier presented by the plant cell wall. In addition to being a preformed, passive barrier limiting access of pathogens to plant cells, the cell wall is actively remodeled and reinforced specifically at discrete sites of interaction with potentially pathogenic microbes. Active reinforcement of the cell wall through the deposition of cell wall appositions, referred to as papillae, is an early response to perception of numerous categories of pathogens including fungi and bacteria. Rapid deposition of papillae is generally correlated with resistance to fungal pathogens that attempt to penetrate plant cell walls for the establishment of feeding structures. Despite the ubiquity and apparent importance of this early defense response, relatively little is known about the underlying molecular mechanisms and cellular processes involved in the targeting and assembly of papillae. This review summarizes recent advances in our understanding of cell wall-associated defenses induced by pathogen perception as well as the impact of changes in cell wall polymers on interactions with pathogens and highlights significant unanswered questions driving future research in the area.

  11. Microtubule organization and microtubule-associated proteins in plant cells.

    PubMed

    Hamada, Takahiro

    2014-01-01

    Plants have unique microtubule (MT) arrays, cortical MTs, preprophase band, mitotic spindle, and phragmoplast, in the processes of evolution. These MT arrays control the directions of cell division and expansion especially in plants and are essential for plant morphogenesis and developments. Organizations and functions of these MT arrays are accomplished by diverse MT-associated proteins (MAPs). This review introduces 10 of conserved MAPs in eukaryote such as γ-TuC, augmin, katanin, kinesin, EB1, CLASP, MOR1/MAP215, MAP65, TPX2, formin, and several plant-specific MAPs such as CSI1, SPR2, MAP70, WVD2/WDL, RIP/MIDD, SPR1, MAP18/PCaP, EDE1, and MAP190. Most of the studies cited in this review have been analyzed in the particular model plant, Arabidopsis thaliana. The significant knowledge of A. thaliana is the important established base to understand MT organizations and functions in plants. PMID:25262237

  12. Development of water buffalo (Bubalus bubalis) embryos from in vitro matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei.

    PubMed

    Meena, C R; Das, S K

    2006-07-01

    The present study was carried out to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to find out the developmental competence of embryos following transfer of these nuclei to in vitro matured enucleated buffalo oocytes. Skin cells were isolated from 1 to 2-month-old fetuses obtained from slaughterhouse, by enzymatic digestion (0.5% w/v trypsin +0.05% w/v collagenase in Dulbecco's PBS) for 15-20 min. The cells were washed 4 times with Dulbecco's PBS and then once with RPMI-1640+10% FBS by centrifugation at 600 x g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 2-3 days. Cumulus-oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 5 microg/ml FSH-P + 10 microg/ml LH+10% FBS) for 20-22 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body with 10-15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electro fused and incubated in the activation medium (TCM-199 + 8 microg/ml cytochalasin-B+10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199+10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 48 h. The cleaved embryos were then co-cultured with buffalo oviduct cells in embryo development media (EDM). Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electro fused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to 2-cell stage, 3 (3.8%) reached to 4-cell stage and 3 (3.8%) reached to 8-cell stage. In the synchronized group, out of 62 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40

  13. Structure and function of endosomes in plant cells.

    PubMed

    Contento, Anthony L; Bassham, Diane C

    2012-08-01

    Endosomes are a heterogeneous collection of organelles that function in the sorting and delivery of internalized material from the cell surface and the transport of materials from the Golgi to the lysosome or vacuole. Plant endosomes have some unique features, with an organization distinct from that of yeast or animal cells. Two clearly defined endosomal compartments have been studied in plant cells, the trans-Golgi network (equivalent to the early endosome) and the multivesicular body (equivalent to the late endosome), with additional endosome types (recycling endosome, late prevacuolar compartment) also a possibility. A model has been proposed in which the trans-Golgi network matures into a multivesicular body, which then fuses with the vacuole to release its cargo. In addition to basic trafficking functions, endosomes in plant cells are known to function in maintenance of cell polarity by polar localization of hormone transporters and in signaling pathways after internalization of ligand-bound receptors. These signaling functions are exemplified by the BRI1 brassinosteroid hormone receptor and by receptors for pathogen elicitors that activate defense responses. After endocytosis of these receptors from the plasma membrane, endosomes act as a signaling platform, thus playing an essential role in plant growth, development and defense responses. Here we describe the key features of plant endosomes and their differences from those of other organisms and discuss the role of these organelles in cell polarity and signaling pathways.

  14. Space radiation effects on plant and mammalian cells

    NASA Astrophysics Data System (ADS)

    Arena, C.; De Micco, V.; Macaeva, E.; Quintens, R.

    2014-11-01

    The study of the effects of ionizing radiation on organisms is related to different research aims. The current review emphasizes the studies on the effects of different doses of sparsely and densely ionizing radiation on living organisms, with the final purpose of highlighting specific and common effects of space radiation in mammals and plants. This topic is extremely relevant in the context of radiation protection from space environment. The response of different organisms to ionizing radiation depends on the radiation quality/dose and/or the intrinsic characteristics of the living system. Macromolecules, in particular DNA, are the critical targets of radiation, even if there is a strong difference between damages encountered by plant and mammalian cells. The differences in structure and metabolism between the two cell types are responsible for the higher resistance of the plant cell compared with its animal counterpart. In this review, we report some recent findings from studies performed in Space or on Earth, simulating space-like levels of radiation with ground-based facilities, to understand the effect of ionizing radiation on mammalian and plant cells. In particular, our attention is focused on genetic alterations and repair mechanisms in mammalian cells and on structures and mechanisms conferring radioresistance to plant cells.

  15. Fluorescence lifetime imaging of DAPI-stained nuclei as a novel diagnostic tool for the detection and classification of B-cell chronic lymphocytic leukemia.

    PubMed

    Yahav, Gilad; Hirshberg, Abraham; Salomon, Ophira; Amariglio, Ninette; Trakhtenbrot, Luba; Fixler, Dror

    2016-07-01

    B-cell chronic lymphocytic leukaemia (B-CLL) and B-cell precursor acute lymphoblastic leukaemia (B-ALL) are the most common type of leukaemia in adults and children, respectively. Today, fluorescence in situ hybridization (FISH) is the standard for detecting chromosomal aberrations that reflect adverse and favorable outcome. This study revealed a new, simple, and fast diagnostic tool to detect pathological cells by measuring and imaging the fluorescence lifetime (FLT) using FLT imaging microscopy (FLIM) of the peripheral blood (PB) cells of B-CLL samples that were labeled with the DNA binder, DAPI. The FLT of DAPI in healthy individuals was found to be 2.66 ± 0.12 ns. In contrast, PB cells of B-CLL and BM cells of B-ALL patients were characterized by a specific group distribution of the FLT values. The FLT of DAPI was divided into four subgroups, relative to 2.66 ns: short+, normal, prolonged, and prolonged+. These alterations could be related to different chromatin arrangements of B-CLL and B-ALL interphase nuclei. Notably, extremely long FLT of nuclear DAPI correlate with the presence of extra chromosome 12, while moderate increases compared to normal characterize the deletion of p53. Such correlations potentially enable a FLT-based rapid automatic diagnosis and classification of B-CLL even when the frequency of genetic and chromosomal abnormalities is low. © 2016 International Society for Advancement of Cytometry. PMID:27315046

  16. Advanced coal gasifier-fuel cell power plant systems design

    NASA Technical Reports Server (NTRS)

    Heller, M. E.

    1983-01-01

    Two advanced, high efficiency coal-fired power plants were designed, one utilizing a phosphoric acid fuel cell and one utilizing a molten carbonate fuel cell. Both incorporate a TRW Catalytic Hydrogen Process gasifier and regenerator. Both plants operate without an oxygen plant and without requiring water feed; they, instead, require makeup dolomite. Neither plant requires a shift converter; neither plant has heat exchangers operating above 1250 F. Both plants have attractive efficiencies and costs. While the molten carbonate version has a higher (52%) efficiency than the phosphoric acid version (48%), it also has a higher ($0.078/kWh versus $0.072/kWh) ten-year levelized cost of electricity. The phosphoric acid fuel cell power plant is probably feasible to build in the near term: questions about the TRW process need to be answered experimentally, such as weather it can operate on caking coals, and how effective the catalyzed carbon-dioxide acceptor will be at pilot scale, both in removing carbon dioxide and in removing sulfur from the gasifier.

  17. Prospects for advanced coal-fuelled fuel cell power plants

    NASA Astrophysics Data System (ADS)

    Jansen, D.; Vanderlaag, P. C.; Oudhuis, A. B. J.; Ribberink, J. S.

    1994-04-01

    As part of ECN's in-house R&D programs on clean energy conversion systems with high efficiencies and low emissions, system assessment studies have been carried out on coal gasification power plants integrated with high-temperature fuel cells (IGFC). The studies also included the potential to reduce CO2 emissions, and to find possible ways for CO2 extraction and sequestration. The development of this new type of clean coal technology for large-scale power generation is still far off. A significant market share is not envisaged before the year 2015. To assess the future market potential of coal-fueled fuel cell power plants, the promise of this fuel cell technology was assessed against the performance and the development of current state-of-the-art large-scale power generation systems, namely the pulverized coal-fired power plants and the integrated coal gasification combined cycle (IGCC) power plants. With the anticipated progress in gas turbine and gas clean-up technology, coal-fueled fuel cell power plants will have to face severe competition from advanced IGCC power plants, despite their higher efficiency.

  18. Polyphosphoinositides are present in plant tissue culture cells

    SciTech Connect

    Boss, W.F.; Massel, M.O.

    1985-11-15

    Polyphosphoinositides have been isolated from wild carrot cells grown in suspension culture. This is the first report of polyphosphoinositides in plant cells. The phospholipids were identified by comigration with known standards on thin-layer plates. After overnight labeling of the cells with myo-(2-/sup 3/H) inositol, the phosphoinositides as percent recovered inositol were 93% phosphatidylinositol., 3.7% lysophosphatidylinositol, 1.7% phosphatidylinositol monophosphate, 0.8% phosphatidylinositol bisphosphate.

  19. Screening and characterization of plant cell walls using carbohydrate microarrays.

    PubMed

    Sørensen, Iben; Willats, William G T

    2011-01-01

    Plant cells are surrounded by cell walls built largely from complex carbohydrates. The primary walls of growing plant cells consist of interdependent networks of three polysaccharide classes: cellulose, cross-linking glycans (also known as hemicelluloses), and pectins. Cellulose microfibrils are tethered together by cross-linking glycans, and this assembly forms the major load-bearing component of primary walls, which is infiltrated with pectic polymers. In the secondary walls of woody tissues, pectins are much reduced and walls are reinforced with the phenolic polymer lignin. Plant cell walls are essential for plant life and also have numerous industrial applications, ranging from wood to nutraceuticals. Enhancing our knowledge of cell wall biology and the effective use of cell wall materials is dependent to a large extent on being able to analyse their fine structures. We have developed a suite of techniques based on microarrays probed with monoclonal antibodies with specificity for cell wall components, and here we present practical protocols for this type of analysis.

  20. (Study of plant cells and tumors): Progress report

    SciTech Connect

    Not Available

    1989-01-01

    Studies of the cell and molecular biology of animal cell tumors has long been recognized as a fertile and productive area for obtaining new and fundamental insights into mechanisms regulating the growth and differentiation of animal cells. As a novel approach to studying similar phenomena in plant cells, we have isolated a number of tumors in the small cruciferous plant Arabidopsis thaliana and have begun to characterize these at the cellular and molecular levels. Studies at the cellular level should lead to new insights into the relationships between hormones, cell growth and cell differentiation, while studies at the molecular level may reveal and allow us to isolate genes involved either in the hormone response, or in other important aspects of the cells' growth regulatory network. Tumors were induced on the plant by irradiation of seed or seedlings with Co-60 gamma rays. When placed in culture, these tumors were able to grow on hormone-free medium, in contrast to normal plant tissues which requires both an auxin and a cytokinin for growth. In the first phase of this project, we have concentrated on characterizing the growth, general phenotype, and hormonal sensitivity of the tumors. These studies will lead into a molecular analysis of the changes expressed in each tumor which may be responsible for the altered phenotype. 7 refs., 1 tab.

  1. Metabolism of histones and nonhistone proteins of the nuclei and chromatin of liver cells in rats of different ages

    SciTech Connect

    Klimenko, A.I.; Malyshev, A.B.; Kulachenko, B.V.

    1986-10-10

    The metabolism of various classes of histones and nonhistone proteins in whole nuclei and liver chromatin of albino Wistar rats 1, 3, 12, and 24 months of age was studied. It was shown that in the course of postnatal ontogenesis, the metabolism of nonhistone proteins, extractable by a 0.14 M solution of NaCl, is increased in the animals. The incorporation of labeled precursors into the HMG 14 and HMG 17 proteins decreases with age of the animals; a higher level of specific radioactivity was established for the HMG 1+2 proteins in the 3- and 24-month old animals. The intensity of the metabolism of nonhistone proteins and histones is higher in the chromatin complex than in the whole nucleus at all stages of postnatal development of the animals. Among the histone proteins, H1 histones possess a higher level of specific radioactivity in animals of all age groups.

  2. 3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography

    PubMed Central

    Reichelt, Mike; Joubert, Lydia; Perrino, John; Koh, Ai Leen; Phanwar, Ibanri; Arvin, Ann M.

    2012-01-01

    Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell

  3. The culture of single plant cells: a historical view.

    PubMed

    Pennazio, Sergio

    2002-01-01

    Plant tissue culture, introduced unsuccessfully at the beginning of the twentieth century by Haberlandt, received full confirmation in the late Thirties by the works of Gautheret and Nobécourt, thanks to the discovery of auxin. A further special improvement--the free cell culture--, already fore-told by Haberlandt, was successfully achieved towards the mid-1950s by several physiologists thanks to coconut milk (cytokinin). The English physiologist Frederick Steward (who grouped an excellent American team of research during his twenty years stay at the Cornell University in Ithaca) was able to obtain complete cell differentiation from single cells cultured in vitro and demonstrate the totipotence of plant cells at any stage of development. The historical meaning of the research of Steward's team, accomplished between 1958 and 1970, rests on the concept of plant hormones as regulators of gene activity. In other terms, organogenesis was conceived as an epigenetically controlled series of events in which plant genes were "switched on" or "switched off" by special biomolecules. Steward's research paved the way for molecular plant physiology and inspired future research on the relation between cell receptors and specific hormones. PMID:12680309

  4. Structure of Plant Cell Walls 1

    PubMed Central

    Ishii, Tadashi; Thomas, Jerry; Darvill, Alan; Albersheim, Peter

    1989-01-01

    Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-α-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-α-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na2CO3 at 1 and 22°C. These previously uncharacterized polysaccharides accounted for ∼4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO3-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na2CO3 at 1°C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells. PMID:16666559

  5. The DOF protein, SAD, interacts with GAMYB in plant nuclei and activates transcription of endosperm-specific genes during barley seed development.

    PubMed

    Diaz, Isabel; Martinez, Manuel; Isabel-LaMoneda, Ines; Rubio-Somoza, Ignacio; Carbonero, Pilar

    2005-06-01

    The DOF protein, SAD, previously shown to be a transcriptional activator in barley aleurone cells upon seed germination, also has an important role in gene regulation during endosperm development. mRNA was detected in early (10 days after flowering) developing barley seeds where it accumulated in the starchy endosperm, aleurone cells, nucellar projection, vascular tissues and the immature embryo, as shown by RT-PCR and in situ hybridization analyses. The SAD protein, expressed in bacteria, binds to oligonucleotides containing the prolamine box, 5'-A/TAAAG-3'sequence, derived from the promoter regions of the endosperm-specific genes Hor2 and Itr1, encoding a B-hordein and trypsin-inhibitor BTI-CMe, respectively. SAD competed for the same binding sites with another endosperm-expressed DOF protein, BPBF. Transient expression experiments in co-bombarded developing endosperms demonstrated that SAD trans-activated transcription from Hor2 and Itr1 promoters through binding to the intact DOF motifs. When the two DOF factors are co-bombarded together an additive effect was observed upon the expression of the Itr1 gene. In-frame fusion of the Sad ORF to the reporter green fluorescent protein gene directs the fluorescence expression to the nucleus in transiently transformed onion epidermal layers. The visualization of fluorescence in the nucleus of onion cells, using the bimolecular fluorescent complex (BiFC) approach, has shown the in vivo interaction between SAD and the R2R3MYB protein GAMYB. The interaction in plant cells has also been documented for the DOF protein BPBF and GAMYB, but nuclear interaction could not be detected between BPBF and SAD by this procedure. PMID:15918880

  6. Nanosecond electric pulses trigger actin responses in plant cells

    SciTech Connect

    Berghoefer, Thomas; Eing, Christian; Flickinger, Bianca; Hohenberger, Petra; Wegner, Lars H.; Frey, Wolfgang; Nick, Peter

    2009-09-25

    We have analyzed the cellular effects of nanosecond pulsed electrical fields on plant cells using fluorescently tagged marker lines in the tobacco cell line BY-2 and confocal laser scanning microscopy. We observe a disintegration of the cytoskeleton in the cell cortex, followed by contraction of actin filaments towards the nucleus, and disintegration of the nuclear envelope. These responses are accompanied by irreversible permeabilization of the plasma membrane manifest as uptake of Trypan Blue. By pretreatment with the actin-stabilizing drug phalloidin, the detachment of transvacuolar actin from the cell periphery can be suppressed, and this treatment can also suppress the irreversible perforation of the plasma membrane. We discuss these findings in terms of a model, where nanosecond pulsed electric fields trigger actin responses that are key events in the plant-specific form of programmed cell death.

  7. Optical Property Analyses of Plant Cells for Adaptive Optics Microscopy

    NASA Astrophysics Data System (ADS)

    Tamada, Yosuke; Murata, Takashi; Hattori, Masayuki; Oya, Shin; Hayano, Yutaka; Kamei, Yasuhiro; Hasebe, Mitsuyasu

    2014-04-01

    In astronomy, adaptive optics (AO) can be used to cancel aberrations caused by atmospheric turbulence and to perform diffraction-limited observation of astronomical objects from the ground. AO can also be applied to microscopy, to cancel aberrations caused by cellular structures and to perform high-resolution live imaging. As a step toward the application of AO to microscopy, here we analyzed the optical properties of plant cells. We used leaves of the moss Physcomitrella patens, which have a single layer of cells and are thus suitable for optical analysis. Observation of the cells with bright field and phase contrast microscopy, and image degradation analysis using fluorescent beads demonstrated that chloroplasts provide the main source of optical degradations. Unexpectedly, the cell wall, which was thought to be a major obstacle, has only a minor effect. Such information provides the basis for the application of AO to microscopy for the observation of plant cells.

  8. Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells

    PubMed Central

    Kwon, Kwang-Chul; Daniell, Henry

    2016-01-01

    Plants cells are now approved by the FDA for cost-effective production of protein drugs (PDs) in large-scale current Good Manufacturing Practice (cGMP) hydroponic growth facilities. In lyophilized plant cells, PDs are stable at ambient temperature for several years, maintaining their folding and efficacy. Upon oral delivery, PDs bioencapsulated in plant cells are protected in the stomach from acids and enzymes but are subsequently released into the gut lumen by microbes that digest the plant cell wall. The large mucosal area of the human intestine offers an ideal system for oral drug delivery. When tags (receptor-binding proteins or cell-penetrating peptides) are fused to PDs, they efficiently cross the intestinal epithelium and are delivered to the circulatory or immune system. Unique tags to deliver PDs to human immune or nonimmune cells have been developed recently. After crossing the epithelium, ubiquitous proteases cleave off tags at engineered sites. PDs are also delivered to the brain or retina by crossing the blood–brain or retinal barriers. This review highlights recent advances in PD delivery to treat Alzheimer's disease, diabetes, hypertension, Gaucher's or ocular diseases, as well as the development of affordable drugs by eliminating prohibitively expensive purification, cold chain and sterile delivery. PMID:27378236

  9. Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells.

    PubMed

    Kwon, Kwang-Chul; Daniell, Henry

    2016-08-01

    Plants cells are now approved by the FDA for cost-effective production of protein drugs (PDs) in large-scale current Good Manufacturing Practice (cGMP) hydroponic growth facilities. In lyophilized plant cells, PDs are stable at ambient temperature for several years, maintaining their folding and efficacy. Upon oral delivery, PDs bioencapsulated in plant cells are protected in the stomach from acids and enzymes but are subsequently released into the gut lumen by microbes that digest the plant cell wall. The large mucosal area of the human intestine offers an ideal system for oral drug delivery. When tags (receptor-binding proteins or cell-penetrating peptides) are fused to PDs, they efficiently cross the intestinal epithelium and are delivered to the circulatory or immune system. Unique tags to deliver PDs to human immune or nonimmune cells have been developed recently. After crossing the epithelium, ubiquitous proteases cleave off tags at engineered sites. PDs are also delivered to the brain or retina by crossing the blood-brain or retinal barriers. This review highlights recent advances in PD delivery to treat Alzheimer's disease, diabetes, hypertension, Gaucher's or ocular diseases, as well as the development of affordable drugs by eliminating prohibitively expensive purification, cold chain and sterile delivery. PMID:27378236

  10. The endoplasmic reticulum: a social network in plant cells.

    PubMed

    Chen, Jun; Doyle, Caitlin; Qi, Xingyun; Zheng, Huanquan

    2012-11-01

    The endoplasmic reticulum (ER) is an interconnected network comprised of ribosome-studded sheets and smooth tubules. The ER plays crucial roles in the biosynthesis and transport of proteins and lipids, and in calcium (Ca(2+) ) regulation in compartmentalized eukaryotic cells including plant cells. To support its well-segregated functions, the shape of the ER undergoes notable changes in response to both developmental cues and outside influences. In this review, we will discuss recent findings on molecular mechanisms underlying the unique morphology and dynamics of the ER, and the importance of the interconnected ER network in cell polarity. In animal and yeast cells, two family proteins, the reticulons and DP1/Yop1, are required for shaping high-curvature ER tubules, while members of the atlastin family of dynamin-like GTPases are involved in the fusion of ER tubules to make an interconnected ER network. In plant cells, recent data also indicate that the reticulons are involved in shaping ER tubules, while RHD3, a plant member of the atlastin GTPases, is required for the generation of an interconnected ER network. We will also summarize the current knowledge on how the ER interacts with other membrane-bound organelles, with a focus on how the ER and Golgi interplay in plant cells. PMID:23046093

  11. Gravity, chromosomes, and organized development in aseptically cultured plant cells

    NASA Technical Reports Server (NTRS)

    Krikorian, Abraham D.

    1993-01-01

    The objectives of the PCR experiment are: to test the hypothesis that microgravity will in fact affect the pattern and developmental progression of embryogenically competent plant cells from one well-defined, critical stage to another; to determine the effects of microgravity in growth and differentiation of embryogenic carrot cells grown in cell culture; to determine whether microgravity or the space environment fosters an instability of the differentiated state; and to determine whether mitosis and chromosome behavior are adversely affected by microgravity. The methods employed will consist of the following: special embryogenically competent carrot cell cultures will be grown in cell culture chambers provided by NASDA; four cell culture chambers will be used to grow cells in liquid medium; two dishes (plant cell culture dishes) will be used to grow cells on a semi-solid agar support; progression to later embryonic stages will be induced in space via crew intervention and by media manipulation in the case of liquid grown cell cultures; progression to later stages in case of semi-solid cultures will not need crew intervention; embryo stages will be fixed at a specific interval (day 6) in flight only in the case of liquid-grown cultures; and some living cells and somatic embryos will be returned for continued post-flight development and 'grown-out.' These will derive from the semi-solid grown cultures.

  12. Plant Cell Division Analyzed by Transient Agrobacterium-Mediated Transformation of Tobacco BY-2 Cells.

    PubMed

    Buschmann, Henrik

    2016-01-01

    The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.

  13. Do cancer cells in human and meristematic cells in plant exhibit similar responses toward plant extracts with cytotoxic activities?

    PubMed

    Khalifa, Noha S; Barakat, Hoda S; Elhallouty, Salwa; Salem, Dina

    2015-01-01

    We examined the effect of water extracts of Persea americana fruit, and of the leaves of Tabernamontana divericata, Nerium oleander and Annona cherimolia (positive control) on Vicia faba root cells. We had confirmed in our previously published data the cytotoxicity of these plant extracts on four human cancer cell lines: liver (HepG-2), lung (A549), colon (HT-29) and breast (MCF-7). Vicia faba roots were soaked in plant extracts at dilutions of 100, 1,250, 2,500, 5,000, 10,000, 20,000 ppm for 4 and 24 h. All treatments resulted in a significant reduction in the mitotic index in a dose dependant manner. Root cells treated with T. divericata, N. oleander and A. cherimolia exhibited a decrease in prophase cell percentage, increase in micronuclei and chromosomal abnormalities as concentration increased. The P. americana treatment showed the highest cytotoxic effect on cancer cells, prophase cell percentage increased linearly with the applied concentration and no micronuclei were detected. This study shows that root tip assay of beans can be used in initial screening for new plant extracts to validate their use as candidates for containing active cytotoxic agents against malignant cells. This will greatly help in exploring new plant extracts as drugs for cancer treatment. PMID:24705601

  14. Roles and regulation of plant cell walls surrounding plasmodesmata.

    PubMed

    Knox, J Paul; Benitez-Alfonso, Yoselin

    2014-12-01

    In plants, the intercellular transport of simple and complex molecules can occur symplastically through plasmodesmata. These are membranous channels embedded in cell walls that connect neighbouring cells. The properties of the cell walls surrounding plasmodesmata determine their transport capacity and permeability. These cell wall micro-domains are enriched in callose and have a characteristic pectin distribution. Cell wall modifications, leading to changes in plasmodesmata structure, have been reported to occur during development and in response to environmental signals. Cell wall remodelling enzymes target plasmodesmata to rapidly control intercellular communication in situ. Here we describe current knowledge on the composition of cell walls at plasmodesmata sites and on the proteins and signals that modify cell walls to regulate plasmodesmata aperture.

  15. The Structure of Plant Cell Walls

    PubMed Central

    Bauer, Wolfgang D.; Talmadge, Kenneth W.; Keegstra, Kenneth; Albersheim, Peter

    1973-01-01

    The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed “amyloid” xyloglucans. Xyloglucan—or fragments of xyloglucan—and acidic fragments of the pectic polysaccharides are released from endopolygalacturonase-pretreated sycamore walls by treatment of these walls with 8 m urea, endoglucanase, or 0.5 n NaOH. Some of the xyloglucan thus released is found to cochromatograph with the acidic pectic fragments on diethylaminoethyl Sephadex. The chemical or enzymic treatments required for the release of xyloglucan from the walls and the cochromatography of xyloglucan with the acidic pectic fragments indicate that xyloglucan is covalently linked to the pectic polysaccharides and is noncovalently bound to the cellulose fibrils of the sycamore cell wall. The molecular structure of sycamore xyloglucan was characterized by methylation analysis of the oligosaccharides obtained by endoglucanase treatment of the polymer. The structure of the polymer is based on a repeating heptasaccharide unit which consists of 4 residues of β-1-4-linked glucose and 3 residues of terminal xylose. A single xylose residue is glycosidically linked to carbon 6 of 3 of the glucosyl residues. PMID:16658281

  16. Shuttle orbter fuel cell power plant

    NASA Technical Reports Server (NTRS)

    1983-01-01

    This is one of the three fuel cells that make up the generating system which provides electrical power to the space shuttle orbiter. Each unit measures 14 inches (35 centimeters) high, 15 inches (38 centimeters) wide, 40 inches (101 centimeters) long and weighs 200 pounds.

  17. Puzzling Out the Cell's Power Plant.

    ERIC Educational Resources Information Center

    Miller, Julie Ann

    1979-01-01

    The biological research, of Gottfried Schatz at the University of Basel and Gunter Blobel at Rockefeller University, which explains a mechanism by which mitochondrial proteins are transported across membranes is described. Results indicate that the construction and heredity of mitochondria have surprising differences from other cell processes. (BT)

  18. Role of the plant cell wall in gravity resistance.

    PubMed

    Hoson, Takayuki; Wakabayashi, Kazuyuki

    2015-04-01

    Gravity resistance, mechanical resistance to the gravitational force, is a principal graviresponse in plants, comparable to gravitropism. The cell wall is responsible for the final step of gravity resistance. The gravity signal increases the rigidity of the cell wall via the accumulation of its constituents, polymerization of certain matrix polysaccharides due to the suppression of breakdown, stimulation of cross-link formation, and modifications to the wall environment, in a wide range of situations from microgravity in space to hypergravity. Plants thus develop a tough body to resist the gravitational force via an increase in cell wall rigidity and the modification of growth anisotropy. The development of gravity resistance mechanisms has played an important role in the acquisition of responses to various mechanical stresses and the evolution of land plants.

  19. Compost in plant microbial fuel cell for bioelectricity generation.

    PubMed

    Moqsud, M A; Yoshitake, J; Bushra, Q S; Hyodo, M; Omine, K; Strik, David

    2015-02-01

    Recycling of organic waste is an important topic in developing countries as well as developed countries. Compost from organic waste has been used for soil conditioner. In this study, an experiment has been carried out to produce green energy (bioelectricity) by using paddy plant microbial fuel cells (PMFCs) in soil mixed with compost. A total of six buckets filled with the same soil were used with carbon fiber as the electrodes for the test. Rice plants were planted in five of the buckets, with the sixth bucket containing only soil and an external resistance of 100 ohm was used for all cases. It was observed that the cells with rice plants and compost showed higher values of voltage and power density with time. The highest value of voltage showed around 700 mV when a rice plant with 1% compost mixed soil was used, however it was more than 95% less in the case of no rice plant and without compost. Comparing cases with and without compost but with the same number of rice plants, cases with compost depicted higher voltage to as much as 2 times. The power density was also 3 times higher when the compost was used in the paddy PMFCs which indicated the influence of compost on bio-electricity generation.

  20. Gene Delivery into Plant Cells for Recombinant Protein Production

    PubMed Central

    Chen, Qiang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  1. Gene delivery into plant cells for recombinant protein production.

    PubMed

    Chen, Qiang; Lai, Huafang

    2015-01-01

    Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275

  2. Calcium signaling in plant cells in altered gravity

    NASA Astrophysics Data System (ADS)

    Kordyum, E. L.

    2003-10-01

    Changes in the intracellular Ca 2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca 2+ signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus - response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in 80 th, a review highlighting the performed research and the possible significance of such Ca 2+ changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumebly specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca 2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca 2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravisensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface

  3. Stress induced by premature chromatin condensation triggers chromosome shattering and chromothripsis at DNA sites still replicating in micronuclei or multinucleate cells when primary nuclei enter mitosis.

    PubMed

    Terzoudi, Georgia I; Karakosta, Maria; Pantelias, Antonio; Hatzi, Vasiliki I; Karachristou, Ioanna; Pantelias, Gabriel

    2015-11-01

    Combination of next-generation DNA sequencing, single nucleotide polymorphism array analyses and bioinformatics has revealed the striking phenomenon of chromothripsis, described as complex genomic rearrangements acquired in a single catastrophic event affecting one or a few chromosomes. Via an unproven mechanism, it is postulated that mechanical stress causes chromosome shattering into small lengths of DNA, which are then randomly reassembled by DNA repair machinery. Chromothripsis is currently examined as an alternative mechanism of oncogenesis, in contrast to the present paradigm that considers a stepwise development of cancer. While evidence for the mechanism(s) underlying chromosome shattering during cancer development remains elusive, a number of hypotheses have been proposed to explain chromothripsis, including ionizing radiation, DNA replication stress, breakage-fusion-bridge cycles, micronuclei formation and premature chromosome compaction. In the present work, we provide experimental evidence on the mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single catastrophic event. Considering the dynamic nature of chromatin nucleoprotein complex, capable of rapid unfolding, disassembling, assembling and refolding, we first show that chromatin condensation at repairing or replicating DNA sites induces the mechanical stress needed for chromosome shattering to ensue. Premature chromosome condensation is then used to visualize the dynamic nature of interphase chromatin and demonstrate that such mechanical stress and chromosome shattering can also occur in chromosomes within micronuclei or asynchronous multinucleate cells when primary nuclei enter mitosis. Following an aberrant mitosis, chromosomes could find themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and rearrangement in a single catastrophic event. Specifically, our results support the hypothesis that premature chromosome

  4. Stress induced by premature chromatin condensation triggers chromosome shattering and chromothripsis at DNA sites still replicating in micronuclei or multinucleate cells when primary nuclei enter mitosis.

    PubMed

    Terzoudi, Georgia I; Karakosta, Maria; Pantelias, Antonio; Hatzi, Vasiliki I; Karachristou, Ioanna; Pantelias, Gabriel

    2015-11-01

    Combination of next-generation DNA sequencing, single nucleotide polymorphism array analyses and bioinformatics has revealed the striking phenomenon of chromothripsis, described as complex genomic rearrangements acquired in a single catastrophic event affecting one or a few chromosomes. Via an unproven mechanism, it is postulated that mechanical stress causes chromosome shattering into small lengths of DNA, which are then randomly reassembled by DNA repair machinery. Chromothripsis is currently examined as an alternative mechanism of oncogenesis, in contrast to the present paradigm that considers a stepwise development of cancer. While evidence for the mechanism(s) underlying chromosome shattering during cancer development remains elusive, a number of hypotheses have been proposed to explain chromothripsis, including ionizing radiation, DNA replication stress, breakage-fusion-bridge cycles, micronuclei formation and premature chromosome compaction. In the present work, we provide experimental evidence on the mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single catastrophic event. Considering the dynamic nature of chromatin nucleoprotein complex, capable of rapid unfolding, disassembling, assembling and refolding, we first show that chromatin condensation at repairing or replicating DNA sites induces the mechanical stress needed for chromosome shattering to ensue. Premature chromosome condensation is then used to visualize the dynamic nature of interphase chromatin and demonstrate that such mechanical stress and chromosome shattering can also occur in chromosomes within micronuclei or asynchronous multinucleate cells when primary nuclei enter mitosis. Following an aberrant mitosis, chromosomes could find themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and rearrangement in a single catastrophic event. Specifically, our results support the hypothesis that premature chromosome

  5. Plastids: dynamic components of plant cell development.

    PubMed

    Guikema, J A; Gallegos, G L

    1992-01-01

    The gravitropic bending of maize roots, as a response to reorientation of the root within a gravitational field, was examined for sensitivity to exogenous applications of the cytoskeletal inhibitor, cytochalasin D. Agar blocks were impregnated with this inhibitor, and were applied either to the root cap or to the zone of root cell elongation. Root growth was normal with either treatment, if the roots were not repositioned with respect to the gravitational vector. When untreated roots were placed in a horizontal position with respect to gravity, a 40 degree bending response was observed within one hour. This bending also occurred when cytochalasin D was applied at high concentrations to the zone of root cell elongation. However, when cytochalasin D above 40 micrograms/ml was applied to the root cap, roots lost the ability of directional reorientation within the gravitational field, causing a random bending. PMID:11537985

  6. Plastids: dynamic components of plant cell development

    NASA Technical Reports Server (NTRS)

    Guikema, J. A.; Gallegos, G. L.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The gravitropic bending of maize roots, as a response to reorientation of the root within a gravitational field, was examined for sensitivity to exogenous applications of the cytoskeletal inhibitor, cytochalasin D. Agar blocks were impregnated with this inhibitor, and were applied either to the root cap or to the zone of root cell elongation. Root growth was normal with either treatment, if the roots were not repositioned with respect to the gravitational vector. When untreated roots were placed in a horizontal position with respect to gravity, a 40 degree bending response was observed within one hour. This bending also occurred when cytochalasin D was applied at high concentrations to the zone of root cell elongation. However, when cytochalasin D above 40 micrograms/ml was applied to the root cap, roots lost the ability of directional reorientation within the gravitational field, causing a random bending.

  7. The cell biology of lignification in higher plants

    PubMed Central

    Barros, Jaime; Serk, Henrik; Granlund, Irene; Pesquet, Edouard

    2015-01-01

    Background Lignin is a polyphenolic polymer that strengthens and waterproofs the cell wall of specialized plant cell types. Lignification is part of the normal differentiation programme and functioning of specific cell types, but can also be triggered as a response to various biotic and abiotic stresses in cells that would not otherwise be lignifying. Scope Cell wall lignification exhibits specific characteristics depending on the cell type being considered. These characteristics include the timing of lignification during cell differentiation, the palette of associated enzymes and substrates, the sub-cellular deposition sites, the monomeric composition and the cellular autonomy for lignin monomer production. This review provides an overview of the current understanding of lignin biosynthesis and polymerization at the cell biology level. Conclusions The lignification process ranges from full autonomy to complete co-operation depending on the cell type. The different roles of lignin for the function of each specific plant cell type are clearly illustrated by the multiple phenotypic defects exhibited by knock-out mutants in lignin synthesis, which may explain why no general mechanism for lignification has yet been defined. The range of phenotypic effects observed include altered xylem sap transport, loss of mechanical support, reduced seed protection and dispersion, and/or increased pest and disease susceptibility. PMID:25878140

  8. Differential scanning calorimetry of plant cell walls

    SciTech Connect

    Lin, Liangshiou; Varner, J.E. ); Yuen, H.K. )

    1991-03-15

    High-sensitivity differential scanning calorimetry has been used to study the phase transition of cell wall preparations of the elongating and mature regions of soybean hypocotyls and of celery epidermis and collenchyma strands. A step-like transition believed to be glass transition was observed in walls isolated from the elongating region of soybean hypocotyls at 52.9C. Addition of 1 mM CaCl{sub 2} to the cell wall preparation increased the transition temperature to 60.8C and greatly reduced the transition magnitude. In walls from the mature region, the transition was small and occurred at a higher temperature (60.1C). Addition of calcium to the mature region cell wall had little effect on the transition. Based on the known interactions between calcium and pectin, the authors propose that calcium affects the glass transition by binding to the polygalacturonate backbone of wall pectin, resulting in a more rigid wall with a smaller transition at a higher temperature. The mature region either has more calcium in the wall or has more methyl-esterified pectin, making it less responsive to added calcium.

  9. Cell proliferation and plant development under novel altered gravity environments.

    PubMed

    Herranz, R; Medina, F J

    2014-01-01

    Gravity is a key factor for life on Earth. It is the only environmental factor that has remained constant throughout evolution, and plants use it to modulate important physiological activities; gravity removal or alteration produces substantial changes in essential functions. For root gravitropism, gravity is sensed in specialised cells, which are capable of detecting magnitudes of the g vector lower than 10(-3) . Then, the mechanosignal is transduced to upper zones of the root, resulting in changes in the lateral distribution of auxin and in the rate of auxin polar transport. Gravity alteration has consequences for cell growth and proliferation rates in root meristems, which are the basis of the developmental programme of a plant, in which regulation via auxin is involved. The effect is disruption of meristematic competence, i.e. the strict coordination between cell proliferation and growth, which characterises meristematic cells. This effect can be related to changes in the transport and distribution of auxin throughout the root. However, similar effects of gravity alteration have been found in plant cell cultures in vitro, in which neither specialised structures for gravity sensing and signal transduction, nor apparent gravitropism have been described. We postulate that gravity resistance, a general mechanism of cellular origin for developing rigid structures in plants capable of resisting the gravity force, could also be responsible for the changes in cell growth and proliferation parameters detected in non-specialised cells. The mechanisms of gravitropism and graviresistance are complementary, the first being mostly sensitive to the direction of the gravity vector, and the second to its magnitude. At a global molecular level, the consequence of gravity alteration is that the genome should be finely tuned to counteract a type of stress that plants have never encountered before throughout evolution. Multigene families and redundant genes present an advantage in

  10. Programmed cell death in plants: A chloroplastic connection

    PubMed Central

    Ambastha, Vivek; Tripathy, Baishnab C; Tiwari, Budhi Sagar

    2015-01-01

    Programmed cell death (PCD) is an integral cellular program by which targeted cells culminate to demise under certain developmental and pathological conditions. It is essential for controlling cell number, removing unwanted diseased or damaged cells and maintaining the cellular homeostasis. The details of PCD process has been very well elucidated and characterized in animals but similar understanding of the process in plants has not been achieved rather the field is still in its infancy that sees some sporadic reports every now and then. The plants have 2 energy generating sub-cellular organelles- mitochondria and chloroplasts unlike animals that just have mitochondria. The presence of chloroplast as an additional energy transducing and ROS generating compartment in a plant cell inclines to advocate the involvement of chloroplasts in PCD execution process. As chloroplasts are supposed to be progenies of unicellular photosynthetic organisms that evolved as a result of endosymbiosis, the possibility of retaining some of the components involved in bacterial PCD by chloroplasts cannot be ruled out. Despite several excellent reviews on PCD in plants, there is a void on an update of information at a place on the regulation of PCD by chloroplast. This review has been written to provide an update on the information supporting the involvement of chloroplast in PCD process and the possible future course of the field. PMID:25760871

  11. The mysterious microcircuitry of the cerebellar nuclei

    PubMed Central

    Uusisaari, Marylka; De Schutter, Erik

    2011-01-01

    Abstract The microcircuitry of cerebellar cortex and, in particular, the physiology of its main element, the Purkinje neuron, has been extensively investigated and described. However, activity in Purkinje neurons, either as single cells or populations, does not directly mediate the cerebellar effects on the motor effector systems. Rather, the result of the entire cerebellar cortical computation is passed to the relatively small cerebellar nuclei that act as the final, integrative processing unit in the cerebellar circuitry. The nuclei ultimately control the temporal and spatial features of the cerebellar output. Given this key role, it is striking that the internal organization and the connectivity with afferent and efferent pathways in the cerebellar nuclei are rather poorly known. In the present review, we discuss some of the many critical shortcomings in the understanding of cerebellar nuclei microcircuitry: the extent of convergence and divergence of the cerebellar cortical pathway to the various cerebellar nuclei neurons and subareas, the possible (lack of) conservation of the finely-divided topographical organization in the cerebellar cortex at the level of the nuclei, as well as the absence of knowledge of the synaptic circuitry within the cerebellar nuclei. All these issues are important for predicting the pattern-extraction and encoding capabilities of the cerebellar nuclei and, until resolved, theories and models of cerebellar motor control and learning may err considerably. PMID:21521761

  12. Organization of projections from the raphe nuclei to the vestibular nuclei in rats

    NASA Technical Reports Server (NTRS)

    Halberstadt, A. L.; Balaban, C. D.

    2003-01-01

    Previous anatomic and electrophysiological evidence suggests that serotonin modulates processing in the vestibular nuclei. This study examined the organization of projections from serotonergic raphe nuclei to the vestibular nuclei in rats. The distribution of serotonergic axons in the vestibular nuclei was visualized immunohistochemically in rat brain slices using antisera directed against the serotonin transporter. The density of serotonin transporter-immunopositive fibers is greatest in the superior vestibular nucleus and the medial vestibular nucleus, especially along the border of the fourth ventricle; it declines in more lateral and caudal regions of the vestibular nuclear complex. After unilateral iontophoretic injections of Fluoro-Gold into the vestibular nuclei, retrogradely labeled neurons were found in the dorsal raphe nucleus (including the dorsomedial, ventromedial and lateral subdivisions) and nucleus raphe obscurus, and to a minor extent in nucleus raphe pallidus and nucleus raphe magnus. The combination of retrograde tracing with serotonin immunohistofluorescence in additional experiments revealed that the vestibular nuclei receive both serotonergic and non-serotonergic projections from raphe nuclei. Tracer injections in densely innervated regions (especially the medial and superior vestibular nuclei) were associated with the largest numbers of Fluoro-Gold-labeled cells. Differences were observed in the termination patterns of projections from the individual raphe nuclei. Thus, the dorsal raphe nucleus sends projections that terminate predominantly in the rostral and medial aspects of the vestibular nuclear complex, while nucleus raphe obscurus projects relatively uniformly throughout the vestibular nuclei. Based on the topographical organization of raphe input to the vestibular nuclei, it appears that dense projections from raphe nuclei are colocalized with terminal fields of flocculo-nodular lobe and uvula Purkinje cells. It is hypothesized that

  13. Plant cell, tissue and organ culture: the most flexible foundations for plant metabolic engineering applications.

    PubMed

    Ogita, Shinjiro

    2015-05-01

    Significant advances in plant cell, tissue and organ culture (PCTOC) have been made in the last five decades. PCTOC is now thought to be the underlying technique for understanding general or specific biological functions of the plant kingdom, and it is one of the most flexible foundations for morphological, physiological and molecular biological applications of plants. Furthermore, the recent advances in the field of information technology (IT) have enabled access to a large amount of information regarding all aspects of plant biology. For example, sequencing information is stored in mega repositories such as the National Center for Biotechnology Information (NCBI), which can be easily accessed by researchers worldwide. To date, the PCTOC and IT combination strategy for regulation of target plant metabolism and the utilization of bioactive plant metabolites for commercial purposes is essential. In this review, the advantages and the limitations of these methodologies, especially regarding the production of bioactive plant secondary metabolites and metabolic engineering in target plants are discussed mainly from the phenotypic view point.

  14. Neat methanol fuel cell power plant

    NASA Astrophysics Data System (ADS)

    Abens, S.; Farooque, M.

    1985-12-01

    Attention is given to a fuel cell development effort which has been directed, by ease-of-supply, low weight, and low volume criteria toward the use of undiluted methanol. Partial oxidation and internal water recovery concepts are incorporated, allowing the onboard dilution of methanol fuel through mixing with exhaust-recovered water. This scheme is successfully demonstrated for the case of a 3 kW unit employing commercial cross flow heat exchangers, as well as for a 5 kW reformer flue exhaust water recovery design with U.S. Air force baseload stationary applications. The USAF powerplant has an overall thermal efficiency of 32 percent at rated load.

  15. Probing visual transduction in a plant cell

    PubMed Central

    Uhl, Rainer; Hegemann, Peter

    1990-01-01

    Light scattering studies of vertebrate rod cells have greatly aided our understanding of the visual transduction process. This technique has now been successfully applied to study visual transduction in a unicellular alga. Flash-induced light scattering changes have been recorded which are repeatable, graded with photon exposure, and adaptive. They appear on a timescale of 15-1,000 ms and correlate kinetically with flash-induced movement responses. The responsible photoreceptor is a rhodopsin. Evidence is provided for the ability of the organism to count single photons. PMID:19431775

  16. Cloning higher plants from aseptically cultured tissues and cells

    NASA Technical Reports Server (NTRS)

    Krikorian, A. D.

    1982-01-01

    A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

  17. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1990-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells. 10 figs., 2 tabs.

  18. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1991-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells.

  19. Cell-to-cell movement of mitochondria in plants.

    PubMed

    Gurdon, Csanad; Svab, Zora; Feng, Yaping; Kumar, Dibyendu; Maliga, Pal

    2016-03-22

    We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer from Nicotiana sylvestris into Nicotiana tabacum cells. The alloplasmic N. tabacum line we used carries Nicotiana undulata cytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer from N. sylvestris to N. tabacum could be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility to orf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation. PMID:26951647

  20. Cell-to-cell movement of mitochondria in plants

    PubMed Central

    Gurdon, Csanad; Svab, Zora; Feng, Yaping; Kumar, Dibyendu; Maliga, Pal

    2016-01-01

    We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer from Nicotiana sylvestris into Nicotiana tabacum cells. The alloplasmic N. tabacum line we used carries Nicotiana undulata cytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer from N. sylvestris to N. tabacum could be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility to orf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation. PMID:26951647

  1. Tubulin tyrosine nitration regulates microtubule organization in plant cells

    PubMed Central

    Blume, Yaroslav B.; Krasylenko, Yuliya A.; Demchuk, Oleh M.; Yemets, Alla I.

    2013-01-01

    During last years, selective tyrosine nitration of plant proteins gains importance as well-recognized pathway of direct nitric oxide (NO) signal transduction. Plant microtubules are one of the intracellular signaling targets for NO, however, the molecular mechanisms of NO signal transduction with the involvement of cytoskeletal proteins remain to be elucidated. Since biochemical evidence of plant α-tubulin tyrosine nitration has been obtained recently, potential role of this posttranslational modification in regulation of microtubules organization in plant cell is estimated in current paper. It was shown that 3-nitrotyrosine (3-NO2-Tyr) induced partially reversible Arabidopsis primary root growth inhibition, alterations of root hairs morphology and organization of microtubules in root cells. It was also revealed that 3-NO2-Tyr intensively decorates such highly dynamic microtubular arrays as preprophase bands, mitotic spindles and phragmoplasts of Nicotiana tabacum Bright Yellow-2 (BY-2) cells under physiological conditions. Moreover, 3D models of the mitotic kinesin-8 complexes with the tail of detyrosinated, tyrosinated and tyrosine nitrated α-tubulin (on C-terminal Tyr 450 residue) from Arabidopsis were reconstructed in silico to investigate the potential influence of tubulin nitrotyrosination on the molecular dynamics of α-tubulin and kinesin-8 interaction. Generally, presented data suggest that plant α-tubulin tyrosine nitration can be considered as its common posttranslational modification, the direct mechanism of NO signal transduction with the participation of microtubules under physiological conditions and one of the hallmarks of the increased microtubule dynamics. PMID:24421781

  2. Plant cell walls: Protecting the barrier from degradation by microbial enzymes.

    PubMed

    Lagaert, Stijn; Beliën, Tim; Volckaert, Guido

    2009-12-01

    Plant cell walls are predominantly composed of polysaccharides, which are connected in a strong, yet resilient network. They determine the size and shape of plant cells and form the interface between the cell and its often hostile environment. To penetrate the cell wall and thus infect plants, most phytopathogens secrete numerous cell wall degrading enzymes. Conversely, as a first line of defense, plant cell walls contain an array of inhibitors of these enzymes. Scientific knowledge on these inhibitors significantly progressed in the past years and this review is meant to give a comprehensive overview of plant inhibitors against microbial cell wall degrading enzymes and their role in plant protection.

  3. JC virus inclusions in progressive multifocal leukoencephalopathy: scaffolding promyelocytic leukemia nuclear bodies grow with cell cycle transition through an S-to-G2-like state in enlarging oligodendrocyte nuclei.

    PubMed

    Shishido-Hara, Yukiko; Yazawa, Takuya; Nagane, Motoo; Higuchi, Kayoko; Abe-Suzuki, Shiho; Kurata, Morito; Kitagawa, Masanobu; Kamma, Hiroshi; Uchihara, Toshiki

    2014-05-01

    In progressive multifocal leukoencephalopathy, JC virus-infected oligodendroglia display 2 distinct patterns of intranuclear viral inclusions: full inclusions in which progeny virions are present throughout enlarged nuclei and dot-shaped inclusions in which virions are clustered in subnuclear domains termed "promyelocytic leukemia nuclear bodies" (PML-NBs). Promyelocytic leukemia nuclear bodies may serve a scaffolding role in viral progeny production. We analyzed the formation process of intranuclear viral inclusions by morphometry and assessed PML-NB alterations in the brains of 2 patients with progressive multifocal leukoencephalopathy. By immunohistochemistry, proliferating cell nuclear antigen was most frequently detected in smaller nuclei; cyclin A was detected in larger nuclei. This suggests an S-to-G2 cell cycle transition in infected cells associated with nuclear enlargement. Sizes of PML-NBs were variable, but they were usually either small speckles 200 to 400 nm in diameter or distinct spherical shells with a diameter of 1 μm or more. By confocal microscopy, JC virus capsid proteins were associated with both small and large PML-NBs, but disruption of large PML-NBs was observed by ground-state depletion fluorescence nanoscopy. Clusters of progeny virions were also detected by electron microscopy. Our data suggest that, in progressive multifocal leukoencephalopathy, JC virus produces progeny virions in enlarging oligodendrocyte nuclei in association with growing PML-NBs and with cell cycle transition through an S-to-G2-like state.

  4. The Structure of Plant Cell Walls

    PubMed Central

    Talmadge, Kenneth W.; Keegstra, Kenneth; Bauer, Wolfgang D.; Albersheim, Peter

    1973-01-01

    This is the first in a series of papers dealing with the structure of cell walls isolated from suspension-cultured sycamore cells (Acer pseudoplatanus). These studies have been made possible by the availability of purified hydrolytic enzymes and by recent improvements in the techniques of methylation analysis. These techniques have permitted us to identify and quantitate the macromolecular components of sycamore cell walls. These walls are composed of 10% arabinan, 2% 3,6-linked arabinogalactan, 23% cellulose, 9% oligo-arabinosides (attached to hydroxyproline), 8% 4-linked galactan, 10% hydroxyproline-rich protein, 16% rhamnogalacturonan, and 21% xyloglucan. The structures of the pectic polymers (the neutral arabinan, the neutral galactan, and the acidic rhamnogalacturonan) were obtained, in part, by methylation analysis of fragments of these polymers which were released from the sycamore walls by the action of a highly purified endopolygalacturonase. The data suggest a branched arabinan and a linear 4-linked galactan occurring as side chains on the rhamnogalacturonan. Small amounts or pieces of a xyloglucan, the wall hemicellulose, appear to be covalently linked to some of the galactan chains. Thus, the galactan appears to serve as a bridge between the xyloglucan and rhamnogalacturonan components of the wall. The rhamnogalacturonan consists of an α-(1 → 4)-linked galacturonan chain which is interspersed with 2-linked rhamnosyl residues. The rhamnosyl residues are not randomly distributed in the chain but probably occur in units of rhamnosyl- (1 → 4)-galacturonosyl- (1 → 2)-rhamnosyl. This sequence appears to alternate with a homogalacturonan sequence containing approximately 8 residues of 4-linked galacturonic acid. About half of the rhamnosyl residues are branched, having a substituent attached to carbon 4. This is likely to be the site of attachment of the 4-linked galactan. The hydroxyprolyl oligo-arabinosides of the hydroxyproline-rich glycoprotein

  5. Accumulation of acidic SK₃ dehydrins in phloem cells of cold- and drought-stressed plants of the Solanaceae.

    PubMed

    Szabala, Bartosz Mieczyslaw; Fudali, Sylwia; Rorat, Tadeusz

    2014-04-01

    The role of acidic SK(n) dehydrins in stress tolerance of important crop and model species of the Solanaceae remains unknown. We have previously shown that the acidic SK₃ dehydrin DHN24 from Solanum sogarandinum is constitutively expressed and its expression is associated with cold acclimation. Here we found that DHN24 is specifically localized to phloem cells of vegetative organs of non-acclimated plants. More precise localization of DHN24 revealed that it is primarily found in sieve elements (SEs) and companion cells (CCs) of roots and stems. In cold-acclimated plants, DHN24 is mainly present in all cell types of the phloem. Dhn24 transcripts are also predominantly localized to phloem cells of cold-acclimated stems. Immunoelectron microscopy localized DHN24 to the cytosol and close to organelle membranes of phloem cells, the lumen with phloem protein filaments, parietal cytoplasm of SEs and the nucleoplasm of some nuclei. Cell fractionation experiments revealed that DHN24 was detected in the cytosolic, nuclear and microsomal fractions. We also determined whether homologous members of the acidic subclass dehydrins from Capsicum annuum and Lycopersicon chilense share the characteristics of DHN24. We showed that they are also constitutively expressed, but their protein level is upregulated preferentially by drought stress. Immunofluorescent localization revealed that they are detected in SEs and CCs of unstressed plants and throughout the phloem in drought-stressed plants. These results suggest that one of the primary roles of DHN24 and its homologs may be the protection of the phloem region from adverse effects of abiotic stresses.

  6. The plant cell nucleus: a true arena for the fight between plants and pathogens.

    PubMed

    Deslandes, Laurent; Rivas, Susana

    2011-01-01

    Communication between the cytoplasm and the nucleus is a fundamental feature shared by both plant and animal cells. Cellular factors involved in the transport of macromolecules through the nuclear envelope, including nucleoporins, importins and Ran-GTP related components, are conserved among a variety of eukaryotic systems. Interestingly, mutations in these nuclear components compromise resistance signalling, illustrating the importance of nucleocytoplasmic trafficking in plant innate immunity. Indeed, spatial restriction of defence regulators by the nuclear envelope and stimulus-induced nuclear translocation constitute an important level of defence-associated gene regulation in plants. A significant number of effectors from different microbial pathogens are targeted to the plant cell nucleus. In addition, key host factors, including resistance proteins, immunity components, transcription factors and transcriptional regulators shuttle between the cytoplasm and the nucleus, and their level of nuclear accumulation determines the output of the defence response, further confirming the crucial role played by the nucleus during the interaction between plants and pathogens. Here, we discuss recent findings that situate the nucleus at the frontline of the mutual recognition between plants and invading microbes.

  7. A multi-model approach to simultaneous segmentation and classification of heterogeneous populations of cell nuclei in 3D confocal microscope images.

    PubMed

    Lin, Gang; Chawla, Monica K; Olson, Kathy; Barnes, Carol A; Guzowski, John F; Bjornsson, Christopher; Shain, William; Roysam, Badrinath

    2007-09-01

    Automated segmentation and morphometry of fluorescently labeled cell nuclei in batches of 3D confocal stacks is essential for quantitative studies. Model-based segmentation algorithms are attractive due to their robustness. Previous methods incorporated a single nuclear model. This is a limitation for tissues containing multiple cell types with different nuclear features. Improved segmentation for such tissues requires algorithms that permit multiple models to be used simultaneously. This requires a tight integration of classification and segmentation algorithms. Two or more nuclear models are constructed semiautomatically from user-provided training examples. Starting with an initial over-segmentation produced by a gradient-weighted watershed algorithm, a hierarchical fragment merging tree rooted at each object is built. Linear discriminant analysis is used to classify each candidate using multiple object models. On the basis of the selected class, a Bayesian score is computed. Fragment merging decisions are made by comparing the score with that of other candidates, and the scores of constituent fragments of each candidate. The overall segmentation accuracy was 93.7% and classification accuracy was 93.5%, respectively, on a diverse collection of images drawn from five different regions of the rat brain. The multi-model method was found to achieve high accuracy on nuclear segmentation and classification by correctly resolving ambiguities in clustered regions containing heterogeneous cell populations.

  8. Programmed cell death in plants: lessons from bacteria?

    PubMed Central

    Wang, Junhui; Bayles, Kenneth W.

    2012-01-01

    Programmed cell death (PCD) has well-established roles in the development and physiology of animals, plants, and fungi. Although aspects of PCD control appear evolutionarily conserved between these organisms, the extent of conservation remains controversial. Recently, a putative bacterial PCD protein homolog in plants was found to play a significant role in cell death control, indicating a conservation of function between these highly divergent organisms. Interestingly, these bacterial proteins are thought to be evolutionarily linked to the Bcl-2 family of proteins. In this Opinion article, we propose a new unifying model to describe the relationship between bacterial and plant PCD systems and propose that the underlying control of PCD is conserved across at least three Kingdoms of life. PMID:23083702

  9. Introducing the Cell Concept with Both Animal and Plant Cells: A Historical and Didactic Approach

    ERIC Educational Resources Information Center

    Clement, Pierre

    2007-01-01

    In France, as well as in several other countries, the cell concept is introduced at school by two juxtaposed drawings, a plant cell and an animal cell. After indicating the didactic obstacles associated with this presentation, this paper focuses on the reasons underlying the persistence of these two prototypes, through three complementary…

  10. Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

    PubMed Central

    Wang, Shu-Long; Zhao, Ge; Zhu, Wei; Dong, Xiao-Meng; Liu, Ting; Li, Yuan-Yuan; Song, Wen-Gang; Wang, Yi-Qiang

    2015-01-01

    AIM To investigate into the potential involvement of pyrin containing 3 gene (NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses. METHODS The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1 (HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40 (SV40)-immortalized human corneal epithelial cell line were also examined. Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β. RESULTS The NLRP3 activation induced by HSV-1 infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore, in the SV40-immortalized human corneal epithelial cells, NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium (known as an inhibitor of NLRP3 activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot. CONCLUSION It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study. PMID:25709906

  11. Measuring the Mechanical Properties of Plant Cell Walls.

    PubMed

    Vogler, Hannes; Felekis, Dimitrios; Nelson, Bradley J; Grossniklaus, Ueli

    2015-03-25

    The size, shape and stability of a plant depend on the flexibility and integrity of its cell walls, which, at the same time, need to allow cell expansion for growth, while maintaining mechanical stability. Biomechanical studies largely vanished from the focus of plant science with the rapid progress of genetics and molecular biology since the mid-twentieth century. However, the development of more sensitive measurement tools renewed the interest in plant biomechanics in recent years, not only to understand the fundamental concepts of growth and morphogenesis, but also with regard to economically important areas in agriculture, forestry and the paper industry. Recent advances have clearly demonstrated that mechanical forces play a crucial role in cell and organ morphogenesis, which ultimately define plant morphology. In this article, we will briefly review the available methods to determine the mechanical properties of cell walls, such as atomic force microscopy (AFM) and microindentation assays, and discuss their advantages and disadvantages. But we will focus on a novel methodological approach, called cellular force microscopy (CFM), and its automated successor, real-time CFM (RT-CFM).

  12. Measuring the Mechanical Properties of Plant Cell Walls

    PubMed Central

    Vogler, Hannes; Felekis, Dimitrios; Nelson, Bradley J.; Grossniklaus, Ueli

    2015-01-01

    The size, shape and stability of a plant depend on the flexibility and integrity of its cell walls, which, at the same time, need to allow cell expansion for growth, while maintaining mechanical stability. Biomechanical studies largely vanished from the focus of plant science with the rapid progress of genetics and molecular biology since the mid-twentieth century. However, the development of more sensitive measurement tools renewed the interest in plant biomechanics in recent years, not only to understand the fundamental concepts of growth and morphogenesis, but also with regard to economically important areas in agriculture, forestry and the paper industry. Recent advances have clearly demonstrated that mechanical forces play a crucial role in cell and organ morphogenesis, which ultimately define plant morphology. In this article, we will briefly review the available methods to determine the mechanical properties of cell walls, such as atomic force microscopy (AFM) and microindentation assays, and discuss their advantages and disadvantages. But we will focus on a novel methodological approach, called cellular force microscopy (CFM), and its automated successor, real-time CFM (RT-CFM). PMID:27135321

  13. Measuring the Mechanical Properties of Plant Cell Walls.

    PubMed

    Vogler, Hannes; Felekis, Dimitrios; Nelson, Bradley J; Grossniklaus, Ueli

    2015-01-01

    The size, shape and stability of a plant depend on the flexibility and integrity of its cell walls, which, at the same time, need to allow cell expansion for growth, while maintaining mechanical stability. Biomechanical studies largely vanished from the focus of plant science with the rapid progress of genetics and molecular biology since the mid-twentieth century. However, the development of more sensitive measurement tools renewed the interest in plant biomechanics in recent years, not only to understand the fundamental concepts of growth and morphogenesis, but also with regard to economically important areas in agriculture, forestry and the paper industry. Recent advances have clearly demonstrated that mechanical forces play a crucial role in cell and organ morphogenesis, which ultimately define plant morphology. In this article, we will briefly review the available methods to determine the mechanical properties of cell walls, such as atomic force microscopy (AFM) and microindentation assays, and discuss their advantages and disadvantages. But we will focus on a novel methodological approach, called cellular force microscopy (CFM), and its automated successor, real-time CFM (RT-CFM). PMID:27135321

  14. Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly c...

  15. Importin β1 mediates nuclear factor-κB signal transduction into the nuclei of myeloma cells and affects their proliferation and apoptosis.

    PubMed

    Yan, Wenqing; Li, Rong; He, Jie; Du, Juan; Hou, Jian

    2015-04-01

    Multiple myeloma (MM) is a plasma cell neoplasm that is currently incurable. The activation of nuclear factor-κB (NF-κB) signalling plays a crucial role in the immortalisation of MM cells. As the most important transcription factor of the canonical NF-κB pathway, the p50/p65 heterodimer requires transportation into the nucleus for its successful signal transduction. Importin β1 is the key transport receptor that mediates p50/p65 nuclear import. Currently, it remains unclear whether the regulation of importin β1 function affects the biological behaviour of MM cells. In the present study, we investigated the changes in p65 translocation and the proliferation and apoptosis of MM cells after treatment with small interfering RNA (siRNA) or an importin β1 inhibitor. The underlying mechanisms were also investigated. We found importin β1 over-expression and the excessive nuclear transport of p65 in myeloma cells. Confocal laser scanning microscopy and Western blot analysis results indicated that p65 nuclear transport was blocked after inhibiting importin β1 expression with siRNA and the importin β1-specific inhibitor importazole (IPZ). Importantly, electronic mobility shift assay results also verified that p65 nuclear transport was dramatically reduced. Moreover, the expression of the NF-κB signalling target genes involved in MM cell apoptosis, such as BCL-2, c-IAP1 and XIAP, were markedly reduced, as demonstrated by the RT-PCR results. Furthermore, the proliferation of MM cells was inhibited, as demonstrated by MTT assay results, and the MM cell apoptosis rate was higher, as demonstrated by the annexin V/propidium iodide (PI) double-staining assay results. Additionally, the percentage of S phase cells in the myeloma cell lines treated with IPZ was dramatically reduced. In conclusion, our results clearly show that importin β1 mediates the translocation of NF-κB into the nuclei of myeloma cells, thereby regulating proliferation and blocking apoptosis, which

  16. Active galactic nuclei

    PubMed Central

    Fabian, Andrew C.

    1999-01-01

    Active galactic nuclei are the most powerful, long-lived objects in the Universe. Recent data confirm the theoretical idea that the power source is accretion into a massive black hole. The common occurrence of obscuration and outflows probably means that the contribution of active galactic nuclei to the power density of the Universe has been generally underestimated. PMID:10220363

  17. (Rapid regulatory control of plant cell expansion and wall relaxation)

    SciTech Connect

    Cosgrove, D.J.

    1990-01-01

    This section presents a brief overview of accomplishments related to this project in the past 3-year period. Our work has focused on the basic mechanisms of plant cell expansion, particularly on the interrelations of water and solute transport with cell wall relaxation and expansion. To study these processes, we have developed new methods and used these methods to analyze the dynamic behavior of growth processes and to examine how various agents (GA, drought, light, genetic lesions) alter the growth machinery of the cell.

  18. Measurement of pectin methylation in plant cell walls

    SciTech Connect

    McFeeters, R.F.; Armstrong, S.A.

    1984-01-01

    A procedure was developed to measure the degree of pectin methylation in small samples of isolated cell walls from nonlignified plant tissues or pectin solutions. Galacturonic acid was determined colorimetrically with the 3,5-dimethylphenol reagent. Methylation was measured by base hydrolysis of galacturonic acid methyl esters, followed by gas chromatographic determination of released methanol. Estimates of the precision of analysis of pectin and cell wall samples were made. The coefficient of variation for estimates of the pectin esterification in cell walls isolated from 10-g samples of cucumber tissue ranged from 7.7 to 13.2%.

  19. Determining the polysaccharide composition of plant cell walls.

    PubMed

    Pettolino, Filomena A; Walsh, Cherie; Fincher, Geoffrey B; Bacic, Antony

    2012-09-01

    The plant cell wall is a chemically complex structure composed mostly of polysaccharides. Detailed analyses of these cell wall polysaccharides are essential for our understanding of plant development and for our use of plant biomass (largely wall material) in the food, agriculture, fabric, timber, biofuel and biocomposite industries. We present analytical techniques not only to define the fine chemical structures of individual cell wall polysaccharides but also to estimate the overall polysaccharide composition of cell wall preparations. The procedure covers the preparation of cell walls, together with gas chromatography-mass spectrometry (GC-MS)-based methods, for both the analysis of monosaccharides as their volatile alditol acetate derivatives and for methylation analysis to determine linkage positions between monosaccharide residues as their volatile partially methylated alditol acetate derivatives. Analysis time will vary depending on both the method used and the tissue type, and ranges from 2 d for a simple neutral sugar composition to 2 weeks for a carboxyl reduction/methylation linkage analysis. PMID:22864200

  20. Micrasterias as a Model System in Plant Cell Biology.

    PubMed

    Lütz-Meindl, Ursula

    2016-01-01

    The unicellular freshwater alga Micrasterias denticulata is an exceptional organism due to its complex star-shaped, highly symmetric morphology and has thus attracted the interest of researchers for many decades. As a member of the Streptophyta, Micrasterias is not only genetically closely related to higher land plants but shares common features with them in many physiological and cell biological aspects. These facts, together with its considerable cell size of about 200 μm, its modest cultivation conditions and the uncomplicated accessibility particularly to any microscopic techniques, make Micrasterias a very well suited cell biological plant model system. The review focuses particularly on cell wall formation and composition, dictyosomal structure and function, cytoskeleton control of growth and morphogenesis as well as on ionic regulation and signal transduction. It has been also shown in the recent years that Micrasterias is a highly sensitive indicator for environmental stress impact such as heavy metals, high salinity, oxidative stress or starvation. Stress induced organelle degradation, autophagy, adaption and detoxification mechanisms have moved in the center of interest and have been investigated with modern microscopic techniques such as 3-D- and analytical electron microscopy as well as with biochemical, physiological and molecular approaches. This review is intended to summarize and discuss the most important results obtained in Micrasterias in the last 20 years and to compare the results to similar processes in higher plant cells. PMID:27462330

  1. Determining the polysaccharide composition of plant cell walls.

    PubMed

    Pettolino, Filomena A; Walsh, Cherie; Fincher, Geoffrey B; Bacic, Antony

    2012-09-01

    The plant cell wall is a chemically complex structure composed mostly of polysaccharides. Detailed analyses of these cell wall polysaccharides are essential for our understanding of plant development and for our use of plant biomass (largely wall material) in the food, agriculture, fabric, timber, biofuel and biocomposite industries. We present analytical techniques not only to define the fine chemical structures of individual cell wall polysaccharides but also to estimate the overall polysaccharide composition of cell wall preparations. The procedure covers the preparation of cell walls, together with gas chromatography-mass spectrometry (GC-MS)-based methods, for both the analysis of monosaccharides as their volatile alditol acetate derivatives and for methylation analysis to determine linkage positions between monosaccharide residues as their volatile partially methylated alditol acetate derivatives. Analysis time will vary depending on both the method used and the tissue type, and ranges from 2 d for a simple neutral sugar composition to 2 weeks for a carboxyl reduction/methylation linkage analysis.

  2. Regulation of cell division in higher plants. Final technical report

    SciTech Connect

    Jacobs, Thomas W.

    2000-02-29

    Research in the latter part of the grant period was divided into two parts: (1) expansion of the macromolecular tool kit for studying plant cell division; (2) experiments in which the roles played by plant cell cycle regulators were to be cast in the light of the emerging yeast and animal cell paradigm for molecular control of the mitotic cycle. The first objectives were accomplished to a very satisfactory degree. With regard to the second part of the project, we were driven to change our objectives for two reasons. First, the families of cell cycle control genes that we cloned encoded such closely related members that the prospects for success at raising distinguishing antisera against each were sufficiently dubious as to be impractical. Epitope tagging is not feasible in Pisum sativum, our experimental system, as this species is not realistically transformable. Therefore, differentiating the roles of diverse cyclins and cyclin-dependent kinases was problematic. Secondly, our procedure for generating mitotically synchronized pea root meristems for biochemical studies was far too labor intensive for the proposed experiments. We therefore shifted our objectives to identifying connections between the conserved proteins of the cell cycle engine and factors that interface it with plant physiology and development. In this, we have obtained some very exciting results.

  3. Micrasterias as a Model System in Plant Cell Biology.

    PubMed

    Lütz-Meindl, Ursula

    2016-01-01

    The unicellular freshwater alga Micrasterias denticulata is an exceptional organism due to its complex star-shaped, highly symmetric morphology and has thus attracted the interest of researchers for many decades. As a member of the Streptophyta, Micrasterias is not only genetically closely related to higher land plants but shares common features with them in many physiological and cell biological aspects. These facts, together with its considerable cell size of about 200 μm, its modest cultivation conditions and the uncomplicated accessibility particularly to any microscopic techniques, make Micrasterias a very well suited cell biological plant model system. The review focuses particularly on cell wall formation and composition, dictyosomal structure and function, cytoskeleton control of growth and morphogenesis as well as on ionic regulation and signal transduction. It has been also shown in the recent years that Micrasterias is a highly sensitive indicator for environmental stress impact such as heavy metals, high salinity, oxidative stress or starvation. Stress induced organelle degradation, autophagy, adaption and detoxification mechanisms have moved in the center of interest and have been investigated with modern microscopic techniques such as 3-D- and analytical electron microscopy as well as with biochemical, physiological and molecular approaches. This review is intended to summarize and discuss the most important results obtained in Micrasterias in the last 20 years and to compare the results to similar processes in higher plant cells.

  4. Micrasterias as a Model System in Plant Cell Biology

    PubMed Central

    Lütz-Meindl, Ursula

    2016-01-01

    The unicellular freshwater alga Micrasterias denticulata is an exceptional organism due to its complex star-shaped, highly symmetric morphology and has thus attracted the interest of researchers for many decades. As a member of the Streptophyta, Micrasterias is not only genetically closely related to higher land plants but shares common features with them in many physiological and cell biological aspects. These facts, together with its considerable cell size of about 200 μm, its modest cultivation conditions and the uncomplicated accessibility particularly to any microscopic techniques, make Micrasterias a very well suited cell biological plant model system. The review focuses particularly on cell wall formation and composition, dictyosomal structure and function, cytoskeleton control of growth and morphogenesis as well as on ionic regulation and signal transduction. It has been also shown in the recent years that Micrasterias is a highly sensitive indicator for environmental stress impact such as heavy metals, high salinity, oxidative stress or starvation. Stress induced organelle degradation, autophagy, adaption and detoxification mechanisms have moved in the center of interest and have been investigated with modern microscopic techniques such as 3-D- and analytical electron microscopy as well as with biochemical, physiological and molecular approaches. This review is intended to summarize and discuss the most important results obtained in Micrasterias in the last 20 years and to compare the results to similar processes in higher plant cells. PMID:27462330

  5. Atomic force microscope observation on ultrastructures in plant cells.

    PubMed

    Wang, Xin; Zhang, Yuliang; Du, Kaihe; Fang, Xiaohong

    2010-10-01

    AFM is being applied in increasingly wide research fields and extracting more biochemical/biophysical information that is beyond the capability of traditional SEM and TEM. Due to its inherent features, AFM is rarely used to observe the subcellular details within cells. Although subcellular features were recently observed on thin sections of plant tissues using AFM, this method might introduce unexpected artifacts during sample processing. Here we try to observe plant cells still embedded in resin block. This modified method minimizes the possibility of artifacts. The comparison among outcomes of AFM, SEM, TEM and LM on the same single cell suggest that this modified method is a good, applicable, efficient and faithful way applying AFM on biological materials.

  6. Using Apple Peel Sections To Study Plant Cells and Water Relations.

    ERIC Educational Resources Information Center

    Silvius, John E.; Eckart, Christopher P.

    1997-01-01

    Suggests the cells of an apple peel as a plant species that can further enhance the plant cell laboratory. Describes the structure of apple peel cells and the benefits of including them in studies of plant cells. Suggests questions to stimulate further investigations for open-ended laboratories or independent studies. (PVD)

  7. Plant Cell Cancer: May Natural Phenolic Compounds Prevent Onset and Development of Plant Cell Malignancy? A Literature Review.

    PubMed

    Rasouli, Hassan; Farzaei, Mohammad Hosein; Mansouri, Kamran; Mohammadzadeh, Sara; Khodarahmi, Reza

    2016-01-01

    Phenolic compounds (PCs) are known as a chemically diverse category of secondary and reactive metabolites which are produced in plants via the shikimate-phenylpropanoid pathways. These compounds-ubiquitous in plants-are an essential part of the human diet, and are of considerable interest due to their antioxidant properties. Phenolic compounds are essential for plant functions, because they are involved in oxidative stress reactions, defensive systems, growth, and development. A large body of cellular and animal evidence carried out in recent decades has confirmed the anticancer role of PCs. Phytohormones-especially auxins and cytokinins-are key contributors to uncontrolled growth and tumor formation. Phenolic compounds can prevent plant growth by the endogenous regulation of auxin transport and enzymatic performance, resulting in the prevention of tumorigenesis. To conclude, polyphenols can reduce plant over-growth rate and the development of tumors in plant cells by regulating phytohormones. Future mechanistic studies are necessary to reveal intracellular transcription and transduction agents associated with the preventive role of phenolics versus plant pathological malignancy cascades. PMID:27563858

  8. Lignin variability in plant cell walls: contribution of new models.

    PubMed

    Neutelings, Godfrey

    2011-10-01

    Lignin is a major component of certain plant cell walls. The enzymes and corresponding genes associated with the metabolic pathway leading to the production of this complex phenolic polymer have been studied for many years now and are relatively well characterized. The use of genetically modified model plants (Arabidopsis, tobacco, poplar.) and mutants has contributed greatly to our current understanding of this process. The recent utilisation and/or development of a number of dedicated genomic and transcriptomic tools for other species opens new perspectives for advancing our knowledge of the biological role of this important polymer in less typical situations and/or species. In this context, studies on the formation of hypolignified G-type fibres in angiosperm tension wood, and the natural hypolignification of secondary cell walls in plant bast fibre species such as hemp (Cannabis sativa), flax (Linum usitatissimum) or ramie (Boehmeria nivea) are starting to provide novel information about how plants control secondary cell wall formation. Finally, other biologically interesting species for which few molecular resources currently exist could also represent interesting future models.

  9. Membrane Targeting of P-type ATPases in Plant Cells

    SciTech Connect

    Jeffrey F. Harper, Ph.D.

    2004-06-30

    How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems.

  10. Mass Spectrometry for Characterizing Plant Cell Wall Polysaccharides

    PubMed Central

    Bauer, Stefan

    2012-01-01

    Mass spectrometry is a selective and powerful technique to obtain identification and structural information on compounds present in complex mixtures. Since it requires only small sample amount it is an excellent tool for researchers interested in detecting changes in composition of complex carbohydrates of plants. This mini-review gives an overview of common mass spectrometry techniques applied to the analysis of plant cell wall carbohydrates. It presents examples in which mass spectrometry has been used to elucidate the structure of oligosaccharides derived from hemicelluloses and pectins and illustrates how information on sequence, linkages, branching, and modifications are obtained from characteristic fragmentation patterns. PMID:22645587

  11. How to let go: pectin and plant cell adhesion.

    PubMed

    Daher, Firas Bou; Braybrook, Siobhan A

    2015-01-01

    Plant cells do not, in general, migrate. They maintain a fixed position relative to their neighbors, intimately linked through growth and differentiation. The mediator of this connection, the pectin-rich middle lamella, is deposited during cell division and maintained throughout the cell's life to protect tissue integrity. The maintenance of adhesion requires cell wall modification and is dependent on the actin cytoskeleton. There are developmental processes that require cell separation, such as organ abscission, dehiscence, and ripening. In these instances, the pectin-rich middle lamella must be actively altered to allow cell separation, a process which also requires cell wall modification. In this review, we will focus on the role of pectin and its modification in cell adhesion and separation. Recent insights gained in pectin gel mechanics will be discussed in relation to existing knowledge of pectin chemistry as it relates to cell adhesion. As a whole, we hope to begin defining the physical mechanisms behind a cells' ability to hang on, and how it lets go.

  12. Quality analysis of buffalo blastocysts derived from oocytes vitrified before or after enucleation and reconstructed with somatic cell nuclei.

    PubMed

    Muenthaisong, S; Laowtammathron, C; Ketudat-Cairns, M; Parnpai, R; Hochi, S

    2007-03-01

    We investigated the potential of vitrified-warmed buffalo oocytes to develop to blastocysts after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). In vitro-matured oocytes before and after enucleation (M-II oocytes and enucleated oocytes, respectively) were put in 7.5% DMSO and 7.5% ethylene glycol (EG) for 4, 7 and 10 min, and then vitrified (Cryotop device) after 1-min equilibration in 15% DMSO, 15% EG and 0.5M sucrose. Following 4-, 7- and 10-min exposure, proportions of the post-warm oocytes with a normal vitelline membrane were similar (66-71% in M-II oocytes and 69-71% in enucleated oocytes). However, 18-20% of the normal M-II oocytes had no detectable first polar body in their perivitelline space (no potential for subsequent enucleation). When the post-warm M-II oocytes were treated for PA by 7% ethanol, 10 microg/mL cycloheximide and 1.25 microg/mL cytochalasin-D, parthenogenetic development into Day-7 blastocysts occurred in 10-13% of cultured oocytes, lower (P<0.05) than fresh (control) oocytes (24%). In the absence of the cooling and warming, blastocyst rates in the 4-min exposure group (22%), but not in the 7-min and 10-min exposure groups (14-15%), were similar to that in the fresh group (23%). The total cell number (group average 117-132 cells) and the ICM ratio (22-24%) of the PA blastocysts derived from vitrified M-II oocytes were comparable with fresh oocytes (127 cells and 25%). After SCNT (with fibroblast cells and vitrified-warmed oocytes), blastocyst rates were similar for the three exposure periods for M-II oocytes (8-10%) and enucleated oocytes (7-9%), but were lower (P<0.05) than in the fresh group (15%). The total cell number of the SCNT blastocysts derived from vitrified M-II and enucleated oocytes (80-90 and 82-101 cells) was smaller (P<0.05) than from fresh oocytes (135 cells); the ICM ratio of blastocysts derived from the M-II and enucleated oocytes after vitrification in 7- or 10-min exposure groups (20

  13. Novel sporophyte-like plants are regenerated from protoplasts fused between sporophytic and gametophytic protoplasts of Bryopsis plumosa.

    PubMed

    Yamagishi, Takahiro; Hishinuma, Tasuku; Kataoka, Hironao

    2004-06-01

    Protoplasts of the marine coenocytic macrophyte Bryopsis plumosa (Hudson) C. Agardh. [Caulerpales] can easily be obtained by cutting gametophytes or sporophytes with sharp scissors. When a protoplast isolated from a gametophyte was fused with a protoplast isolated from a sporophyte of this alga, it germinated and developed into either one of two completely different forms. One plant form, named Type G, appeared quite similar to a gametophyte, and the other, named Type S, looked similar to a sporophyte. While the Type G plant contained many small nuclei of gametophyte origin together with a single giant nucleus of sporophyte origin, the Type S plant contained many large nuclei of uniform size. These large nuclei in the Type S plant had metamorphosed from the gametophytic nuclei, and were not formed through division of the giant nucleus of sporophyte origin. Fragments of the Type S plant, each having such a large nucleus, developed into creeping filaments that look very similar to sporophytes. While cell walls of gametophytes and Type G plants were stained by Congo-red, those of the thalli of regenerated Type S plants and sporophytes were not stained by the dye. This indicated that the large nuclei of the Type S plant did not express genes for xylan synthesis, which are characteristic of gametophytes. Two-dimensional gel electrophoretic analysis revealed that most of the proteins synthesized in the Type S plant were identical to those of sporophytes. These results strongly suggest that in the Type S plant, the gametophytic nuclei are transformed into sporophyte-like nuclei by an unknown factor(s) produced by the giant nucleus of sporophyte origin and that the transformed nuclei express the set of genes characteristic of sporophytes. Despite morphological similarity, however, the regenerated Type S plant could not produce zoospores, because its large nuclei did not divide normally. The transformed large nuclei of gametophyte origin still seemed to be in the haploid

  14. Identification of a hormone-regulated dynamic nuclear actin network associated with estrogen receptor alpha in human breast cancer cell nuclei.

    PubMed

    Ambrosino, Concetta; Tarallo, Roberta; Bamundo, Angela; Cuomo, Danila; Franci, Gianluigi; Nassa, Giovanni; Paris, Ornella; Ravo, Maria; Giovane, Alfonso; Zambrano, Nicola; Lepikhova, Tatiana; Jänne, Olli A; Baumann, Marc; Nyman, Tuula A; Cicatiello, Luigi; Weisz, Alessandro

    2010-06-01

    Estrogen receptor alpha (ERalpha) is a modular protein of the steroid/nuclear receptor family of transcriptional regulators that upon binding to the hormone undergoes structural changes, resulting in its nuclear translocation and docking to specific chromatin sites. In the nucleus, ERalpha assembles in multiprotein complexes that act as final effectors of estrogen signaling to the genome through chromatin remodeling and epigenetic modifications, leading to dynamic and coordinated regulation of hormone-responsive genes. Identification of the molecular partners of ERalpha and understanding their combinatory interactions within functional complexes is a prerequisite to define the molecular basis of estrogen control of cell functions. To this end, affinity purification was applied to map and characterize the ERalpha interactome in hormone-responsive human breast cancer cell nuclei. MCF-7 cell clones expressing human ERalpha fused to a tandem affinity purification tag were generated and used to purify native nuclear ER-containing complexes by IgG-Sepharose affinity chromatography and glycerol gradient centrifugation. Purified complexes were analyzed by two-dimensional DIGE and mass spectrometry, leading to the identification of a ligand-dependent multiprotein complex comprising beta-actin, myosins, and several proteins involved in actin filament organization and dynamics and/or known to participate in actin-mediated regulation of gene transcription, chromatin dynamics, and ribosome biogenesis. Time course analyses indicated that complexes containing ERalpha and actin are assembled in the nucleus early after receptor activation by ligands, and gene knockdown experiments showed that gelsolin and the nuclear isoform of myosin 1c are key determinants for assembly and/or stability of these complexes. Based on these results, we propose that the actin network plays a role in nuclear ERalpha actions in breast cancer cells, including coordinated regulation of target gene

  15. Roles of cell wall peroxidases in plant development.

    PubMed

    Francoz, Edith; Ranocha, Philippe; Nguyen-Kim, Huan; Jamet, Elisabeth; Burlat, Vincent; Dunand, Christophe

    2015-04-01

    Class III peroxidases (CIII Prxs) are plant specific proteins. Based on in silico prediction and experimental evidence, they are mainly considered as cell wall localized proteins. Thanks to their dual hydroxylic and peroxidative cycles, they can produce ROS as well as oxidize cell wall aromatic compounds within proteins and phenolics that are either free or linked to polysaccharides. Thus, they are tightly associated to cell wall loosening and stiffening. They are members of large multigenic families, mostly due to an elevated rate of gene duplication in higher plants, resulting in a high risk of functional redundancy between them. However, proteomic and (micro)transcriptomic analyses have shown that CIII Prx expression profiles are highly specific. Based on these omic analyses, several reverse genetic studies have demonstrated the importance of the spatio-temporal regulation of their expression and ability to interact with cell wall microdomains in order to achieve specific activity in vivo. Each CIII Prx isoform could have specific functions in muro and this could explain the conservation of a high number of genes in plant genomes.

  16. Somatic embryogenesis - Stress-induced remodeling of plant cell fate.

    PubMed

    Fehér, Attila

    2015-04-01

    Plants as sessile organisms have remarkable developmental plasticity ensuring heir continuous adaptation to the environment. An extreme example is somatic embryogenesis, the initiation of autonomous embryo development in somatic cells in response to exogenous and/or endogenous signals. In this review I briefly overview the various pathways that can lead to embryo development in plants in addition to the fertilization of the egg cell and highlight the importance of the interaction of stress- and hormone-regulated pathways during the induction of somatic embryogenesis. Somatic embryogenesis can be initiated in planta or in vitro, directly or indirectly, and the requirement for dedifferentiation as well as the way to achieve developmental totipotency in the various systems is discussed in light of our present knowledge. The initiation of all forms of the stress/hormone-induced in vitro as well as the genetically provoked in planta somatic embryogenesis requires extensive and coordinated genetic reprogramming that has to take place at the chromatin level, as the embryogenic program is under strong epigenetic repression in vegetative plant cells. Our present knowledge on chromatin-based mechanisms potentially involved in the somatic-to-embryogenic developmental transition is summarized emphasizing the potential role of the chromatin to integrate stress, hormonal, and developmental pathways leading to the activation of the embryogenic program. The role of stress-related chromatin reorganization in the genetic instability of in vitro cultures is also discussed. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.

  17. Cell-Wall Polysaccharides of Developing Flax Plants.

    PubMed Central

    Gorshkova, T. A.; Wyatt, S. E.; Salnikov, V. V.; Gibeaut, D. M.; Ibragimov, M. R.; Lozovaya, V. V.; Carpita, N. C.

    1996-01-01

    Flax (Linum usitatissimum L.) fibers originate from procambial cells of the protophloem and develop in cortical bundles that encircle the vascular cylinder. We determined the polysaccharide composition of the cell walls from various organs of the developing flax plant, from fiber-rich strips peeled from the stem, and from the xylem. Ammonium oxalate-soluble polysaccharides from all tissues contained 5-linked arabinans with low degrees of branching, rhamnogalacturonans, and polygalacturonic acid. The fiber-rich peels contained, in addition, substantial amounts of a buffer-soluble, 4-linked galactan branched at the 0-2 and 0-3 positions with nonreducing terminal-galactosyl units. The cross-linking glycans from all tissues were (fucogalacto)xyloglucan, typical of type-I cell walls, xylans containing (1->)-[beta]-D-xylosyl units branched exclusively at the xylosyl O-2 with t-(4-O-methyl)-glucosyluronic acid units, and (galacto)glucomannans. Tissues containing predominantly primary cell wall contained a larger proportion of xyloglucan. The xylem cells were composed of about 60% 4-xylans, 32% cellulose, and small amounts of pectin and the other cross-linking polysaccharides. The noncellulosic polysaccharides of flax exhibit an uncommonly low degree of branching compared to similar polysaccharides from other flowering plants. Although the relative abundance of the various noncellulosic polysaccharides varies widely among the different cell types, the linkage structure and degree of branching of several of the noncellulosic polysaccharides are invariant. PMID:12226214

  18. Strain differences in pH-sensitive K+ channel-expressing cells in chemosensory and nonchemosensory brain stem nuclei

    PubMed Central

    Martino, Paul F.; Olesiak, S.; Batuuka, D.; Riley, D.; Neumueller, S.; Forster, H. V.

    2014-01-01

    The ventilatory CO2 chemoreflex is inherently low in inbred Brown Norway (BN) rats compared with other strains, including inbred Dahl salt-sensitive (SS) rats. Since the brain stem expression of various pH-sensitive ion channels may be determinants of the CO2 chemoreflex, we tested the hypothesis that there would be fewer pH-sensitive K+ channel-expressing cells in BN relative to SS rats within brain stem sites associated with respiratory chemoreception, such as the nucleus tractus solitarius (NTS), but not within the pre-Bötzinger complex region, nucleus ambiguus or the hypoglossal motor nucleus. Medullary sections (25 μm) from adult male and female BN and SS rats were stained with primary antibodies targeting TASK-1, Kv1.4, or Kir2.3 K+ channels, and the total (Nissl-stained) and K+ channel immunoreactive (-ir) cells counted. For both male and female rats, the numbers of K+ channel-ir cells within the NTS were reduced in the BN compared with SS rats (P < 0.05), despite equal numbers of total NTS cells. In contrast, we found few differences in the numbers of K+ channel-ir cells among the strains within the nucleus ambiguus, hypoglossal motor nucleus, or pre-Bötzinger complex regions in both male and female rats. However, there were no predicted functional mutations in each of the K+ channels studied comparing genomic sequences among these strains. Thus we conclude that the relatively selective reductions in pH-sensitive K+ channel-expressing cells in the NTS of male and female BN rats may contribute to their severely blunted ventilatory CO2 chemoreflex. PMID:25150225

  19. Metabolism of fluoranthene in different plant cell cultures and intact plants

    SciTech Connect

    Kolb, M.; Harms, H.

    2000-05-01

    The metabolism of fluoranthene was investigated in 11 cell cultures of different plant species using a [{sup 14}C]-labeled standard. Most species metabolized less than 5% of fluoranthene to soluble metabolites and formed less than 5% nonextractable residues during the standardized 48-h test procedure. Higher metabolic rates were observed in lettuce (Lactuca sativa, 6%), wheat (Tricitum aestivum, 9%), and tomato (Lycopersicon esculentum, 15%). A special high metabolic rate of nearly 50% was determined for the rose species Paul's Scarlet. Chromatographic analysis of metabolites extracted from aseptically grown tomato plants proved that the metabolites detected in the cell cultures were also formed in the intact plants. Metabolites produced in tomato and rose cells from [{sup 14}C]-fluoranthene were conjugated with glucose, glucuronic acid, and other cell components. After acid hydrolyses, the main metabolite of both species was 1-hydroxyfluoranthene as identified by gas chromatography-mass spectrometry and high-performance liquid chromatography with diode array detection. The second metabolite formed by both species was 8-hydroxyfluoranthene. A third metabolite in tomatoes was 3-hydroxyfluoranthene.

  20. The Quantitative Criteria Based on the Fractal Dimensions, Entropy, and Lacunarity for the Spatial Distribution of Cancer Cell Nuclei Enable Identification of Low or High Aggressive Prostate Carcinomas

    PubMed Central

    Waliszewski, Przemyslaw

    2016-01-01

    Background: Tumor grading, PSA concentration, and stage determine a risk of prostate cancer patients with accuracy of about 70%. An approach based on the fractal geometrical model was proposed to eliminate subjectivity from the evaluation of tumor aggressiveness and to improve the prediction. This study was undertaken to validate classes of equivalence for the spatial distribution of cancer cell nuclei in a larger, independent set of prostate carcinomas. Methods: The global fractal capacity D0, information D1 and correlation D2 dimension, the local fractal dimension (LFD) and the local connected fractal dimension (LCFD), Shannon entropy H and lacunarity λ were measured using computer algorithms in digitalized images of both the reference set (n = 60) and the test set (n = 208) of prostate carcinomas. Results: Prostate carcinomas were re-stratified into seven classes of equivalence. The cut-off D0-values 1.5450, 1.5820, 1.6270, 1.6490, 1.6980, 1.7640 defined the classes from C1 to C7, respectively. The other measures but the D1 failed to define the same classes of equivalence. The pairs (D0, LFD), (D0, H), (D0, λ), (D1, LFD), (D1, H), (D1, λ) characterized the spatial distribution of cancer cell nuclei in each class. The co-application of those measures enabled the subordination of prostate carcinomas to one out of three clusters associated with different tumor aggressiveness. For D0 < 1.5820, LFD < 1.3, LCFD > 1.5, H < 0.7, and λ > 0.8, the class C1 or C2 contains low complexity low aggressive carcinomas exclusively. For D0 > 1.6980, LFD > 1.7644, LCFD > 1.7051, H > 0.9, and λ < 0.7, the class C6 or C7 contains high complexity high aggressive carcinomas. Conclusions: The cut-off D0-values defining the classes of equivalence were validated in this study. The cluster analysis suggested that the number of the subjective Gleason grades and the number of the objective classes of equivalence could be decreased from seven to three without a loss of clinically

  1. Ligand-directed reduction-sensitive shell-sheddable biodegradable micelles actively deliver doxorubicin into the nuclei of target cancer cells.

    PubMed

    Zhong, Yinan; Yang, Weijing; Sun, Huanli; Cheng, Ru; Meng, Fenghua; Deng, Chao; Zhong, Zhiyuan

    2013-10-14

    The therapeutic performance of biodegradable micellar drugs is far from optimal due to existing challenges like poor tumor cell uptake and intracellular drug release. Here, we report on ligand-directed reduction-sensitive shell-sheddable biodegradable micelles based on poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) copolymer actively delivering doxorubicin (DOX) into the nuclei of target cancer cells, inducing superb in vitro antitumor effects. The micelles were constructed from PEG-SS-PCL and galactose-PEG-PCL (Gal-PEG-PCL) block copolymers, in which Gal-PEG-PCL was designed with a longer PEG than that in PEG-SS-PCL (6.0 vs 5.0 kDa) to fully expose Gal ligands onto the surface of micelles for effective targeting to hepatocellular carcinoma cells. PEG-SS-PCL combining with 10 or 20 wt % of Gal-PEG-PCL formed uniform micelles with average sizes of 56.1 and 58.2 nm (denoted as PEG-SS-PCL/Gal10 and PEG-SS-PCL/Gal20, respectively). The in vitro release studies showed that about 81.1 and 75.0% DOX was released in 12 h from PEG-SS-PCL/Gal10 and PEG-SS-PCL/Gal20 micelles under a reducing condition containing 10 mM dithiothreitol (DTT). In contrast, minimal DOX release (<12%) was observed for PEG-SS-PCL/Gal10 and PEG-SS-PCL/Gal20 micelles under nonreducing conditions as well as for reduction-insensitive Gal-PEG-PCL and PEG-PCL/Gal20 micelles in the presence of 10 mM DTT. MTT assays in HeLa and HepG2 cells showed that DOX-loaded PEG-SS-PCL/Gal20 micelles exhibited apparent targetability and significantly enhanced antitumor efficacy toward asialoglycoprotein receptor (ASGP-R)-overexpressing HepG2 cells with a particularly low half maximal inhibitory concentration (IC50) of 1.58 μg DOX equiv/mL, which was comparable to free DOX and approximately six times lower than that for nontargeting PEG-SS-PCL counterparts under otherwise the same conditions. Interestingly, confocal microscopy observations using FITC-labeled PEG-SS-PCL/Gal20 micelles showed that DOX was

  2. Traffic monitors at the cell periphery: the role of cell walls during early female reproductive cell differentiation in plants.

    PubMed

    Tucker, Matthew R; Koltunow, Anna M G

    2014-02-01

    The formation of female gametes in plants occurs within the ovule, a floral organ that is also the precursor of the seed. Unlike animals, plants lack a typical germline separated from the soma early in development and rely on positional signals, including phytohormones, mobile mRNAs and sRNAs, to direct diploid somatic precursor cells onto a reproductive program. In addition, signals moving between plant cells must overcome the architectural limitations of a cell wall which surrounds the plasma membrane. Recent studies have addressed the molecular and histological signatures of young ovule cells and indicate that dynamic cell wall changes occur over a short developmental window. These changes in cell wall properties impact signal flow and ovule cell identity, thereby aiding the establishment of boundaries between reproductive and somatic ovule domains.

  3. Space stress and genome shock in developing plant cells

    NASA Technical Reports Server (NTRS)

    Krikorian, A. D.

    1996-01-01

    In the present paper I review symptoms of stress at the level of the nucleus in cells of plants grown in space under nonoptimized conditions. It remains to be disclosed to what extent gravity "unloading" in the space environment directly contributes to the low mitotic index and the chromosomal anomalies and damage that is frequently, but not invariably, demonstrable in space-grown plants. Evaluation of the available facts indicates that indirect effects play a major role and that there is a significant biological component to the susceptibility to stress damage equation as well. Much remains to be learned on how to provide strictly controlled, optimal environments for plant growth in space. Only after optimized controls become possible will one be able to attribute any observed space effects to lowered gravity or to other significant but more indirect effects of the space environment.

  4. Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells.

    PubMed

    Vermeer, Joop E M; Thole, Julie M; Goedhart, Joachim; Nielsen, Erik; Munnik, Teun; Gadella, Theodorus W J

    2009-01-01

    Polyphosphoinositides represent a minor group of phospholipids, accounting for less than 1% of the total. Despite their low abundance, these molecules have been implicated in various signalling and membrane trafficking events. Phosphatidylinositol 4-phosphate (PtdIns4P) is the most abundant polyphosphoinositide. (32)Pi-labelling studies have shown that the turnover of PtdIns4P is rapid, but little is known about where in the cell or plant this occurs. Here, we describe the use of a lipid biosensor that monitors PtdIns4P dynamics in living plant cells. The biosensor consists of a fusion between a fluorescent protein and a lipid-binding domain that specifically binds PtdIns4P, i.e. the pleckstrin homology domain of the human protein phosphatidylinositol-4-phosphate adaptor protein-1 (FAPP1). YFP-PH(FAPP1) was expressed in four plant systems: transiently in cowpea protoplasts, and stably in tobacco BY-2 cells, Medicago truncatula roots and Arabidopsis thaliana seedlings. All systems allowed YFP-PH(FAPP1) expression without detrimental effects. Two distinct fluorescence patterns were observed: labelling of motile punctate structures and the plasma membrane. Co-expression studies with organelle markers revealed strong co-labelling with the Golgi marker STtmd-CFP, but not with the endocytic/pre-vacuolar marker GFP-AtRABF2b. Co-expression with the Ptdins3P biosensor YFP-2 x FYVE revealed totally different localization patterns. During cell division, YFP-PH(FAPP1) showed strong labelling of the cell plate, but PtdIns3P was completely absent from the newly formed cell membrane. In root hairs of M. truncatula and A. thaliana, a clear PtdIns4P gradient was apparent in the plasma membrane, with the highest concentration in the tip. This only occurred in growing root hairs, indicating a role for PtdIns4P in tip growth.

  5. Secondary metabolite localization by autofluorescence in living plant cells.

    PubMed

    Talamond, Pascale; Verdeil, Jean-Luc; Conéjéro, Geneviève

    2015-01-01

    Autofluorescent molecules are abundant in plant cells and spectral images offer means for analyzing their spectra, yielding information on their accumulation and function. Based on their fluorescence characteristics, an imaging approach using multiphoton microscopy was designed to assess localization of the endogenous fluorophores in living plant cells. This method, which requires no previous treatment, provides an effective experimental tool for discriminating between multiple naturally-occurring fluorophores in living-tissues. Combined with advanced Linear Unmixing, the spectral analysis extends the possibilities and enables the simultaneous detection of fluorescent molecules reliably separating overlapping emission spectra. However, as with any technology, the possibility for artifactual results does exist. This methodological article presents an overview of the applications of tissular and intra-cellular localization of these intrinsic fluorophores in leaves and fruits (here for coffee and vanilla). This method will provide new opportunities for studying cellular environments and the behavior of endogenous fluorophores in the intracellular environment. PMID:25808147

  6. Plant Phosphoglycerolipids: The Gatekeepers of Vascular Cell Differentiation.

    PubMed

    Gujas, Bojan; Rodriguez-Villalon, Antia

    2016-01-01

    In higher plants, the plant vascular system has evolved as an inter-organ communication network essential to deliver a wide range of signaling factors among distantly separated organs. To become conductive elements, phloem and xylem cells undergo a drastic differentiation program that involves the degradation of the majority of their organelles. While the molecular mechanisms regulating such complex process remain poorly understood, it is nowadays clear that phosphoglycerolipids display a pivotal role in the regulation of vascular tissue formation. In animal cells, this class of lipids is known to mediate acute responses as signal transducers and also act as constitutive signals that help defining organelle identity. Their rapid turnover, asymmetrical distribution across subcellular compartments as well as their ability to rearrange cytoskeleton fibers make phosphoglycerolipids excellent candidates to regulate complex morphogenetic processes such as vascular differentiation. Therefore, in this review we aim to summarize, emphasize and connect our current understanding about the involvement of phosphoglycerolipids in phloem and xylem differentiation.

  7. Secondary metabolite localization by autofluorescence in living plant cells.

    PubMed

    Talamond, Pascale; Verdeil, Jean-Luc; Conéjéro, Geneviève

    2015-01-01

    Autofluorescent molecules are abundant in plant cells and spectral images offer means for analyzing their spectra, yielding information on their accumulation and function. Based on their fluorescence characteristics, an imaging approach using multiphoton microscopy was designed to assess localization of the endogenous fluorophores in living plant cells. This method, which requires no previous treatment, provides an effective experimental tool for discriminating between multiple naturally-occurring fluorophores in living-tissues. Combined with advanced Linear Unmixing, the spectral analysis extends the possibilities and enables the simultaneous detection of fluorescent molecules reliably separating overlapping emission spectra. However, as with any technology, the possibility for artifactual results does exist. This methodological article presents an overview of the applications of tissular and intra-cellular localization of these intrinsic fluorophores in leaves and fruits (here for coffee and vanilla). This method will provide new opportunities for studying cellular environments and the behavior of endogenous fluorophores in the intracellular environment.

  8. Betacyanins from plants and cell cultures of Phytolacca americana.

    PubMed

    Schliemann, W; Joy, R W; Komamine, A; Metzger, J W; Nimtz, M; Wray, V; Strack, D

    1996-07-01

    Betacyanins from cell cultures of Phytolacca americana were characterized and compared with those of the stems and ripening fruits of the plant. Whereas in fruits prebetanin (betanin 6'-O-sulphate) and its isoform predominate, in the stem and cell cultures feruloylated derivatives occur as the major components. These were rigorously identified by various spectroscopic techniques (DAD-HPLC, NMR, LC-MS and electrospray MS-MS) and carbohydrate analyses as betanidin 5-O-[(5"-O-E-feruloyl)-2'-O-beta-D-apiofuranosyl] -beta-D-glucopyranoside, a new betacyanin of higher plants, and betanidin 5-O-(6'-O-E-feruloyl)-beta-D-glucopyranoside (lampranthin II), together with their isoforms.

  9. Plant Phosphoglycerolipids: The Gatekeepers of Vascular Cell Differentiation

    PubMed Central

    Gujas, Bojan; Rodriguez-Villalon, Antia

    2016-01-01

    In higher plants, the plant vascular system has evolved as an inter-organ communication network essential to deliver a wide range of signaling factors among distantly separated organs. To become conductive elements, phloem and xylem cells undergo a drastic differentiation program that involves the degradation of the majority of their organelles. While the molecular mechanisms regulating such complex process remain poorly understood, it is nowadays clear that phosphoglycerolipids display a pivotal role in the regulation of vascular tissue formation. In animal cells, this class of lipids is known to mediate acute responses as signal transducers and also act as constitutive signals that help defining organelle identity. Their rapid turnover, asymmetrical distribution across subcellular compartments as well as their ability to rearrange cytoskeleton fibers make phosphoglycerolipids excellent candidates to regulate complex morphogenetic processes such as vascular differentiation. Therefore, in this review we aim to summarize, emphasize and connect our current understanding about the involvement of phosphoglycerolipids in phloem and xylem differentiation. PMID:26904069

  10. Commercial ballard PEM fuel cell natural gas power plant development

    SciTech Connect

    Watkins, D.S.; Dunnison, D.; Cohen, R.

    1996-12-31

    The electric utility industry is in a period of rapid change. Deregulation, wholesale and retail wheeling, and corporate restructuring are forcing utilities to adopt new techniques for conducting their business. The advent of a more customer oriented service business with tailored solutions addressing such needs as power quality is a certain product of the deregulation of the electric utility industry. Distributed and dispersed power are fundamental requirements for such tailored solutions. Because of their modularity, efficiency and environmental benefits, fuel cells are a favored solution to implement distributed and dispersed power concepts. Ballard Power Systems has been working to develop and commercialize Proton Exchange Membrane (PEM) fuel cell power plants for stationary power markets. PEM`s capabilities of flexible operation and multiple market platforms bodes well for success in the stationary power market. Ballard`s stationary commercialization program is now in its second phase. The construction and successful operation of a 10 kW natural gas fueled, proof-of-concept power plant marked the completion of phase one. In the second phase, we are developing a 250 kW market entry power plant. This paper discusses Ballard`s power plant development plan philosophy, the benefits from this approach, and our current status.

  11. Metal-accelerated oxidation in plant cell death

    SciTech Connect

    Czuba, M. )

    1993-05-01

    Cadmium and mercury toxicity is further enhanced by external oxidizing conditions O[sub 3] or inherent plant processes. Lepidium sativum L, Lycopersicon esculentum Mill., or Phaseolus vulgaris L, were grown inpeat-lite to maturity under continuous cadmium exposure followed by one oxidant (O[sub 3]-6 hr. 30 pphm) exposure, with or without foliar calcium pretreatments. In comparison, Daucus carota, L and other species grown in a 71-V suspension, with or without 2,4-D were exposed continuously to low levels of methylmercury during exponential growth and analyzed in aggregates of distinct populations. Proteins were extracted and analyzed. Mechanisms of toxicity and eventual cell death are Ca-mediated and involve chloroplast, stomatal-water relations and changes in oxidant-anti-oxidant components in cells. Whether the metal-accelerated oxidative damage proceeds to cell death, depends on the species and its differential biotransformation system and cell association component.

  12. Local biofuels power plants with fuel cell generators

    SciTech Connect

    Lindstroem, O.

    1996-12-31

    The fuel cell should be a most important option for Asian countries now building up their electricity networks. The fuel cell is ideal for the schemes for distributed generation which are more reliable and efficient than the centralized schemes so far favoured by the industrialized countries in the West. Not yet developed small combined cycle power plants with advanced radial gas turbines and compact steam turbines will be the competition. Hot combustion is favoured today but cold combustion may win in the long run thanks to its environmental advantages. Emission standards are in general determined by what is feasible with available technology. The simple conclusion is that the fuel cell has to prove that it is competitive to the turbines in cost engineering terms. A second most important requirement is that the fuel cell option has to be superior with respect to electrical efficiency.

  13. Integrating fuel cell power systems into building physical plants

    SciTech Connect

    Carson, J.

    1996-12-31

    This paper discusses the integration of fuel cell power plants and absorption chillers to cogenerate chilled water or hot water/steam for all weather air conditioning as one possible approach to building system applications. Absorption chillers utilize thermal energy in an absorption based cycle to chill water. It is feasible to use waste heat from fuel cells to provide hydronic heating and cooling. Performance regimes will vary as a function of the supply and quality of waste heat. Respective performance characteristics of fuel cells, absorption chillers and air conditioning systems will define relationships between thermal and electrical load capacities for the combined systems. Specifically, this paper develops thermodynamic relationships between bulk electrical power and cooling/heating capacities for combined fuel cell and absorption chiller system in building applications.

  14. Intracellular localization of mevalonate-activating enzymes in plant cells

    PubMed Central

    Rogers, L. J.; Shah, S. P. J.; Goodwin, T. W.

    1966-01-01

    Mevalonate-activating enzymes are shown to be present in the chloroplasts of French-bean leaves. The chloroplast membrane is impermeable to mevalonic acid. Mevalonate-activating enzymes also appear to be found outside the chloroplast. These results support the view that terpenoid biosynthesis in the plant cell is controlled by a combination of enzyme segregation and specific membrane permeability. ImagesFig. 1.Fig. 2. PMID:5947149

  15. Cell physiology of plants growing in cold environments.

    PubMed

    Lütz, Cornelius

    2010-08-01

    The life of plants growing in cold extreme environments has been well investigated in terms of morphological, anatomical, and ecophysiological adaptations. In contrast, long-term cellular or metabolic studies have been performed by only a few groups. Moreover, a number of single reports exist, which often represent just a glimpse of plant behavior. The review draws together the literature which has focused on tissue and cellular adaptations mainly to low temperatures and high light. Most studies have been done with European alpine plants; comparably well studied are only two phanerogams found in the coastal Antarctic. Plant adaptation in northern polar regions has always been of interest in terms of ecophysiology and plant propagation, but nowadays, this interest extends to the effects of global warming. More recently, metabolic and cellular investigations have included cold and UV resistance mechanisms. Low-temperature stress resistance in plants from cold environments reflects the climate conditions at the growth sites. It is now a matter of molecular analyses to find the induced genes and their products such as chaperones or dehydrins responsible for this resistance. Development of plants under snow or pollen tube growth at 0 degrees C shows that cell biology is needed to explain the stability and function of the cytoskeleton. Many results in this field are based on laboratory studies, but several publications show that it is not difficult to study cellular mechanisms with the plants adapted to a natural stress. Studies on high light and UV loads may be split in two parts. Many reports describe natural UV as harmful for the plants, but these studies were mainly conducted by shielding off natural UV (as controls). Other experiments apply additional UV in the field and have had practically no negative impact on metabolism. The latter group is supported by the observations that green overwintering plants increase their flavonoids under snow even in the absence of

  16. Cell physiology of plants growing in cold environments.

    PubMed

    Lütz, Cornelius

    2010-08-01

    The life of plants growing in cold extreme environments has been well investigated in terms of morphological, anatomical, and ecophysiological adaptations. In contrast, long-term cellular or metabolic studies have been performed by only a few groups. Moreover, a number of single reports exist, which often represent just a glimpse of plant behavior. The review draws together the literature which has focused on tissue and cellular adaptations mainly to low temperatures and high light. Most studies have been done with European alpine plants; comparably well studied are only two phanerogams found in the coastal Antarctic. Plant adaptation in northern polar regions has always been of interest in terms of ecophysiology and plant propagation, but nowadays, this interest extends to the effects of global warming. More recently, metabolic and cellular investigations have included cold and UV resistance mechanisms. Low-temperature stress resistance in plants from cold environments reflects the climate conditions at the growth sites. It is now a matter of molecular analyses to find the induced genes and their products such as chaperones or dehydrins responsible for this resistance. Development of plants under snow or pollen tube growth at 0 degrees C shows that cell biology is needed to explain the stability and function of the cytoskeleton. Many results in this field are based on laboratory studies, but several publications show that it is not difficult to study cellular mechanisms with the plants adapted to a natural stress. Studies on high light and UV loads may be split in two parts. Many reports describe natural UV as harmful for the plants, but these studies were mainly conducted by shielding off natural UV (as controls). Other experiments apply additional UV in the field and have had practically no negative impact on metabolism. The latter group is supported by the observations that green overwintering plants increase their flavonoids under snow even in the absence of

  17. Two endogenous proteins that induce cell wall extension in plants

    NASA Technical Reports Server (NTRS)

    McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.

    1992-01-01

    Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

  18. A high-rate perfusion bioreactor for plant cells.

    PubMed

    De Dobbeleer, C; Cloutier, M; Fouilland, M; Legros, R; Jolicoeur, M

    2006-12-20

    A perfusion bioreactor allowing continuous extraction of secondary metabolites was designed and challenged for Eschscholtzia californica plant cell suspensions. Four sedimentation columns mounted inside a 2.5-L bioreactor separated single cells and cell aggregates from the culture medium. Cells were elicited with chitin at day 4 and the liquid medium free of cells and debris was then continuously pumped to the extraction columns containing fluidized XAD-7 resins, and then recirculated back to the cell suspension. A medium upward velocity corresponding to cell sedimentation velocity maintained a stable cell/medium separation front in the columns for sedimented cell volume (SCV) of 90% (70% packed cell volume, PCV). Two perfusion bioreactor cultures of 10 and 14 days were performed. A maximum dilution rate of 20.4/day was reached from day 4 to day 6, and was then reduced to 5/day at day 9 for 55% SCV. Control cultures were performed without and with free extraction resins into the cell suspension. Perfusion cultures showed similar specific growth rates of 0.24 +/- 0.04/day before and after elicitation. However, production level in the perfusion cultures was similar to that from the culture without resins with a maximum of 2.06 micromole/gDW total alkaloids, with 1.54 micromole/gDW in the resins. Cultures with free resins resulted in 30.94 micromole/gDW with 28.4 +/- 8.8 micromole/gDW in the resins. Difference in the cells nutritional state from elicitation was identified as a major cause in the production reduction. However, pathway to chelilutine was favored in the continuous extraction culture.

  19. ERC product improvement activities for direct fuel cell power plants

    SciTech Connect

    Bentley, C.; Carlson, G.; Doyon, J.

    1995-08-01

    This program is designed to advance the carbonate fuel cell technology from the current power plant demonstration status to the commercial design in an approximately five-year period. The specific objectives which will allow attainment of the overall program goal are: (1) Define market-responsive power plant requirements and specifications, (2) Establish the design for a multifuel, low-cost, modular, market-responsive power plant, (3) Resolve power plant manufacturing issues and define the design for the commercial manufacturing facility, (4) Define the stack and BOP equipment packaging arrangement and define module designs, (5) Acquire capability to support developmental testing of stacks and BOP equipment as required to prepare for commercial design, and (6) Resolve stack and BOP equipment technology issues and design, build, and field test a modular commercial prototype power plant to demonstrate readiness for commercial entry. A seven-task program, dedicated to attaining objective(s) in the areas noted above, was initiated in December 1994. Accomplishments of the first six months are discussed in this paper.

  20. ERC product improvement activities for direct fuel cell power plants

    SciTech Connect

    Maru, H.C.; Farooque, M.; Bentley, C.

    1995-12-01

    This program is designed to advance the carbonate fuel cell technology from the current power plant demonstration status to the commercial design in an approximately five-year period. The specific objectives which will allow attainment of the overall program goal are: (1) Define market-responsive power plant requirements and specifications, (2) Establish the design for a multifuel, low-cost, modular, market-responsive power plant, (3) Resolve power plant manufacturing issues and define the design for the commercial manufacturing facility, (4) Define the stack and BOP equipment packaging arrangement and define module designs, (5) Acquire capability to support developmental testing of stacks and BOP equipment as required to prepare for commercial design, and (6) Resolve stack and BOP equipment technology issues and design, build, and field test a modular commercial prototype power plant to demonstrate readiness for commercial entry. A seven-task program, dedicated to attaining objective(s) in the areas noted above, was initiated in December 1994. Accomplishments of the first six months are discussed in this paper.