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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. 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

  3. 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

  4. 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

  5. Stem cell mechanics: Auxetic nuclei

    NASA Astrophysics Data System (ADS)

    Wang, Ning

    2014-06-01

    The nuclei of naive mouse embryonic stem cells that are transitioning towards differentiation expand when the cells are stretched and contract when they are compressed. What drives this auxetic phenotype is, however, unclear.

  6. 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.

  7. Nuclei in motion: movement and positioning of plant nuclei in development, signaling, symbiosis, and disease

    PubMed Central

    Griffis, Anna H. N.; Groves, Norman R.; Zhou, Xiao; Meier, Iris

    2014-01-01

    While textbook figures imply nuclei as resting spheres at the center of idealized cells, this picture fits few real situations. Plant nuclei come in many shapes and sizes, and can be actively transported within the cell. In several contexts, this nuclear movement is tightly coupled to a developmental program, the response to an abiotic signal, or a cellular reprogramming during either mutualistic or parasitic plant–microbe interactions. While many such phenomena have been observed and carefully described, the underlying molecular mechanism and the functional significance of the nuclear movement are typically unknown. Here, we survey recent as well as older literature to provide a concise starting point for applying contemporary molecular, genetic and biochemical approaches to this fascinating, yet poorly understood phenomenon. PMID:24772115

  8. RBP45 and RBP47, two oligouridylate-specific hnRNP-like proteins interacting with poly(A)+ RNA in nuclei of plant cells.

    PubMed Central

    Lorković, Z J; Wieczorek Kirk, D A; Klahre, U; Hemmings-Mieszczak, M; Filipowicz, W

    2000-01-01

    Introns in plant nuclear pre-mRNAs are highly enriched in U or U + A residues and this property is essential for efficient splicing. Moreover, 3'-untranslated regions (3'-UTRs) in plant pre-mRNAs are generally UA-rich and contain sequences that are important for the polyadenylation reaction. Here, we characterize two structurally related RNA-binding proteins (RBPs) from Nicotiana plumbaginifolia, referred to as RBP45 and RBP47, having specificity for oligouridylates. Both proteins contain three RBD-type RNA-binding domains and a glutamine-rich N-terminus, and share similarity with Nam8p, a protein associated with U1 snRNP in the yeast Saccharomyces cerevisiae. Deletion analysis of RBP45 and RBP47 indicated that the presence of at least two RBD are required for interaction with RNA and that domains other than RBD do not significantly contribute to binding. mRNAs for RBP45 and RBP47 and mRNAs encoding six related proteins in Arabidopsis thaliana are constitutively expressed in different plant organs. Indirect immunofluorescence and fractionation of cell extracts showed that RBP45 and RBP47 are localized in the nucleus. In vivo UV crosslinking experiments demonstrated their association with the nuclear poly(A)+ RNA. In contrast to UBP1, another oligouridylate-binding nuclear three-RBD protein of N. plumbaginifolia (Lambermon et al., EMBO J, 2000, 19:1638-1649), RBP45 and RBP47 do not stimulate mRNA splicing and accumulation when transiently overexpressed in protoplasts. Properties of RBP45 and RBP47 suggest they represent hnRNP-proteins participating in still undefined steps of pre-mRNA maturation in plant cell nuclei. PMID:11105760

  9. Extended chromatin and DNA fibers from active plant nuclei for high-resolution FISH.

    PubMed

    Lavania, U C; Yamamoto, M; Mukai, Y

    2003-10-01

    The conventional protocol for isolation of cell wall free nuclei for release of DNA fibers for plants involves mechanical removal of the cell wall and separation of debris by sieve filtration. The mechanical grinding pressure applied during the process leaves only the more tolerant G(1) nuclei intact, and all other states of active nuclei that may be present in the target tissues (e.g., leaf) are simply crushed/disrupted during the isolation process. Here we describe an alternative enzymatic protocol for isolation of nuclei from root tip tissue. Cell wall free nuclei at a given stage of cell cycle, free of any cell debris, could be realized in suspension that are fit for preparation of extended fibers suitable for fiber FISH applications. The protocol utilizes selective harvest of active nuclei from root tip tissue in liquid suspension under the influence of cell wall-degrading enzymes, and provides opportunities to target cell cycle-specific nuclei from interphase through division phase for the release of extended DNA fibers. Availability of cell cycle-specific fibers may have added value in transcriptional analysis, DNA:RNA hybridization, visualization of DNA replication and replication forks, and improved FISH efficiency. PMID:14500692

  10. 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.

  11. Plant nuclei can contain extensive grooves and invaginations.

    PubMed

    Collings, D A; Carter, C N; Rink, J C; Scott, A C; Wyatt, S E; Allen, N S

    2000-12-01

    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. PMID:11148288

  12. Plant cell membranes

    SciTech Connect

    Packer, L.; Douce, R.

    1987-01-01

    The contents of this book are: Cells, Protoplasts, Vacuoles and Liposomes; Tonoplasts; Nuclei, Endolplasmic Reticulum, and Plasma Membrane; Peroxisomes; Plastids; Teneral Physical and Biochemical Methods; and Mitochondira.

  13. 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. PMID:26886972

  14. Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT)

    PubMed Central

    Amin, Nirav M.; Greco, Todd M.; Kuchenbrod, Lauren M.; Rigney, Maggie M.; Chung, Mei-I; Wallingford, John B.; Cristea, Ileana M.; Conlon, Frank L.

    2014-01-01

    The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development. PMID:24496632

  15. 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

  16. 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

  17. Computational hepatocellular carcinoma tumor grading based on cell nuclei classification

    PubMed Central

    Atupelage, Chamidu; Nagahashi, Hiroshi; Kimura, Fumikazu; Yamaguchi, Masahiro; Tokiya, Abe; Hashiguchi, Akinori; Sakamoto, Michiie

    2014-01-01

    Abstract. Hepatocellular carcinoma (HCC) is the most common histological type of primary liver cancer. HCC is graded according to the malignancy of the tissues. It is important to diagnose low-grade HCC tumors because these tissues have good prognosis. Image interpretation-based computer-aided diagnosis (CAD) systems have been developed to automate the HCC grading process. Generally, the HCC grade is determined by the characteristics of liver cell nuclei. Therefore, it is preferable that CAD systems utilize only liver cell nuclei for HCC grading. This paper proposes an automated HCC diagnosing method. In particular, it defines a pipeline-path that excludes nonliver cell nuclei in two consequent pipeline-modules and utilizes the liver cell nuclear features for HCC grading. The significance of excluding the nonliver cell nuclei for HCC grading is experimentally evaluated. Four categories of liver cell nuclear features were utilized for classifying the HCC tumors. Results indicated that nuclear texture is the dominant feature for HCC grading and others contribute to increase the classification accuracy. The proposed method was employed to classify a set of regions of interest selected from HCC whole slide images into five classes and resulted in a 95.97% correct classification rate. PMID:26158066

  18. Mucopolysaccharides associated with nuclei of cultured mammalian cells.

    PubMed Central

    Bhavanandan, V P; Davidson, E A

    1975-01-01

    Mucopolysaccharides have been isolated, fractionated, and characterized from the nuclei of cultured B16 mouse melanoma cells grown in the presence of (3-H)-glucosamine and (35-S)sulfate. Digestion of the nuclei with DNase followed by Pronase gave a mixture of complex carbohydrates from which the mucopolysaccharides were isolated by precipitation with cetylpyridinium chloride. After fractionation by differential salt extraction and chromatography on controlled pore glass bead columns, the components were identified by chemical and enzymatic methods. The major polysaccharide components were a family of high-molecular-weight chondroitin sulfates with different degrees of sulfation; a minor component has been characterized as heparan sulfate. PMID:124440

  19. 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

  20. 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.

  1. Plant Nuclei Can Contain Extensive Grooves and InvaginationsW⃞W⃞

    PubMed Central

    Collings, David A.; Carter, Crystal N.; Rink, Jochen C.; Scott, Amie C.; Wyatt, Sarah E.; Allen, Nina Strömgren

    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. PMID:11148288

  2. 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. PMID:22404469

  3. Mapping eGFP Oligomer Mobility in Living Cell Nuclei

    PubMed Central

    Zwerger, Monika; Müller, Gabriele; Waldeck, Waldemar; Langowski, Jörg

    2009-01-01

    Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers. PMID:19347038

  4. Automated segmentation of cancer cell nuclei in complex tissue sections

    NASA Astrophysics Data System (ADS)

    Loukas, Constantinos G.; Wilson, George D.; Vojnovic, Borivoj

    2001-01-01

    Characterization of the proliferative activity of a tumor has been the subject of research for many years. The majority of the studies presented so far in the field of cytology and histology relates to the analysis of information from a limited number of cells, which are often easily distinguishable from the background and as well as from each other. The present paper introduces an automated image analysis technique for classification of cancer cell nuclei stained with proliferative markers. The images under processing were characterized by a high degree of complexity, containing considerable histological noise. The first step of the method aims to identify nuclear features of proliferating cells only, contained in large-scale histological images, using Principal Components Analysis (PCA). The histogram of the component that demonstrates the best contrast is processed appropriately for generating a binary image. Some standard morphological operations are then applied to remove any irrelevant structures and detect touching and/or overlapping nuclei. Two separate methods, Skeleton by Influence Zone and heuristic processing, are presented for segmentation of clustered cells. The algorithm was tested on tissue section images encountered in routine clinical practice with very encouraging results, after comparing image analysis and human observer cell counting.

  5. 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

  6. An automated image segmentation and classification algorithm for immunohistochemically stained tumor cell nuclei

    NASA Astrophysics Data System (ADS)

    Yeo, Hangu; Sheinin, Vadim; Sheinin, Yuri

    2009-02-01

    As medical image data sets are digitized and the number of data sets is increasing exponentially, there is a need for automated image processing and analysis technique. Most medical imaging methods require human visual inspection and manual measurement which are labor intensive and often produce inconsistent results. In this paper, we propose an automated image segmentation and classification method that identifies tumor cell nuclei in medical images and classifies these nuclei into two categories, stained and unstained tumor cell nuclei. The proposed method segments and labels individual tumor cell nuclei, separates nuclei clusters, and produces stained and unstained tumor cell nuclei counts. The representative fields of view have been chosen by a pathologist from a known diagnosis (clear cell renal cell carcinoma), and the automated results are compared with the hand-counted results by a pathologist.

  7. The illuminated plant cell.

    PubMed

    Mathur, Jaideep

    2007-11-01

    The past decade has provided biologists with a palette of genetically encoded, multicolored fluorescent proteins. The living plant cell turned into a 'coloring book' and today, nearly every text-book organelle has been highlighted in scintillating fluorescent colors. This review provides a concise listing of the earliest representative fluorescent-protein probes used to highlight various targets within the plant cell, and introduces the idea of using the numerous multicolor, subcellular probes for the development of an early intracellular response profile of plants. PMID:17933577

  8. 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.

  9. 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.

  10. Visualization of actin filaments and monomers in somatic cell nuclei.

    PubMed

    Belin, Brittany J; Cimini, Beth A; Blackburn, Elizabeth H; Mullins, R Dyche

    2013-04-01

    In addition to its long-studied presence in the cytoplasm, actin is also found in the nuclei of eukaryotic cells. The function and form (monomer, filament, or noncanonical oligomer) of nuclear actin are hotly debated, and its localization and dynamics are largely unknown. To determine the distribution of nuclear actin in live somatic cells and evaluate its potential functions, we constructed and validated fluorescent nuclear actin probes. Monomeric actin probes concentrate in nuclear speckles, suggesting an interaction of monomers with RNA-processing factors. Filamentous actin probes recognize discrete structures with submicron lengths that are excluded from chromatin-rich regions. In time-lapse movies, these actin filament structures exhibit one of two types of mobility: 1) diffusive, with an average diffusion coefficient of 0.06-0.08 μm(2)/s, or (2) subdiffusive, with a mobility coefficient of 0.015 μm(2)/s. Individual filament trajectories exhibit features of particles moving within a viscoelastic mesh. The small size of nuclear actin filaments is inconsistent with a role in micron-scale intranuclear transport, and their localization suggests that they do not participate directly in chromatin-based processes. Our results instead suggest that actin filaments form part of a large, viscoelastic structure in the nucleoplasm and may act as scaffolds that help organize nuclear contents. PMID:23447706

  11. Bulk isolation in nonaqueous media of nuclei from lyophilized cells.

    PubMed

    Kirsch, W M; Leitner, J W; Gainey, M; Schulz, D; Lasher, R; Nakane, P

    1970-06-26

    Intact lyophilized nuclei are obtainable from a variety of tissues, either in situ or in culture, by freezing at -156 degrees C, drying at -25 degrees C, and mechanical disassociation in glycerol at 2 degrees C. Centrifugal separation of nuclei is accomplished in an 85 : 15 by volume mixture of glycerol and 3-chloro-1,2 propanediol at 2 degrees C. The method gives homogeneous nuclear preparations in high yield with preservation of labile and water-soluble constituents. PMID:4316024

  12. 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

  13. 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. PMID:25855323

  14. 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.

  15. Segmentation of Whole Cells and Cell Nuclei From 3-D Optical Microscope Images Using Dynamic Programming

    PubMed Central

    McCullough, Dean P.; Gudla, Prabhakar R.; Harris, Bradley S.; Collins, Jason A.; Meaburn, Karen J.; Nakaya, Masa-Aki; Yamaguchi, Terry P.; Misteli, Tom; Lockett, Stephen J.

    2009-01-01

    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. PMID:18450544

  16. Statistical Analysis of 3D Images Detects Regular Spatial Distributions of Centromeres and Chromocenters in Animal and Plant Nuclei

    PubMed Central

    Biot, Eric; Adenot, Pierre-Gaël; Hue-Beauvais, Cathy; Houba-Hérin, Nicole; Duranthon, Véronique; Devinoy, Eve; Beaujean, Nathalie; Gaudin, Valérie; Maurin, Yves; Debey, Pascale

    2010-01-01

    In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear “compartments”. Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types. PMID:20628576

  17. Computational efficient segmentation of cell nuclei in 2D and 3D fluorescent micrographs

    NASA Astrophysics Data System (ADS)

    De Vylder, Jonas; Philips, Wilfried

    2011-02-01

    This paper proposes a new segmentation technique developed for the segmentation of cell nuclei in both 2D and 3D fluorescent micrographs. The proposed method can deal with both blurred edges as with touching nuclei. Using a dual scan line algorithm its both memory as computational efficient, making it interesting for the analysis of images coming from high throughput systems or the analysis of 3D microscopic images. Experiments show good results, i.e. recall of over 0.98.

  18. 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

  19. 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

  20. 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. PMID:26503330

  1. 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...

  2. Radiation-induced association of beta-glucuronidase with purified nuclei from irradiated MOLT-4 and HeLa cells

    SciTech Connect

    McClain, D.E.; Kalinich, J.F.; Poplack, J.K.; Snyder, S.L.

    1989-02-01

    Beta-glucuronidase, a lysosomal marker enzyme, associates with purified nuclei from HeLa and MOLT-4 cell lines in a radiation dose-dependent manner, up to 300 cGy in MOLT-4 cells, and 1000 cGy in HeLa cells. In MOLT-4 cells (200-cGy exposure), there is a significant increase in beta-glucuronidase activity detected in the nuclear fraction 24 h postirradiation with a maximum association occurring at 72 h. In HeLa cells (1000-cGy exposure), a significant association is first detected 24 h postirradiation with a maximum association at 48 h. The association is not the result of nonspecific contamination occurring during nuclei purification since nuclei from irradiated cells show no greater levels of plasma membrane marker and mitochondrial marker than controls. The nature of the association remains unclear, but activity is not removed by detergents used in the nuclei isolation procedure, and incubation of the nuclei with EDTA reverses the association only modestly. Exposure of nuclei from irradiated cells to anisotonic buffers also results in only a small decrease in beta-glucuronidase activity associated with the nuclei. These observations suggest that lysosomal hydrolases become intimately associated with the nuclei of irradiated cells.

  3. Automated detection of cells from immunohistochemically-stained tissues: application to Ki-67 nuclei staining

    NASA Astrophysics Data System (ADS)

    Cinar Akakin, Hatice; Kong, Hui; Elkins, Camille; Hemminger, Jessica; Miller, Barrie; Ming, Jin; Plocharczyk, Elizabeth; Roth, Rachel; Weinberg, Mitchell; Ziegler, Rebecca; Lozanski, Gerard; Gurcan, Metin N.

    2012-03-01

    An automated cell nuclei detection algorithm is described to be used for the quantification of immunohistochemicallystained tissues. Detection and segmentation of positively stained cells and their separation from the background and negatively-stained cells is crucial for fast, accurate, consistent and objective analysis of pathology images. One of the major challenges is the identification, hence accurate counting of individual cells, when these cells form clusters. To identify individual cell nuclei within clusters, we propose a new cell nuclei detection method based on the well-known watershed segmentation, which can lead to under- or over-segmentation for this problem. Our algorithm handles oversegmentation by combining H-minima transformed watershed algorithm with a novel region merging technique. To handle under-segmentation problem, we develop a Laplacian-of-Gaussian (LoG) filtering based blob detection algorithm, which estimates the range of the scales from the image adaptively. An SVM classifier was trained in order to separate non-touching single cells and touching cell clusters with five features representing connected region properties such as eccentricity, area, perimeter, convex area and perimeter-to-area ratio. Classified touching cell clusters are segmented with the H-minima based watershed algorithm. The resulting over-segmented regions are improved with the merging algorithm. The remaining under-segmented cell clusters are convolved with LoG filters to detect the cells within them. Cell-by-cell nucleus detection performance is evaluated by comparing computer detections with cell locations manually marked by eight pathology residents. The sensitivity is 89% when the cells are marked as positive at least by one resident and it increases to 99% when the evaluated cells are marked by all eight residents. In comparison, the average reader sensitivity varies between 70% +/- 18% and 95% +/- 11%.

  4. 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.

  5. Diagnostic features in two-dimensional light scattering patterns of normal and dysplastic cervical cell nuclei

    NASA Astrophysics Data System (ADS)

    Arifler, Dizem; MacAulay, Calum; Follen, Michele; Guillaud, Martial

    2014-03-01

    Dysplastic progression in epithelial tissues is linked to changes in morphology and internal structure of cell nuclei. These changes lead to alterations in nuclear light scattering profiles that can potentially be monitored for diagnostic purposes. Numerical tools allow for simulation of complex nuclear models and are particularly useful for quantifying the optical response of cell nuclei as dysplasia progresses. In this study, we first analyze a set of quantitative histopathology images from twenty cervical biopsy sections stained with Feulgen-thionin. Since Feulgen-thionin is stoichiometric for DNA, the images enable us to obtain detailed information on size, shape, and chromatin content of all the segmented nuclei. We use this extensive data set to construct realistic three-dimensional computational models of cervical cell nuclei that are representative of four diagnostic categories, namely normal or negative for dysplasia, mild dysplasia, moderate dysplasia, and severe dysplasia or carcinoma in situ (CIS). We then carry out finite-difference time-domain simulations to compute the light scattering response of the constructed models as a function of the polar scattering angle and the azimuthal scattering angle. The results show that these two-dimensional scattering patterns exhibit characteristic intensity ridges that change form with progression of dysplasia; pattern processing reveals that Haralick features can be used to distinguish moderately and severely dysplastic or CIS nuclei from normal and mildly dysplastic nuclei. Our numerical study also suggests that different angular ranges need to be considered separately to fully exploit the diagnostic potential of two-dimensional light scattering measurements.

  6. Cytophotometric investigation of DNA and RNA content in nuclei of active Strasburger cells in Pinus nigra var. austriaca (Hoess) Badoux.

    PubMed

    Sauter, J J; Ulrich, H

    1977-01-01

    The nuclei of active, sieve cell-associated Strasburger cells in the secondary phloem of Pinus nigra var. austriaca (Hoess) Badoux have been studied for their structure and DNA and RNA content. No difference in size compared to those of ordinary ray cells was found. The nuclear surface is often increased by an ameboid or lobed shape. The amount of highly decondensed chromatin is greatly increased. Cytophotometric measurements of DNA content of both Feulgen and gallocyanine chromalum-stained nuclei showed normal DNA levels and proved absence of endomitotic polyploidization. RNA content, however, was significantly increased as compared to nuclei of young Strasburger cells and of ordinary ray parenchyma cells. PMID:24420510

  7. 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. PMID:19884070

  8. Metabolic pathways for the degradation of phosphatidic acid in isolated nuclei from cerebellar cells.

    PubMed

    Gaveglio, Virginia L; Pasquaré, Susana J; Giusto, Norma M

    2011-03-15

    The aim of the present research was to analyse the pathways for phosphatidic acid metabolism in purified nuclei from cerebellar cells. Lipid phosphate phosphatase and diacylglyceride lipase activities were detected in nuclei from cerebellar cells. It was observed that DAGL activity makes up 50% of LPP activity and that PtdOH can also be metabolised to lysophosphatidic acid. With a nuclear protein content of approximately 40 μg, the production of diacylglycerol and monoacylglycerol was linear for 30 min and 5 min, respectively, whereas it increased with PtdOH concentrations of up to 250 μM. LysoPtdOH, sphingosine 1-phosphate and ceramide 1-phosphate, which are alternative substrates for LPP, significantly reduced DAG production from PA. DAG and MAG production increased in the presence of Triton X-100 (1 mM) whereas no modifications were observed in the presence of ionic detergent sodium deoxycholate. Ca²+ and Mg²+ stimulated MAG production without affecting DAG formation whereas fluoride and vanadate inhibited the generation of both products. Specific PtdOH-phospholipase A1 and PtdOH-phospholipase A2 were also detected in nuclei. Our findings constitute the first reported evidence of active PtdOH metabolism involving LPP, DAGL and PtdOH-selective PLA activities in purified nuclei prepared from cerebellar cells. PMID:21216221

  9. 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

  10. ADP-ribosyltransferase in isolated nuclei during the cell cycle of Physarum polycephalum.

    PubMed Central

    Gröbner, P; Loidl, P

    1985-01-01

    ADP-ribosyltransferase was measured in isolated nuclei of Physarum polycephalum. Activity was determined with and without exogenous DNA and histones. During the synchronous cell cycle the activity measured with exogenous substrates exhibited a typical peak enzyme pattern with a maximum of activity in S-phase, whereas activity measured without exogenous substrates displayed a step enzyme pattern. Both activities doubled in each cell cycle. PMID:3002325

  11. Isolation of Cell Nuclei Using Inert Macromolecules to Mimic the Crowded Cytoplasm

    PubMed Central

    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 µM 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

  12. 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

  13. Plant cell remodeling by autophagy

    PubMed Central

    Kim, Jimi; Lee, Han Nim; Chung, Taijoon

    2014-01-01

    Plant seedlings are not photoautotrophs until they are equipped with photosynthetic machinery. Some plant cells are remodeled after being exposed to light, and a group of peroxisomal proteins are degraded during the remodeling. Autophagy was proposed as one of the mechanisms for the degradation of peroxisomal proteins. We recently showed that ATG7-dependent autophagy is partially responsible for the degradation of obsolete peroxisomal proteins during Arabidopsis seedling growth. PMID:24492493

  14. The absorption of ultraviolet light by cell nuclei. A technique for identifying neoplastic change

    SciTech Connect

    Baisden, C.R.; Booker, D.; Wright, R.D. )

    1989-11-01

    A technique for measuring the absorption of 260-nm ultraviolet light by cell nuclei is described. The results of such measurements of normal thyroid epithelial cells and benign and malignant thyroid neoplastic cells demonstrate a progressive increase in absorbance that correlates with the histologic appearance of neoplasia. The possible theoretic basis for this phenomenon is explored. The increased nuclear absorbance observed in neoplastic cells is hypothesized to result from the disruption of hydrogen bonds between the DNA base pairs, which allows unwinding of the double helix and loss of the normal control of mitosis.

  15. Macrophages do not inhibit the participation of the nuclei of nonmalignant proliferating cells in DNA synthesis in heterokaryons

    SciTech Connect

    Egorov, E.E.; Prudovskii, I.A.; Zelenin, A.V.

    1985-07-01

    The authors continue their investigations into types of heterokaryons in an effort to detect an inhibition of nondividing macrophages (differentiated cells) on the entry of the nuclei of proliferating cells into replication. For the experiments described in this paper, the authors used asynchronous cultures of mouse diploid fibroblasts (MDF), 3T3 mouse cells from continuous culture, and malignant SV3T3 cells (3T3 cells transformed by SV40). Fusion of the cells of the cultures with macrophages was performed using PEG at various periods after deposition (2, 8, 12, and 20 h). The authors used double isotope marking to identify DNA synthesis in the heterokaryons. For this purpose, the nuclei of the culture cells were labeled with (/sup 3/H)thymidine before fusion with macrophages. All the nuclei of the culture cells intensively incorporated the label. After fusion, (/sup 14/C)thymidine was introduced into the incubation medium. If the cell nucleus began to synthesize DNA, it incorporated (/sup 14/C)thymidine, and a supplementary relatively weak label appeared on the auto-radiographic preparations, both above the nuclei themselves and next to them. The nuclei of macrophages in which DNA synthesis was reactivated contained only the (/sup 14/C)label. Fixation was performed 26 h after stimulation (in the case of 3T3) or 35 h after stimulation (for MDF). The percentages of nuclei of culture cells labeled with (/sup 14/C)thymidine were determined in the heterokaryons and free-lying cells.

  16. 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

  17. 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.

  18. 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

  19. 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

  20. Isolation of Nuclei from Skeletal Muscle Satellite Cells and Myofibers for Use in Chromatin lmmunoprecipitation Assays

    PubMed Central

    Ohkawa, Yasuyuki; Mallappa, Chandrashekara; Dacwag Vallaster, Caroline S.; lmbalzano, Anthony N.

    2014-01-01

    Studies investigating mechanisms controlling gene regulation frequently examine specific DNA sequences using chromatin immunoprecipitation (ChIP) assays to determine whether specific regulatory factors or modified histones are present. While use of primary cells or cell line models for differentiating or differentiated tissue is widespread, the ability to assess factor binding and histone modification in tissue defines the events that occur in vivo and provides corroboration for studies in cultured cells. Many tissues can be analyzed with minimal modification to existing ChIP protocols that are designed for cultured cells; however, some tissues, such as skeletal muscle, are problematic in that accessibility of the cross-linking agent is limited. We describe a method to isolate skeletal muscle tissue nuclei suitable for use in ChIP protocols. Furthermore, we utilize a simple fractionation of digested skeletal muscle tissue that can separate mature myofibers from satellite cells, which are responsible for postnatal skeletal muscle regeneration, thereby allowing simultaneous preparation of nuclei from both cell types. PMID:22130858

  1. 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…

  2. 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.

  3. A rapid method for the isolation of DNA-binding proteins from purified nuclei of tissues and cells in culture.

    PubMed Central

    Hagenbüchle, O; Wellauer, P K

    1992-01-01

    We describe a rapid and general method for isolating DNA-binding proteins in high yield from purified nuclei of animal cells. The method has been tested for the isolation of a series of different DNA-binding activities including those of transcription factors PTF1 and SP1. The rationale consists of first preparing purified nuclei from tissue or cells in culture by centrifugation over sucrose cushions. A synthetic, biotinylated oligonucleotide bearing the binding site for the protein of interest is then added directly to nuclei resuspended in binding buffer. At the end of the binding reaction, nuclei are removed by centrifugation; and protein-DNA complexes present in the postnuclear supernatant are attached to streptavidin-agarose. Two rounds of DNA-affinity chromatography are carried out to yield highly purified preparations of DNA-binding proteins. Images PMID:1641323

  4. 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

  5. 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.

  6. 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. PMID:26855123

  7. 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

  8. High resolution image analysis of cell nuclei in tissue sections of primary and metastatic carcinomas.

    PubMed

    Theissig, F; Dimmer, V; Kunze, K D

    1986-01-01

    The present study examines whether certain histological tumour types can be differentiated on account of their nuclear image with the aid of automated image analysis. For karyometric investigations three tumour types (adenocarcinomas, squamous cell carcinomas and mammary carcinomas) were chosen, which occur frequently as occult primary tumours. From each type ten primary tumours with their corresponding lymph node metastases were examined. 100 cell nuclei were measured from each case using 4 micron thick paraffin sections stained with gallocyanin-chromalum. For each cell nucleus 21 contour and texture features were determined. Through the application of linear classifiers 41 out of 52 cases (25 primary tumours, 27 metastases) of these three tumour types were correctly classified. Eight cases could not be classified with certainty and only three cases were wrongly classified. In addition, within the group of adenocarcinomas differences due to localisation were detected which allow us to draw conclusions on the seat of the primary tumour. PMID:3019272

  9. Polynucleotide phosphorylase from plant cells.

    PubMed

    Schumacher-Wittkopf, E; Richter, G; Schulze, S

    1984-06-01

    The isolation of polynucleotide phosphorylase (EC 2. 7. 7. 8) from suspension cultured plant cells of parsley (Petroselinum sativum) and from tomato seedlings (Lycopersicon esculentum) is described. The procedure includes an ultracentrifugation step, a glycerol density gradient centrifugation and preparative gel electrophoresis under nondenaturing conditions. Isoelectric focusing gives rise to a major component (pI ≈ 7.5) and to a minor one (pI ≈ 5). The enzyme contains five subunits with apparent Mr values of 160 000, 140 000, 70 000, 34 000 and 12 000, the 70 000-dalton one being a glycoprotein. PMID:24253429

  10. 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.

  11. Mature microRNAs identified in highly purified nuclei from HCT116 colon cancer cells

    PubMed Central

    Park, Chang Won; Zeng, Yan; Zhang, Xiaoxiao; Subramanian, Subbaya

    2010-01-01

    MicroRNAs (miRNAs) have emerged as one of the major regulatory mechanisms of gene expression. A major function of miRNAs involves the post-transcriptional regulation of target mRNAs, which is reported to occur primarily in the cytoplasm. However, there is a significant amount of evidence demonstrating the existence of small non-coding RNAs, including small-interfering RNA (siRNA), miRNA and Piwi-interacting RNA (piRNA) in the nucleus. In order to elucidate the potential subcellular localizations and functions of miRNAs, we have identified numerous miRNAs that are present in isolated nuclei from human colon cancer HCT116 cells. MicroRNA profiles were compared between cytoplasmic and nuclear fractions of the HCT116 cell line on the basis of multiple microarray analyses. MicroRNA species showing significant existence in isolated and highly purified populations of nuclei were selected and further tested with RT-PCR. The nuclear localization of the mature form of miRNAs was verified again by control RT-PCR excluding the detection of premature forms of miRNA, such as pri-miRNA or pre-miRNA. The elevated levels of representative miRNAs identified in purified nuclei were confirmed by northern blot analysis, supporting the notion that significant numbers of mature miRNAs exist not only in the cytoplasm but also in the nucleus. These results will likely provide a basis for further studies concerning the intracellular trafficking and nuclear location of miRNAs. PMID:20864815

  12. 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

  13. 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.

  14. Aptamer-mediated delivery of splice-switching oligonucleotides to the nuclei of cancer cells.

    PubMed

    Kotula, Jonathan W; Pratico, Elizabeth D; Ming, Xin; Nakagawa, Osamu; Juliano, Rudolph L; Sullenger, Bruce A

    2012-06-01

    To reduce the adverse effects of cancer therapies and increase their efficacy, new delivery agents that specifically target cancer cells are needed. We and others have shown that aptamers can selectively deliver therapeutic oligonucleotides to the endosome and cytoplasm of cancer cells that express a particular cell surface receptor. Identifying a single aptamer that can internalize into many different cancer cell-types would increase the utility of aptamer-mediated delivery of therapeutic agents. We investigated the ability of the nucleolin aptamer (AS1411) to internalize into multiple cancer cell types and observed that it internalizes into a wide variety of cancer cells and migrates to the nucleus. To determine if the aptamer could be utilized to deliver therapeutic oligonucleotides to modulate events in the nucleus, we evaluated the ability of the aptamer to deliver splice-switching oligonucleotides. We observed that aptamer-splice-switching oligonucleotide chimeras can alter splicing in the nuclei of treated cells and are effective at lower doses than the splice switching oligonucleotides alone. Our results suggest that aptamers can be utilized to deliver oligonucleotides to the nucleus of a wide variety of cancer cells to modulate nuclear events such as RNA splicing. PMID:22703281

  15. Aptamer-Mediated Delivery of Splice-Switching Oligonucleotides to the Nuclei of Cancer Cells

    PubMed Central

    Kotula, Jonathan W.; Pratico, Elizabeth D.; Ming, Xin; Nakagawa, Osamu; Juliano, Rudolph L.

    2012-01-01

    To reduce the adverse effects of cancer therapies and increase their efficacy, new delivery agents that specifically target cancer cells are needed. We and others have shown that aptamers can selectively deliver therapeutic oligonucleotides to the endosome and cytoplasm of cancer cells that express a particular cell surface receptor. Identifying a single aptamer that can internalize into many different cancer cell-types would increase the utility of aptamer-mediated delivery of therapeutic agents. We investigated the ability of the nucleolin aptamer (AS1411) to internalize into multiple cancer cell types and observed that it internalizes into a wide variety of cancer cells and migrates to the nucleus. To determine if the aptamer could be utilized to deliver therapeutic oligonucleotides to modulate events in the nucleus, we evaluated the ability of the aptamer to deliver splice-switching oligonucleotides. We observed that aptamer-splice-switching oligonucleotide chimeras can alter splicing in the nuclei of treated cells and are effective at lower doses than the splice switching oligonucleotides alone. Our results suggest that aptamers can be utilized to deliver oligonucleotides to the nucleus of a wide variety of cancer cells to modulate nuclear events such as RNA splicing. PMID:22703281

  16. 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. PMID:27008552

  17. 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

  18. 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

  19. 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

  20. FISH and chips: automation of fluorescent dot counting in interphase cell nuclei.

    PubMed

    Netten, H; Young, I T; van Vliet, L J; Tanke, H J; Vroljik, H; Sloos, W C

    1997-05-01

    Fluorescence in situ hybridization allows the enumeration of chromosomal abnormalities in interphase cell nuclei. This process is called dot counting. To estimate the distribution of chromosomes per cell, a large number of cells have to be analyzed, especially when the frequency of aberrant cells is low. Automation of dot counting is required because manual counting is tedious, fatiguing, and time-consuming. We developed a completely automated fluorescence microscope system that can examine 500 cells in approximately 15 min to determine the number of labeled chromosomes (seen as dots) in each cell nucleus. This system works with two fluorescent dyes, one for the DNA hybridization dots and one for the cell nucleus. After the stage has moved to a new field, the image is automatically focused, acquired by a Photometrics KAF 1400 camera (Photometrics Ltd., Tuscon, AZ, USA), and then analyzed on a Macintosh Quadra 840AV (Apple Computer, Inc., Cupertino, CA, USA) computer. After the required number of cells has been analyzed, the user may interact to correct the computer by working with a gallery of the cell images. The automated dot counter has been tested on a number of normal specimens where 4,'6-diamidino-2-phenylindole (DAPI) was used for the nucleus counterstain and a centromeric 8 probe was used to mark the desired chromosome. The slides contained lymphocytes from cultured blood. We compared the results of the dot counter with manual counting. Manually obtained results, published in the literature, were used as the "ground truth." For a normal specimen, 97.5% of cells will have two dots. Fully automated scanning of 13 slides showed that an average of 89% of all nuclei were counted correctly. In other words, an average of 11% has to be interactively corrected, using a monitor display. The machine accuracies, after interactive correction, are comparable to panels of human experts (manual). The fully automatically obtained results are biased with respect to manual

  1. 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.

  2. 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.

  3. Plant Proteases Involved in Regulated Cell Death.

    PubMed

    Zamyatnin, A A

    2015-12-01

    Each plant genome encodes hundreds of proteolytic enzymes. These enzymes can be divided into five distinct classes: cysteine-, serine-, aspartic-, threonine-, and metalloproteinases. Despite the differences in their structural properties and activities, members of all of these classes in plants are involved in the processes of regulated cell death - a basic feature of eukaryotic organisms. Regulated cell death in plants is an indispensable mechanism supporting plant development, survival, stress responses, and defense against pathogens. This review summarizes recent advances in studies of plant proteolytic enzymes functioning in the initiation and execution of distinct types of regulated cell death. PMID:26878575

  4. 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

  5. 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. PMID:22544390

  6. Epigenome profiling of specific plant cell types using a streamlined INTACT protocol and ChIP-seq.

    PubMed

    Wang, Dongxue; Deal, Roger B

    2015-01-01

    Plants consist of many functionally specialized cell types, each with its own unique epigenome, transcriptome, and proteome. Characterization of these cell type-specific properties is essential to understanding cell fate specification and the responses of individual cell types to the environment. In this chapter we describe an approach to map chromatin features in specific cell types of Arabidopsis thaliana using nuclei purification from individual cell types with the INTACT method (isolation of nuclei tagged in specific cell types) followed by chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq). The INTACT system employs two transgenes to generate affinity-labeled nuclei in the cell type of interest, and these tagged nuclei can then be selectively purified from tissue homogenates. The primary transgene encodes the nuclear tagging fusion protein (NTF), which consists of a nuclear envelope-targeting domain, the green fluorescent protein, and a biotin ligase recognition peptide, while the second transgene encodes the E. coli biotin ligase (BirA), which selectively biotinylates NTF. Expression of NTF and BirA in a specific cell type thus yields nuclei that are coated with biotin and can be purified by virtue of their affinity for streptavidin-coated magnetic beads. Compared with the original INTACT nuclei purification protocol, the procedure presented here is greatly simplified and shortened. After nuclei purification, we provide detailed instructions for chromatin isolation, shearing, and immunoprecipitation. Finally, we present a low input ChIP-seq library preparation protocol based on the nano-ChIP-seq method of Adli and Bernstein, and we describe multiplex Illumina sequencing of these libraries to produce high quality, cell type-specific epigenome profiles at a relatively low cost. The procedures given here are optimized for Arabidopsis but should be easily adaptable to other plant species. PMID:25757765

  7. [Rhythmic nuclear growth of adequately stimulated ganglia cells of acoustic nuclei (rat)].

    PubMed

    Köpf-Maier, P; Wüstenfeld, E

    1975-01-01

    Ganglia cells of the dorsal and ventral cochlear nuclei of white rats were irritated adequately for different periods or left untreated, respectively, and investigated karyometrically. The frequency distribution curves of the nuclear volumes were separated by means of an electronic curve resolver into the component curves, i.e. into groups of nuclei obeying exactly a Gaussian normal distribution and thus representing biologically uniform populations. The analysis of the mean values of the component curves led to the following results: 1. The mean values of the component curves can be arranged in 2 series having the pattern V1, V1 square root 2, V2, V2 square root 2, V4, V4 square root 2...2. The series V1, V1 square root 2, V2, V2 square root 2...is based on a geometrical series of the general formula an = k-qn. 3. It follows from these results that the nuclear volumes grow rhythmically by a factor of square root 2 and, consequently, that there is a periodical doubling in in the growth of the surface. PMID:1200386

  8. 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. PMID:24296078

  9. Methods to isolate a large amount of generative cells, sperm cells and vegetative nuclei from tomato pollen for “omics” analysis

    PubMed Central

    Lu, Yunlong; Wei, Liqin; Wang, Tai

    2015-01-01

    The development of sperm cells (SCs) from microspores involves a set of finely regulated molecular and cellular events and the coordination of these events. The mechanisms underlying these events and their interconnections remain a major challenge. Systems analysis of genome-wide molecular networks and functional modules with high-throughput “omics” approaches is crucial for understanding the mechanisms; however, this study is hindered because of the difficulty in isolating a large amount of cells of different types, especially generative cells (GCs), from the pollen. Here, we optimized the conditions of tomato pollen germination and pollen tube growth to allow for long-term growth of pollen tubes in vitro with SCs generated in the tube. Using this culture system, we developed methods for isolating GCs, SCs and vegetative cell nuclei (VN) from just-germinated tomato pollen grains and growing pollen tubes and their purification by Percoll density gradient centrifugation. The purity and viability of isolated GCs and SCs were confirmed by microscopy examination and fluorescein diacetate staining, respectively, and the integrity of VN was confirmed by propidium iodide staining. We could obtain about 1.5 million GCs and 2.0 million SCs each from 180 mg initiated pollen grains, and 10 million VN from 270 mg initiated pollen grains germinated in vitro in each experiment. These methods provide the necessary preconditions for systematic biology studies of SC development and differentiation in higher plants. PMID:26082789

  10. Estimating Genomic Distance from DNA Sequence Location in Cell Nuclei by a Random Walk Model

    NASA Astrophysics Data System (ADS)

    van den Engh, Ger; Sachs, Rainer; Trask, Barbara J.

    1992-09-01

    The folding of chromatin in interphase cell nuclei was studied by fluorescent in situ hybridization with pairs of unique DNA sequence probes. The sites of DNA sequences separated by 100 to 2000 kilobase pairs (kbp) are distributed in interphase chromatin according to a random walk model. This model provides the basis for calculating the spacing of sequences along the linear DNA molecule from interphase distance measurements. An interphase mapping strategy based on this model was tested with 13 probes from a 4-megabase pair (Mbp) region of chromosome 4 containing the Huntington disease locus. The results confirmed the locations of the probes and showed that the remaining gap in the published maps of this region is negligible in size. Interphase distance measurements should facilitate construction of chromosome maps with an average marker density of one per 100 kbp, approximately ten times greater than that achieved by hybridization to metaphase chromosomes.

  11. Estimating genomic distance from DNA sequence location in cell nuclei by a random walk model

    SciTech Connect

    Engh, G. van den; Trask, B.J. ); Sachs, R. )

    1992-09-04

    The folding of chromatin in interphase cell nuclei was studied by fluorescent in situ hybridization with pairs of unique DNA sequence probes. The sites of DNA sequences separated by 100 to 2000 kilobase pairs (kbp) are distributed in interphase chromatin according to a random walk model. This model provides the basis for calculating the spacing of sequences along the linear DNA molecule from interphase distance measurements. An interphase mapping strategy based on this model was tested with 13 probes from a 4-megabase pair (Mbp) region of chromosome 4 containing the Huntington disease locus. The results confirmed the locations of the probes and showed that the remaining gap in the published maps of this region is negligible in size. Interphase distance measurements should facilitate construction of chromosome maps with an average marker density of one per 100 kbp, approximately ten times greater than that achieved by hybridization to metaphase chromosomes.

  12. Automated recognition of cell phenotypes in histology images based on membrane- and nuclei-targeting biomarkers

    PubMed Central

    Karaçalı, Bilge; Vamvakidou, Alexandra P; Tözeren, Aydın

    2007-01-01

    Background Three-dimensional in vitro culture of cancer cells are used to predict the effects of prospective anti-cancer drugs in vivo. In this study, we present an automated image analysis protocol for detailed morphological protein marker profiling of tumoroid cross section images. Methods Histologic cross sections of breast tumoroids developed in co-culture suspensions of breast cancer cell lines, stained for E-cadherin and progesterone receptor, were digitized and pixels in these images were classified into five categories using k-means clustering. Automated segmentation was used to identify image regions composed of cells expressing a given biomarker. Synthesized images were created to check the accuracy of the image processing system. Results Accuracy of automated segmentation was over 95% in identifying regions of interest in synthesized images. Image analysis of adjacent histology slides stained, respectively, for Ecad and PR, accurately predicted regions of different cell phenotypes. Image analysis of tumoroid cross sections from different tumoroids obtained under the same co-culture conditions indicated the variation of cellular composition from one tumoroid to another. Variations in the compositions of cross sections obtained from the same tumoroid were established by parallel analysis of Ecad and PR-stained cross section images. Conclusion Proposed image analysis methods offer standardized high throughput profiling of molecular anatomy of tumoroids based on both membrane and nuclei markers that is suitable to rapid large scale investigations of anti-cancer compounds for drug development. PMID:17822559

  13. 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

  14. 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...

  15. Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

    PubMed Central

    Bolden, A; Aucker, J; Weissbach, A

    1975-01-01

    Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha. PMID:172658

  16. 5-Carboxylcytosine is localized to euchromatic regions in the nuclei of follicular cells in axolotl ovary.

    PubMed

    Alioui, Anthony; Wheldon, Lee M; Abakir, Abdulkadir; Ferjentsik, Zoltan; Johnson, Andrew D; Ruzov, Alexey

    2012-01-01

    5-Methylcytosine (5-mC) is an epigenetic modification associated with gene repression. Recent studies demonstrated that 5-mC can be enzymatically oxidised into 5-hydroxymethylcytosine and further into 5-formylcytosine (5-fC) and 5-carboxylcytsine (5-caC). 5-caC has been found in embryonic stem cells and in mouse pre-implantation embryos but no detectable levels of this modification have been reported for somatic tissues to date. Whereas it has been suggested that 5-caC can serve as an intermediate in the process of active demethylation, the function of this form of modified cytosine remains obscure. Here we show that 5-caC is immunochemically detectable in somatic cells of axolotl ovary. We demonstrate that both 5-hmC and 5-caC are localized to the euchromatin in the nuclei of axolotl follicular cells with similar patterns of spatial distribution. Our results suggest that 5-carboxylcytosine may play a distinct functional role in certain biological contexts. PMID:23138778

  17. 5-Carboxylcytosine is localized to euchromatic regions in the nuclei of follicular cells in axolotl ovary

    PubMed Central

    Alioui, Anthony; Wheldon, Lee M.; Abakir, Abdulkadir; Ferjentsik, Zoltan; Johnson, Andrew D.; Ruzov, Alexey

    2012-01-01

    5-Methylcytosine (5-mC) is an epigenetic modification associated with gene repression. Recent studies demonstrated that 5-mC can be enzymatically oxidised into 5-hydroxymethylcytosine and further into 5-formylcytosine (5-fC) and 5-carboxylcytsine (5-caC). 5-caC has been found in embryonic stem cells and in mouse pre-implantation embryos but no detectable levels of this modification have been reported for somatic tissues to date. Whereas it has been suggested that 5-caC can serve as an intermediate in the process of active demethylation, the function of this form of modified cytosine remains obscure. Here we show that 5-caC is immunochemically detectable in somatic cells of axolotl ovary. We demonstrate that both 5-hmC and 5-caC are localized to the euchromatin in the nuclei of axolotl follicular cells with similar patterns of spatial distribution. Our results suggest that 5-carboxylcytosine may play a distinct functional role in certain biological contexts. PMID:23138778

  18. Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts.

    PubMed Central

    Bruno, J; Reich, N; Lucas, J J

    1981-01-01

    The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed. Images PMID:7346715

  19. 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

  20. 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.

  1. Asymmetric cell division in plant development.

    PubMed

    Heidstra, Renze

    2007-01-01

    Plant embryogenesis creates a seedling with a basic body plan. Post-embryonically the seedling elaborates with a lifelong ability to develop new tissues and organs. As a result asymmetric cell divisions serve essential roles during embryonic and postembryonic development to generate cell diversity. This review highlights selective cases of asymmetric division in the model plant Arabidopsis thaliana and describes the current knowledge on fate determinants and mechanisms involved. Common themes that emerge are: 1. role of the plant hormone auxin and its polar transport machinery; 2. a MAP kinase signaling cascade and; 3. asymmetric segregating transcription factors that are involved in several asymmetric cell divisions. PMID:17585494

  2. 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

  3. 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

  4. DIRECT FUEL/CELL/TURBINE POWER PLANT

    SciTech Connect

    Hossein Ghezel-Ayagh

    2004-05-01

    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. FCE successfully completed testing of the pre-alpha DFC/T hybrid power plant. This power plant was constructed by integration of a 250kW fuel cell stack and a microturbine. The tests of the cascaded fuel cell concept for achieving high fuel utilizations were completed. The tests demonstrated that the concept results in higher power plant efficiency. Also, the preliminary design of a 40 MW power plant including the key equipment layout and the site plan was completed.

  5. 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

  6. 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.

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

    PubMed

    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

  8. 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

  9. 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.

  10. Plant Cell Nucleolus as a Hot Spot for Iron*

    PubMed Central

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

    2011-01-01

    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. PMID:21719700

  11. 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. PMID:21719700

  12. 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.

  13. 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

  14. DIRECT FUEL CELL/TURBINE POWER PLANT

    SciTech Connect

    Hossein Ghezel-Ayagh

    2003-05-23

    In this reporting period, a milestone was achieved by commencement of testing and operation of the sub-scale hybrid direct fuel cell/turbine (DFC/T{reg_sign}) power plant. The operation was initiated subsequent to the completion of the construction of the balance-of-plant (BOP) and implementation of process and control tests of the BOP for the subscale DFC/T hybrid system. The construction efforts consisted of finishing the power plant insulation and completion of the plant instrumentation including the wiring and tubing required for process measurement and control. The preparation work also included the development of procedures for facility shake down, conditioning and load testing of the fuel cell, integration of the microturbine, and fuel cell/gas turbine load tests. At conclusion of the construction, the process and control (PAC) tests of BOP, including the microturbine, were initiated.

  15. 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

  16. [Determination of the relative RNA-content of liver cell nuclei with gallocyanin-chromealum-staining (author's transl)].

    PubMed

    Wenzelides, K; Korek, G; Voss, K

    1981-01-01

    The relative RNA-content of liver cell nuclei from histological slides of rat liver was determined by automatical microscope picture analysis. The slides were stained with Gallocyanin-Chromealum. Extinction measurements are made before and after perchloracid extraction of RNA for the same slide. The changes of extinction measurements caused by the changes of slide thickness and of tissue inhomogeneity could be lowered using this method. PMID:6177185

  17. 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)

  18. 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

  19. Laboratory studies with cloud-derived Bacterial Cells acting as Ice Nuclei in the Immersion and Deposition Mode

    NASA Astrophysics Data System (ADS)

    Oehm, C.; Chou, C.; Amato, P.; Attard, E.; Delort, A.-M.; Morris, C.; Kiselev, A.; Stetzer, O.; Möhler, O.; Leisner, T.

    2012-04-01

    Atmospheric aerosol particles play an important role in cloud microphysics. Aerosols of biological origin are a subgroup, and some of them are able to act as heterogeneous ice nuclei and thus influence cloud life cycles and the climate. Some bacteria species have been found to act as ice nuclei at relatively high temperatures up to -2 degree Celsius and are therefore of particular importance as "high temperature" ice nuclei. Recently, ice nucleation experiments with bacterial cells from different sources were performed at the aerosol and cloud simulation chamber AIDA at the Karlsruhe Institute of Technology. At the AIDA facility, microphysical cloud processes can be simulated and investigated in laboratory at realistic atmospheric cloud conditions. Different ice nucleation active (INA) bacteria strains were isolated from cloud water, glacier melt water and phyllosphere and examined in AIDA experiments. The living cells were suspended in nanopure or artificial cloud water and injected into the cloud chamber through a dispersion nozzle. The injected droplets evaporated in the chamber and the bacterial cells were transformed into the aerosol phase. After the spraying, the cloud formation was started by expansion cooling. Experiments were performed in the temperature range from -2 down to -20 degree Celsius. Detailed measurements of the number concentration and size distribution of the aerosol particles as well as of the droplets and ice particles were carried out during the AIDA experiments. A minor fraction of the bacteria cells was observed to act as ice nuclei in the immersion nucleation mode at higher temperatures as well as in the deposition nucleation mode at lower temperatures. The ice activity started at -6 degree Celsius. The most efficient INA bacteria species were Pseudomonas syringae 32b74 and Pseudomonas fluorescens Antarctica1. The ice active number fraction with respect to the cells varied from 0,01 to 0,1, and it does not change at different

  20. Direct FuelCell/Turbine Power Plant

    SciTech Connect

    Hossein Ghezel-Ayagh

    2004-11-19

    This report includes the progress in development of Direct Fuel Cell/Turbine. (DFC/T.) 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. FCE successfully completed testing of the pre-alpha sub-MW DFC/T power plant. This power plant was constructed by integration of a 250kW fuel cell stack and a microturbine. Following these proof-of-concept tests, a stand-alone test of the microturbine verified the turbine power output expectations at an elevated (representative of the packaged unit condition) turbine inlet temperature. Preliminary design of the packaged sub-MW alpha DFC/T unit has been completed and procurement activity has been initiated. The preliminary design of a 40 MW power plant including the key equipment layout and the site plan was completed. A preliminary cost estimate for the 40 MW DFC/T plant has also been prepared. The tests of the cascaded fuel cell concept for achieving high fuel utilizations were completed. The tests demonstrated that the concept results in higher power plant efficiency. Alternate stack flow geometries for increased power output/fuel utilization capabilities are also being evaluated.

  1. 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

  2. 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.

  3. Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen Colletotrichum gloeosporioides

    PubMed Central

    Nesher, Iris; Barhoom, Sima; Sharon, Amir

    2008-01-01

    Background In order to initiate plant infection, fungal spores must germinate and penetrate into the host plant. Many fungal species differentiate specialized infection structures called appressoria on the host surface, which are essential for successful pathogenic development. In the model plant pathogen Magnaporthe grisea completion of mitosis and autophagy cell death of the spore are necessary for appressoria-mediated plant infection; blocking of mitosis prevents appressoria formation, and prevention of autophagy cell death results in non-functional appressoria. Results We found that in the closely related plant pathogen Colletotrichum gloeosporioides, blocking of the cell cycle did not prevent spore germination and appressoria formation. The cell cycle always lagged behind the morphogenetic changes that follow spore germination, including germ tube and appressorium formation, differentiation of the penetrating hypha, and in planta formation of primary hyphae. Nuclear division was arrested following appressorium formation and was resumed in mature appressoria after plant penetration. Unlike in M. grisea, blocking of mitosis had only a marginal effect on appressoria formation; development in hydroxyurea-treated spores continued only for a limited number of cell divisions, but normal numbers of fully developed mature appressoria were formed under conditions that support appressoria formation. Similar results were also observed in other Colletotrichum species. Spores, germ tubes, and appressoria retained intact nuclei and remained viable for several days post plant infection. Conclusion We showed that in C. gloeosporioides the differentiation of infection structures including appressoria precedes mitosis and can occur without nuclear division. This phenomenon was also found to be common in other Colletotrichum species. Spore cell death did not occur during plant infection and the fungus primary infection structures remained viable throughout the infection cycle

  4. Stem cell factors in plants: chromatin connections.

    PubMed

    Kornet, N; Scheres, B

    2008-01-01

    The progression of pluripotent stem cells to differentiated cell lineages requires major shifts in cell differentiation programs. In both mammals and higher plants, this process appears to be controlled by a dedicated set of transcription factors, many of which are kingdom specific. These divergent transcription factors appear to operate, however, together with a shared suite of factors that affect the chromatin state. It is of major importance to investigate whether such shared global control mechanisms indicate a common mechanistic basis for preservation of the stem cell state, initiation of differentiation programs, and coordination of cell state transitions. PMID:19150963

  5. 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. PMID:24995477

  6. 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.

  7. 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. PMID:24677732

  8. 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. PMID:27026270

  9. Tocopherol production in plant cell cultures.

    PubMed

    Caretto, Sofia; Nisi, Rossella; Paradiso, Annalisa; De Gara, Laura

    2010-05-01

    Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, essential dietary components for mammals and exclusively synthesized by photosynthetic organisms. Of the four forms (alpha, beta, gamma and delta), alpha-tocopherol is the major vitamin E form present in green plant tissues, and has the highest vitamin E activity. Synthetic alpha-tocopherol, being a racemic mixture of eight different stereoisomers, always results less effective than the natural form (R,R,R) alpha-tocopherol. This raises interest in obtaining this molecule from natural sources, such as plant cell cultures. Plant cell and tissue cultures are able to produce and accumulate valuable metabolites that can be used as food additives, nutraceuticals and pharmaceuticals. Sunflower cell cultures, growing under heterotrophic conditions, were exploited to establish a suitable in vitro production system of natural alpha-tocopherol. Optimization of culture conditions, precursor feeding and elicitor application were used to improve the tocopherol yields of these cultures. Furthermore, these cell cultures were useful to investigate the relationship between alpha-tocopherol biosynthesis and photomixotrophic culture conditions, revealing the possibility to enhance tocopherol production by favouring sunflower cell photosynthetic properties. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism. PMID:20166145

  10. 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

  11. Nick translation of HeLa cell nuclei as a probe for locating DNase I-sensitive nucleosomes

    SciTech Connect

    Javaherian, K.; Fasman, G.D.

    1984-03-10

    The technique of nick translation of nuclei has been used in HeLa cells to label DNase I-sensitive regions. Micrococcal nuclease digestion of the nick translated nuclei was followed by a low ionic strength gel electrophoresis system which separates different types of mononucleosomes. The major label was observed in the vicinity of high mobility group protein containing mononucleosomes. However, further analysis revealed that the particle does not sediment in the position of mononucleosomes on a sucrose gradient. Two alternative explanations are discussed as the possible source of this particle. It is either a high mobility group protein containing nucleosome in some unfolded conformation or the labeled particle originates from discrete DNA fragments, wrapped around some nonhistone proteins, located in a highly DNase I-sensitive region, which is resistant to micrococcal nuclease digestion. 36 references, 7 figures.

  12. The distribution and cells of origin of ACTH(1-39)-stained varicosities in the paraventricular and supraoptic nuclei.

    PubMed

    Sawchenko, P E; Swanson, L W; Joseph, S A

    1982-01-28

    ACTH(1-39)-immunoreactive fibers and varicosities were localized using indirect immunofluorescence histochemistry in normal rats, and were found to be distributed in specific parts of the parvocellular division of the paraventricular nucleus, and in regions of the magnocellular division of the paraventricular and supraoptic nuclei in which oxytocinergic cells predominate. A combined retrograde transport-immunohistochemical method was used to confirm that these projections arise from a group of ACTH(1-39)-stained cells in the arcuate nucleus (and in adjacent regions along the base of the hypothalamus), and to describe their distribution within this region. PMID:6322913

  13. 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.

  14. 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…

  15. 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

  16. 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

  17. DNA (DEOXYRIBONUCLEIC ACID) SYNTHESIS FOLLOWING MICROINJECTION OF HETEROLOGOUS SPERM AND SOMATIC CELL NUCLEI INTO HAMSTER OOCYTES

    EPA Science Inventory

    The authors have 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 3H-thymidine after being parthenogenetically activated by sha...

  18. 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

  19. A flexible and robust approach for segmenting cell nuclei from 2D microscopy images using supervised learning and template matching

    PubMed Central

    Chen, Cheng; Wang, Wei; Ozolek, John A.; Rohde, Gustavo K.

    2013-01-01

    We describe a new supervised learning-based template matching approach for segmenting cell nuclei from microscopy images. The method uses examples selected by a user for building a statistical model which captures the texture and shape variations of the nuclear structures from a given dataset to be segmented. Segmentation of subsequent, unlabeled, images is then performed by finding the model instance that best matches (in the normalized cross correlation sense) local neighborhood in the input image. We demonstrate the application of our method to segmenting nuclei from a variety of imaging modalities, and quantitatively compare our results to several other methods. Quantitative results using both simulated and real image data show that, while certain methods may work well for certain imaging modalities, our software is able to obtain high accuracy across several imaging modalities studied. Results also demonstrate that, relative to several existing methods, the template-based method we propose presents increased robustness in the sense of better handling variations in illumination, variations in texture from different imaging modalities, providing more smooth and accurate segmentation borders, as well as handling better cluttered nuclei. PMID:23568787

  20. Vacuolar staining methods in plant cells.

    PubMed

    Scheuring, David; Schöller, Maria; Kleine-Vehn, Jürgen; Löfke, Christian

    2015-01-01

    Commercially available fluorescent dyes enable the fast and specific visualization of plant vacuoles, allowing for investigation of membrane dynamics and vacuolar biogenesis in living cells. Here, we describe different approaches tinting the tonoplast or the vacuolar lumen with a range of dyes, and illustrate its utilization with established fluorescent-tagged marker lines. PMID:25408446

  1. Integrated bioprocessing for plant cell cultures.

    PubMed

    Choi, J W; Cho, G H; Byun, S Y; Kim, D I

    2001-01-01

    Plant cell suspension culture has become the focus of much attention as a tool for the production of secondary metabolites including paclitaxel, a well-known anticancer agent. Recently, it has also been regarded as one of the host systems for the production of recombinant proteins. In order to produce phytochemicals using plant cell cultures, efficient processes must be developed with adequate bioreactor design. Most of the plant secondary metabolites are toxic to cells at the high concentrations required during culture. Therefore, if the product could be removed in situ during culture, productivity might be enhanced due to the alleviation of this toxicity. In situ removal or extractive bioconversion of such products can be performed by in situ extraction with various kinds of organic solvents. In situ adsorption using polymeric resins is another possibility. Using the fact that secondary metabolites are generally hydrophobic, various integrated bioprocessing techniques can be designed not only to lower toxicity, but also to enhance productivity. In this article, in situ extraction, in situ adsorption, utilization of cyclodextrins, and the application of aqueous two-phase systems in plant cell cultures are reviewed. PMID:11729756

  2. DIRECT FUEL CELL/TURBINE POWER PLANT

    SciTech Connect

    Hossein Ghezel-Ayagh

    2003-05-22

    Project activities were focused on the design and construction the sub-scale hybrid Direct Fuel Cell/turbine (DFC/T{reg_sign}) power plant and modification of a Capstone Simple Cycle Model 330 microturbine. The power plant design work included preparation of system flow sheet and performing computer simulations based on conservation of mass and energy. The results of the simulation analyses were utilized to prepare data sheets and specifications for balance-of-plant equipment. Process flow diagram (PFD) and piping and instrumentation diagrams (P&ID) were also completed. The steady state simulation results were used to develop design information for modifying the control functions, and for sizing the heat exchangers required for recuperating the waste heat from the power plant. Line and valve sizes for the interconnecting pipes between the microturbine and the heat recuperators were also identified.

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. Fuel cell power plants for transportation applications

    SciTech Connect

    Huff, J.R.

    1991-12-31

    Over the past 35 years, the transportation sector has accounted fr approximately 25% of the total gross energy consumption in the United States. As the largest energy user in the United States, transportation accounts for approximately 66% of the country`s current petroleum consumption. Fuel cell power plants using nonpetroleum fuels such as methanol could significantly reduce US dependency on petroleum resources. They offer the additional advantage of minimal air pollution thereby addressing another issue of major concern in the US fuel cell power plant use in city buses and other vehicles is being explored in a number of US Department of Energy and industrial programs that will be described in this paper. 5 refs.

  14. Modulation of Higher Order Chromatin Conformation in Mammalian Cell Nuclei Can Be Mediated by Polyamines and Divalent Cations

    PubMed Central

    Visvanathan, Ashwat; Ahmed, Kashif; Even-Faitelson, Liron; Lleres, David; Bazett-Jones, David P.; Lamond, Angus I.

    2013-01-01

    The organisation of the large volume of mammalian genomic DNA within cell nuclei requires mechanisms to regulate chromatin compaction involving the reversible formation of higher order structures. The compaction state of chromatin varies between interphase and mitosis and is also subject to rapid and reversible change upon ATP depletion/repletion. In this study we have investigated mechanisms that may be involved in promoting the hyper-condensation of chromatin when ATP levels are depleted by treating cells with sodium azide and 2-deoxyglucose. Chromatin conformation was analysed in both live and permeabilised HeLa cells using FLIM-FRET, high resolution fluorescence microscopy and by electron spectroscopic imaging microscopy. We show that chromatin compaction following ATP depletion is not caused by loss of transcription activity and that it can occur at a similar level in both interphase and mitotic cells. Analysis of both live and permeabilised HeLa cells shows that chromatin conformation within nuclei is strongly influenced by the levels of divalent cations, including calcium and magnesium. While ATP depletion results in an increase in the level of unbound calcium, chromatin condensation still occurs even in the presence of a calcium chelator. Chromatin compaction is shown to be strongly affected by small changes in the levels of polyamines, including spermine and spermidine. The data are consistent with a model in which the increased intracellular pool of polyamines and divalent cations, resulting from depletion of ATP, bind to DNA and contribute to the large scale hyper-compaction of chromatin by a charge neutralisation mechanism. PMID:23840764

  15. 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).

  16. 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

  17. Predictive variables for the biological behaviour of basal cell carcinoma of the face: relevance of morphometry of the nuclei.

    PubMed

    Appel, T; Bierhoff, E; Appel, K; von Lindern, J-J; Bergé, S; Niederhagen, B

    2003-06-01

    We did a morphometric analysis of 130 histological sections of basal cell carcinoma (BCC) of the face to find out whether morphometric variables in the structure of the nuclei of BCC cells could serve as predictors of the biological behaviour. We considered the following variables: maximum and minimum diameters, perimeter, nuclear area and five form factors that characterise and quantify the shape of a structure (axis ratio, shape factor, nuclear contour index, nuclear roundness and circumference ratio). We did a statistical analysis of primary and recurring tumours and four histology-based groups (multifocal superficial BCCs, nodular BCCs, sclerosing BCCs and miscellaneous forms) using a two-sided t test for independent samples. Multifocal superficial BCCs showed significantly smaller values for the directly measured variables (maximum and minimum diameters, perimeter and nuclear area). Morphometry could not distinguish between primary and recurring tumours. PMID:12804537

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

    PubMed Central

    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. PMID:27069754

  19. 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.

  20. 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

  1. 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

  2. Plant cell wall lignification and monolignol metabolism

    PubMed Central

    Wang, Yin; Chantreau, Maxime; Sibout, Richard; Hawkins, Simon

    2013-01-01

    Plants are built of various specialized cell types that differ in their cell wall composition and structure. The cell walls of certain tissues (xylem, sclerenchyma) are characterized by the presence of the heterogeneous lignin polymer that plays an essential role in their physiology. This phenolic polymer is composed of different monomeric units – the monolignols – that are linked together by several covalent bonds. Numerous studies have shown that monolignol biosynthesis and polymerization to form lignin are tightly controlled in different cell types and tissues. However, our understanding of the genetic control of monolignol transport and polymerization remains incomplete, despite some recent promising results. This situation is made more complex since we know that monolignols or related compounds are sometimes produced in non-lignified tissues. In this review, we focus on some key steps of monolignol metabolism including polymerization, transport, and compartmentation. As well as being of fundamental interest, the quantity of lignin and its nature are also known to have a negative effect on the industrial processing of plant lignocellulose biomass. A more complete view of monolignol metabolism and the relationship that exists between lignin and other monolignol-derived compounds thereby appears essential if we wish to improve biomass quality. PMID:23847630

  3. Castration reversibly alters levels of cholecystokinin immunoreactivity within cells of three interconnected sexually dimorphic forebrain nuclei in the rat.

    PubMed Central

    Simerly, R B; Swanson, L W

    1987-01-01

    Three sexually dimorphic cell groups in the forebrain of the rat--the central part of the medial preoptic nucleus, the encapsulated part of the bed nucleus of the stria terminalis, and the posterodorsal part of the medial nucleus of the amygdala--are larger in males, contain a high density of gonadal-steroid-concentrating cells, and are thought to play important roles in the control of reproductive behavior and physiology. Since each of these regions contains a large number of cholecystokinin-immunoreactive cells, we used an indirect immunohistochemical method to examine the possibility that levels of this peptide are modulated by circulating gonadal steroids in adult male rats. Rats were castrated at 60 days of age, and one group each was pretreated with colchicine and then killed 3, 7, and 14 days after gonadectomy. Castration clearly decreased CCK immunoreactivity within cells of each region, with the most dramatic effects occurring 7 and 14 days after gonadectomy, and these effects were reversed by treatment with testosterone over a 14-day period. The results suggest that CCK levels within individual cells in each of the interconnected sexually dimorphic nuclei examined here are regulated by circulating gonadal steroids and may be related to the hormonal modulation of reproductive functions thought to be mediated by these cell groups. Images PMID:3550806

  4. 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.

  5. Localization of genetic elements of intact and derivative chromosome 11 and 22 territories in nuclei of Ewing sarcoma cells.

    PubMed

    Taslerová, Renata; Kozubek, Stanislav; Bártová, Eva; Gajdusková, Pavla; Kodet, Roman; Kozubek, Michal

    2006-09-01

    Recurring chromosomal abnormalities are associated with specific tumour types. The EWSR1 and FLI1 genes are involved in balanced translocation t(11;22)(q24;q12), which is present in more than 85% of Ewing sarcomas. In our previous study, we have found that the fusion genes pertaining to both derivative chromosomes 11 and 22 in Ewing sarcoma cell nuclei are shifted to the midway nuclear position between the native EWSR1 and FLI1 genes. In this contribution we focused our attention at nuclear positioning of other genetic elements of chromosomes 11 and 22 in order to find if the whole derivative chromosomes or only their translocated parts change their nuclear positions in comparison with the native chromosomes. Using repeated fluorescence in situ hybridization and high-resolution cytometry, 2D radial positions of EWSR1, BCR, FLI1, BCL1 genes and fluorescence weight centres of chromosome territories were compared for intact and derivative chromosomes 11 and 22 in nuclei of three Ewing sarcoma samples. Significant radial shift was obtained for the derivative EWSR1, FLI1 and BCL1 genes and for the derivative chromosome 11 compared with the intact ones and not very significant for chromosome 22 and the BCR gene. Our results also suggest that the mean nuclear positions of fusion genes are determined by the final structure of the derivative chromosomes and do not depend on the location of the translocation event. PMID:16837212

  6. [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.

  7. Glypican and Biglycan in the Nuclei of Neurons and Glioma Cells: Presence of Functional Nuclear Localization Signals and Dynamic Changes in Glypican During the Cell Cycle

    PubMed Central

    Liang, Yu; Häring, Monika; Roughley, Peter J.; Margolis, Renée K.; Margolis, Richard U.

    1997-01-01

    We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target β-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican β-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly

  8. Sequence-specific microscopic visualization of DNA methylation status at satellite repeats in individual cell nuclei and chromosomes

    PubMed Central

    Li, Yufeng; Miyanari, Yusuke; Shirane, Kenjiro; Nitta, Hirohisa; Kubota, Takeo; Ohashi, Hirofumi; Okamoto, Akimitsu; Sasaki, Hiroyuki

    2013-01-01

    Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis. PMID:23990328

  9. Sequence-specific microscopic visualization of DNA methylation status at satellite repeats in individual cell nuclei and chromosomes.

    PubMed

    Li, Yufeng; Miyanari, Yusuke; Shirane, Kenjiro; Nitta, Hirohisa; Kubota, Takeo; Ohashi, Hirofumi; Okamoto, Akimitsu; Sasaki, Hiroyuki

    2013-10-01

    Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis. PMID:23990328

  10. 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.

  11. 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. PMID:25640432

  12. Mineralocorticoid hormone action in plant cells.

    PubMed

    Mirshahi, M; Mirshahi, A; Nato, A; Agarwal, M K

    1992-07-31

    The multiplication of Chlamydomonas reinhardtii wild type cells can be arrested by the spirolactone RU 26752 and this is fully reversible by the natural mineralocorticoid aldosterone. Evidence is presented for a 52 kDa protein that possesses functional DNA and ligand binding domains and tests positive for mineralocorticoid receptor-like activity by immuneprecipitation, macroaggregation, and photoaffinity. The regulation of trans-activation by steroid hormones in the animal world would therefore appear to be just as valid for the plant kingdom, thereby providing a new model for genetic analysis. PMID:1323283

  13. [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

  14. 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

  15. 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

  16. Apoptosis induced clustering of IP(3)R1 in nuclei of non-differentiated PC12 cells.

    PubMed

    Ondrias, Karol; Lencesova, Lubomira; Sirova, Marta; Labudova, Martina; Pastorekova, Silvia; Kopacek, Juraj; Krizanova, Olga

    2011-12-01

    Inositol 1,4,5-trisphosphate (IP(3)) receptors are emerging as key sites for regulation by pro- and anti-apoptotic factors. Induction of apoptosis for 3 h increased mRNA and protein levels of type 1 IP(3) receptors in non-differentiated (ND), but not in differentiated (D) PC12 cells. Inhibitors of the IP(3) R's calcium release-2-aminoethoxydiphenyl borate (2-APB) and xestospongin-completely prevented Bax and caspase-3 mRNA increase after treatment with the apoptosis inducer set (AIK), and this reinforces the importance of IP(3) R1 in the apoptosis of ND PC12 cells. Apoptosis induction not only increases the IP(3) R1 protein, but it also causes formation of IP(3) R1 clusters in the nucleus which most likely result from fusion of the nucleoplasmic reticulum and/or IP(3) R1 translocation to the nucleus. This is quite similar to the observations noted after overexpression of IP(3) R1 in PC12 cells. The amount of IP(3) induced calcium release was higher in control than in AIK-treated cells. From our results we propose that after the apoptosis induction the amount of intranuclear calcium decreased dramatically due to the increase of calcium permeability of the nuclear calcium store vesicles. Therefore, increase of the calcium permeability may result from IP(3) receptors translocation to nuclei that can boost the calcium transport through IP(3) receptors. PMID:21302308

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  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. PMID:26599954

  5. 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

  6. 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.

  7. 2C or not 2C: a closer look at cell nuclei and their DNA content.

    PubMed

    Greilhuber, Johann; Dolezel, Jaroslav

    2009-06-01

    The life cycle of animals and plants involves changes in chromosome number (nuclear phase) and sometimes even the karyotype, and consequently the DNA content of a nuclear genome is not static in time. Thus, in order to interpret DNA content data, it is important that the status of the materials from which DNA content is estimated be precisely defined. The previously proposed distinction between "holoploid" (C) and "monoploid" (Cx) genome size covers the most frequent states of plant and animal nuclear genomes. However, restricting nomenclature to just C and Cx still leaves a number of unresolved problems. Here, we propose an extension of the C-value terminology to handle a range of cytogenetic conditions, life cycle segments, and nuclear phases. A set of superscripts and subscripts are used in a formal way to identify life cycle segments and to express the quantitative relationship between these segments. A revision of the current usage of the holoploid chromosome number n was necessary to maintain the intimate link between n and C-value and between the monoploid chromosome number x and Cx-value. In this revision, haplophase individuals (i.e., "haploid" animals and "haploid" spontaneous or experimentally induced land plant sporophytes) have chromosome number n (not 2n, as is the current tradition) and thus nuclear DNA contents based on 1C. However, to avoid an unlimited progression of n levels due to generative polyploidy, zygotic individuals are assigned as 2n starting from the zygote, whatever their ploidy level. Their ploidy is indicated by multiples of the basic chromosome number x. The extended terminology for genome size should eliminate ambiguities in reporting DNA contents in both plants and animals. PMID:19242716

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

    PubMed

    Clark, Benjamin J; Brown, Joel E; Taube, Jeffrey S

    2012-11-01

    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

  9. 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

  10. 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.

  11. 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)

  12. 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

  13. 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)

  14. 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

  15. 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.

  16. 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...

  17. 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

  18. 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.

  19. Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis

    PubMed Central

    Higaki, Takumi; Kutsuna, Natsumaro; Sano, Toshio; Hasezawa, Seiichiro

    2008-01-01

    Background Plant cells divide by the formation of new cross walls, known as cell plates, from the center to periphery of each dividing cell. Formation of the cell plate occurs in the phragmoplast, a complex structure composed of membranes, microtubules (MTs) and actin microfilaments (MFs). Disruption of phragmoplast MTs was previously found to completely inhibit cell plate formation and expansion, indicative of their crucial role in the transport of cell plate membranes and materials. In contrast, disruption of MFs only delays cell plate expansion but does not completely inhibit cell plate formation. Despite such findings, the significance and molecular mechanisms of MTs and MFs remain largely unknown. Results Time-sequential changes in MF-distribution were monitored by live imaging of tobacco BY-2 cells stably expressing the GFP-actin binding domain 2 (GFP-ABD2) fusion protein, which vitally co-stained with the endocytic tracer, FM4-64, that labels the cell plate. During cytokinesis, MFs accumulated near the newly-separated daughter nuclei towards the emerging cell plate, and subsequently approached the expanding cell plate edges. Treatment with an actin polymerization inhibitor caused a decrease in the cell plate expansion rate, which was quantified using time-lapse imaging and regression analysis. Our results demonstrated time-sequential changes in the contribution of MFs to cell plate expansion; MF-disruption caused about a 10% decrease in the cell plate expansion rate at the early phase of cytokinesis, but about 25% at the late phase. MF-disruption also caused malformation of the emerging cell plate at the early phase, indicative of MF involvement in early cell plate formation and expansion. The dynamic movement of endosomes around the cell plate was also inhibited by treatment with an actin polymerization inhibitor and a myosin ATPase inhibitor, respectively. Furthermore, time-lapse imaging of the endoplasmic reticulum (ER) revealed that MFs were involved in

  20. Proliferating or Differentiating Stimuli Act on Different Lipid-dependent Signaling Pathways in Nuclei of Human Leukemia Cells

    PubMed Central

    Neri, Luca M.; Bortul, Roberta; Borgatti, Paola; Tabellini, Giovanna; Baldini, Giovanna; Capitani, Silvano; Martelli, Alberto M.

    2002-01-01

    Previous results have shown that the human promyelocytic leukemia HL-60 cell line responds to either proliferating or differentiating stimuli. When these cells are induced to proliferate, protein kinase C (PKC)-βII migrates toward the nucleus, whereas when they are exposed to differentiating agents, there is a nuclear translocation of the α isoform of PKC. As a step toward the elucidation of the early intranuclear events that regulate the proliferation or the differentiation process, we show that in the HL-60 cells, a proliferating stimulus (i.e., insulin-like growth factor-I [IGF-I]) increased nuclear diacylglycerol (DAG) production derived from phosphatidylinositol (4,5) bisphosphate, as indicated by the inhibition exerted by 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and U-73122 (1-[6((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), which are pharmacological inhibitors of phosphoinositide-specific phospholipase C. In contrast, when HL-60 cells were induced to differentiate along the granulocytic lineage by dimethyl sulfoxide, we observed a rise in the nuclear DAG mass, which was sensitive to either neomycin or propranolol, two compounds with inhibitory effect on phospholipase D (PLD)-mediated DAG generation. In nuclei of dimethyl sulfoxide-treated HL-60 cells, we observed a rise in the amount of a 90-kDa PLD, distinct from PLD1 or PLD2. When a phosphatidylinositol (4,5) bisphosphate-derived DAG pool was generated in the nucleus, a selective translocation of PKC-βII occurred. On the other hand, nuclear DAG derived through PLD, recruited PKC-α to the nucleus. Both of these PKC isoforms were phosphorylated on serine residues. These results provide support for the proposal that in the HL-60 cell nucleus there are two independently regulated sources of DAG, both of which are capable of acting as the driving force that attracts to this organelle distinct, DAG-dependent PKC isozymes. Our results assume a particular

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Spatially Resolved Quantification of Chromatin Condensation through Differential Local Rheology in Cell Nuclei Fluorescence Lifetime Imaging

    PubMed Central

    Spagnol, Stephen T.; Dahl, Kris Noel

    2016-01-01

    The linear sequence of DNA encodes access to the complete set of proteins that carry out cellular functions. Yet, much of the functionality appropriate for each cell is nested within layers of dynamic regulation and organization, including a hierarchy of chromatin structural states and spatial arrangement within the nucleus. There remain limitations in our understanding of gene expression within the context of nuclear organization from an inability to characterize hierarchical chromatin organization in situ. Here we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to quantify and spatially resolve chromatin condensation state using cell-permeable, DNA-binding dyes (Hoechst 33342 and PicoGreen). Through in vitro and in situ experiments we demonstrate the sensitivity of fluorescence lifetime to condensation state through the mechanical effects that accompany the structural changes and are reflected through altered viscosity. The establishment of FLIM for resolving and quantifying chromatin condensation state opens the door for single-measurement mechanical studies of the nucleus and for characterizing the role of genome structure and organization in nuclear processes that accompany physiological and pathological changes. PMID:26765322

  8. Spatially Resolved Quantification of Chromatin Condensation through Differential Local Rheology in Cell Nuclei Fluorescence Lifetime Imaging.

    PubMed

    Spagnol, Stephen T; Dahl, Kris Noel

    2016-01-01

    The linear sequence of DNA encodes access to the complete set of proteins that carry out cellular functions. Yet, much of the functionality appropriate for each cell is nested within layers of dynamic regulation and organization, including a hierarchy of chromatin structural states and spatial arrangement within the nucleus. There remain limitations in our understanding of gene expression within the context of nuclear organization from an inability to characterize hierarchical chromatin organization in situ. Here we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to quantify and spatially resolve chromatin condensation state using cell-permeable, DNA-binding dyes (Hoechst 33342 and PicoGreen). Through in vitro and in situ experiments we demonstrate the sensitivity of fluorescence lifetime to condensation state through the mechanical effects that accompany the structural changes and are reflected through altered viscosity. The establishment of FLIM for resolving and quantifying chromatin condensation state opens the door for single-measurement mechanical studies of the nucleus and for characterizing the role of genome structure and organization in nuclear processes that accompany physiological and pathological changes. PMID:26765322

  9. 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

    2016-01-01

    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

  10. Exotic Nuclei

    SciTech Connect

    Galindo-Uribarri, Alfredo {nmn}

    2010-01-01

    Current experimental developments on the study of exotic nuclei far from the valley of stability are discussed. I start with general aspects related to the production of radioactive beams followed by the description of some of the experimental tools and specialized techniques for studies in reaction spectroscopy, nuclear structure research and nuclear applications with examples from selected topical areas with which I have been involved. I discuss some of the common challenges faced in Accelerator Mass Spectrometry (AMS) and Radioactive Ion Beam (RIB) science.

  11. 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.

  12. Fluorescent probes for exploring plant cell wall deconstruction: a review.

    PubMed

    Paës, Gabriel

    2014-01-01

    Plant biomass is a potential resource of chemicals, new materials and biofuels that could reduce our dependency on fossil carbon, thus decreasing the greenhouse effect. However, due to its chemical and structural complexity, plant biomass is recalcitrant to green biological transformation by enzymes, preventing the establishment of integrated bio-refineries. In order to gain more knowledge in the architecture of plant cell wall to facilitate their deconstruction, many fluorescent probes bearing various fluorophores have been devised and used successfully to reveal the changes in structural motifs during plant biomass deconstruction, and the molecular interactions between enzymes and plant cell wall polymers. Fluorescent probes are thus relevant tools to explore plant cell wall deconstruction. PMID:24995923

  13. Super-resolution Microscopy in Plant Cell Imaging.

    PubMed

    Komis, George; Šamajová, Olga; Ovečka, Miroslav; Šamaj, Jozef

    2015-12-01

    Although the development of super-resolution microscopy methods dates back to 1994, relevant applications in plant cell imaging only started to emerge in 2010. Since then, the principal super-resolution methods, including structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED), have been implemented in plant cell research. However, progress has been limited due to the challenging properties of plant material. Here we summarize the basic principles of existing super-resolution methods and provide examples of applications in plant science. The limitations imposed by the nature of plant material are reviewed and the potential for future applications in plant cell imaging is highlighted. PMID:26482957

  14. 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.

  15. Plant cell cultures for the production of recombinant proteins.

    PubMed

    Hellwig, Stephan; Drossard, Jürgen; Twyman, Richard M; Fischer, Rainer

    2004-11-01

    The use of whole plants for the synthesis of recombinant proteins has received a great deal of attention recently because of advantages in economy, scalability and safety compared with traditional microbial and mammalian production systems. However, production systems that use whole plants lack several of the intrinsic benefits of cultured cells, including the precise control over growth conditions, batch-to-batch product consistency, a high level of containment and the ability to produce recombinant proteins in compliance with good manufacturing practice. Plant cell cultures combine the merits of whole-plant systems with those of microbial and animal cell cultures, and already have an established track record for the production of valuable therapeutic secondary metabolites. Although no recombinant proteins have yet been produced commercially using plant cell cultures, there have been many proof-of-principle studies and several companies are investigating the commercial feasibility of such production systems. PMID:15529167

  16. 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

  17. α-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells

    PubMed Central

    Grula, Marjori A.; Buller, Patricia L.; Weaver, Robert F.

    1981-01-01

    [3H]RNA was synthesized in nuclei isolated at various times postinfection from the fat bodies of Heliothis zea larvae infected with H. zea nuclear polyhedrosis virus and from cultured Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus. To detect virus-specific RNA synthesis, the [3H]RNA was hybridized to denatured viral DNA immobilized on nitrocellulose filters. Nuclear polyhedrosis virus-specific RNA synthesis in the infected nuclei isolated from H. zea larval fat bodies and S. frugiperda cells was only inhibited 20 to 25% by concentrations of α-amanitin sufficient to inhibit the host RNA polymerase II. In addition, a productive nuclear polyhedrosis virus infection was obtained in S. frugiperda cells grown in the presence of an α-amanitin concentration that inhibited 90% of the cellular RNA polymerase II activity. The cellular RNA polymerase II enzyme remained sensitive to α-amanitin during infection, and there was no evidence that a virus-coded, α-amanitin-resistant enzyme was synthesized after the onset of infection. The data suggest that the bulk of nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei is transcribed by an enzyme other than the host RNA polymerase II. PMID:16789208

  18. 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 Central

    Yoder, Ryan M.; Taube, Jeffrey S.

    2010-01-01

    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, since lesions of the PoS disrupt landmark control in head direction (HD) cells and hippocampal place cells (Goodridge and Taube, 1997; Calton et al., 2003). 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 indicate 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 indicate 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. The present study provides the first evidence that the PoS→ADN and PoS→LMN projections arise from distinct, non-overlapping cell layers in PoS. Functionally, the PoS may provide landmark information to HD cells in LMN. PMID:20575008

  19. 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

  20. 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

  1. 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

  2. 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

  3. Nontransgenic Genome Modification in Plant Cells1[W][OA

    PubMed Central

    Marton, Ira; Zuker, Amir; Shklarman, Elena; Zeevi, Vardit; Tovkach, Andrey; Roffe, Suzy; Ovadis, Marianna; Tzfira, Tzvi; Vainstein, Alexander

    2010-01-01

    Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods. PMID:20876340

  4. 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.

  5. 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

  6. 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...

  7. 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

  8. 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

  9. 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

  10. 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)

  11. 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)

  12. Association of guanine nucleotide-exchange protein BIG1 in HepG2 cell nuclei with nucleolin, U3 snoRNA, and fibrillarin.

    PubMed

    Padilla, Philip Ian; Uhart, Marina; Pacheco-Rodriguez, Gustavo; Peculis, Brenda A; Moss, Joel; Vaughan, Martha

    2008-03-01

    BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein, activates class I ADP-ribosylation factors (ARF1-3) by catalyzing the replacement of bound GDP by GTP, an action critical for the regulation of protein transport in eukaryotic cells. Our earlier report [Padilla PI, Pancheco-Rodriguez G, Moss J, Vaughan M (2004) Proc Natl Acad Sci USA 101:2752-2757] that BIG1 concentrated in nucleoli of serum-starved HepG2 cells prompted us to identify molecules associated with BIG1 in dynamic nucleolar structures. Antibodies against BIG1 or nucleolin coprecipitated both proteins from nuclei, which was abolished by the incubation of nuclei with RNase A or DNase, indicating that the interaction depended on nucleic acids. (32)P labeling of RNAs immunoprecipitated with BIG1 or nucleolin from nuclei revealed bands of approximately 210 bases that also hybridized with U3 small nucleolar (sno)RNA-specific oligonucleotides. Clones of U3 snoRNA cDNAs from the material precipitated by antibodies against BIG1 or nucleolin yielded identical nucleotide sequences that also were found in genomic DNA. Later analyses revealed the presence of fibrillarin, nucleoporin p62, and La in BIG1 and nucleolin immunoprecipitates. Our data demonstrate that BIG1, nucleolin, U3, the U3-binding protein fibrillarin, and the RNA-binding protein La may exist together in nuclear complexes, consistent with a potential role for BIG1 in nucleolar processes. Evidence that BIG1 and nucleolin, but not fibrillarin, can be present with p62 at the nuclear envelope confirms the presence of BIG1 and nucleolin in dynamic molecular complexes that change in composition while moving through nuclei. Nuclear functions of BIG1 remain to be determined. PMID:18292223

  13. 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

  14. 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.

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

    PubMed Central

    XUE, TAO; WEI, LI; ZHA, DING-JUN; QIAO, LI; LU, LIAN-JUN; CHEN, FU-QUAN; QIU, JIAN-HUA

    2015-01-01

    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. PMID:25605314

  16. 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.

  17. Auxetic nuclei

    PubMed Central

    Wang, Ning

    2015-01-01

    The nucleus of naïve mouse embryonic stem cells in transition to differentiation expands when the cells are stretched and contracts when they are compressed. What drives this auxetic phenotype is, however, unclear. PMID:24845989

  18. 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.

  19. Setup with Laser Ionization in Gas Cell for Production and Study of Neutron-Rich Heavy Nuclei

    NASA Astrophysics Data System (ADS)

    Zagrebaev, V. I.; Zemlyanoy, S. G.; Kozulin, E. M.; Kudryavtsev, Yu.; Fedosseev, V.; Bark, R.; Janas, Z.; Othman, H. A.

    2015-11-01

    The present limits of the upper part of the nuclear map are very close to stability while the unexplored area of heavy neutron-rich nuclides along the neutron closed shell N=126 is extremely important for nuclear astrophysics investigations and, in particular, for the understanding of the r-process of astrophysical nucleosynthesis. This area of the nuclear map can be reached neither in fusion-fission reactions nor in fragmentation processes widely used nowadays for the production of exotic nuclei. A new way was recently proposed for the production of these nuclei via low-energy multi-nucleon transfer reactions. The estimated yields of neutron-rich nuclei are found to be significantly high in such reactions and several tens of new nuclides can be produced, for example, in the near-barrier collision of 136Xe with 208Pb. A new setup is proposed to produce and study heavy neutron-rich nuclei located along the neutron closed shell N=126.

  20. 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. PMID:16399085

  1. 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

  2. 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.

  3. Confocal imaging of ionised calcium in living plant cells.

    PubMed

    Williams, D A; Cody, S H; Gehring, C A; Parish, R W; Harris, P J

    1990-04-01

    Laser-scanning confocal microscopy has been used in conjunction with Fluo-3, a highly fluorescent visible wavelength probe for Ca2+, to visualize Ca2(+)-dynamics in the function of living plant cells. This combination has overcome many of the problems that have limited the use of fluorescence imaging techniques in the study of the role of cations (Ca2+ and H+) in plant cell physiology and enables these processes to be studied in single cells within intact plant tissue preparations. Maize coleoptiles respond to application of ionophores and plant growth hormones with elevations in cytosolic Ca2+ that can be resolved with a high degree of spatial resolution and can be interpreted quantitatively. PMID:2113832

  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. PMID:14695300

  5. 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.

  6. DIRECT DISMANTLING OF REPROCESSING PLANT CELLS THE EUREX PLANT EXPERIENCEe2d12c

    SciTech Connect

    Gili, M.; Troiani, F.; Risoluti, P.

    2003-02-27

    After finishing the reprocessing campaigns in 1970-1983, the EUREX pilot reprocessing plant of ENEA Saluggia Research Center started into a new phase, aiming to materials and irradiated fuel systemation and radioactive wastes conditioning. In 1997 the project ''CORA'' for a vitrification plant for the high and intermediate liquid radioactive wastes started. The ''CORA'' plant will be hosted in some dismantled cells of the EUREX plant, reusing many of the EUREX plant auxiliary systems, duly refurbished, saving money and construction time and avoiding a new nuclear building in the site. Two of the cells that will be reused were part of the EUREX chemical process (solvent recovery and 2nd extraction cycle) and the components were obviously internally contaminated. In 2000 the direct (hands-on) dismantling of one of them started and has been completed in summer 2002; the second one will be dismantled in the next year and then the ''CORA'' plant will be assembled inside the cells. Special care w as taken to avoid spread of contamination in the cells, where ''CORA'' installation activities will start in the next years, during the dismantling process The analysis of data and results collected during the dismantling of the first cell shows that direct dismantling can be achieved with careful choice of tools, procedures and techniques, to reduce volumes of wastes to be disposed and radiological burden.

  7. 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.

  8. 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

  9. 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.

  10. 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.