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Sample records for cell division dna

  1. DNA synthesis and cell division in the adult primate brain

    International Nuclear Information System (INIS)

    Rakic, P.

    1985-01-01

    It is generally accepted that the adult human brain is incapable of producing new neuron. Even cursory examination of neurologic, neuropathologic, or neurobiological textbooks published during the past 50 years will testify that this belief is deeply entrenched. In his classification of cell populations on the basis of their proliferative behavior, Leblond regarded neurons of the central nervous system as belonging to a category of static, nonrenewing epithelial tissue incapable of expanding or replenishing itself. This belief, however needs to re reexamined for two major reasons: First, as reviewed below, a number of reports have provided evidence of neurogenesis in adult brain of several vertebrate species. Second, the capacity for neurogenesis in the adult primate central nervous system has never been examined by modern methods. In this article the author described recent results from an extensive autoradiographic analysis performed on twelve rhesus monkeys injected with the specific DNA precursor [ 3 H] thymidine at ages ranging from 6 postnatal months to 17 years

  2. CD8 Memory Cells Develop Unique DNA Repair Mechanisms Favoring Productive Division.

    Science.gov (United States)

    Galgano, Alessia; Barinov, Aleksandr; Vasseur, Florence; de Villartay, Jean-Pierre; Rocha, Benedita

    2015-01-01

    Immune responses are efficient because the rare antigen-specific naïve cells are able to proliferate extensively and accumulate upon antigen stimulation. Moreover, differentiation into memory cells actually increases T cell accumulation, indicating improved productive division in secondary immune responses. These properties raise an important paradox: how T cells may survive the DNA lesions necessarily induced during their extensive division without undergoing transformation. We here present the first data addressing the DNA damage responses (DDRs) of CD8 T cells in vivo during exponential expansion in primary and secondary responses in mice. We show that during exponential division CD8 T cells engage unique DDRs, which are not present in other exponentially dividing cells, in T lymphocytes after UV or X irradiation or in non-metastatic tumor cells. While in other cell types a single DDR pathway is affected, all DDR pathways and cell cycle checkpoints are affected in dividing CD8 T cells. All DDR pathways collapse in secondary responses in the absence of CD4 help. CD8 T cells are driven to compulsive suicidal divisions preventing the propagation of DNA lesions. In contrast, in the presence of CD4 help all the DDR pathways are up regulated, resembling those present in metastatic tumors. However, this up regulation is present only during the expansion phase; i.e., their dependence on antigen stimulation prevents CD8 transformation. These results explain how CD8 T cells maintain genome integrity in spite of their extensive division, and highlight the fundamental role of DDRs in the efficiency of CD8 immune responses.

  3. Template DNA-strand co-segregation and asymmetric cell division in skeletal muscle stem cells.

    Science.gov (United States)

    Shinin, Vasily; Gayraud-Morel, Barbara; Tajbakhsh, Shahragim

    2009-01-01

    Stem cells are present in all tissues and organs, and are crucial for normal regulated growth. How the pool size of stem cells and their progeny is regulated to establish the tissue prenatally, then maintain it throughout life, is a key question in biology and medicine. The ability to precisely locate stem and progenitors requires defining lineage progression from stem to differentiated cells, assessing the mode of cell expansion and self-renewal and identifying markers to assess the different cell states within the lineage. We have shown that during lineage progression from a quiescent adult muscle satellite cell to a differentiated myofibre, both symmetric and asymmetric divisions take place. Furthermore, we provide evidence that a sub-population of label retaining satellite cells co-segregate template DNA strands to one daughter cell. These findings provide a means of identifying presumed stem and progenitor cells within the lineage. In addition, asymmetric segregation of template DNA and the cytoplasmic protein Numb provides a landmark to define cell behaviour as self-renewal and differentiation decisions are being executed.

  4. Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

    DEFF Research Database (Denmark)

    Persson, H.; Købler, Carsten; Mølhave, Kristian

    2013-01-01

    beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA......Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule-delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain...... largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion...

  5. Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

    DEFF Research Database (Denmark)

    Persson, H.; Købler, Carsten; Mølhave, Kristian

    2013-01-01

    Mouse fibroblasts cultured on 7-μm-long vertical nanowires are reported on page 4006 by C. N. Prinz and co-workers. Culturing cells on this kind of substrate interferes greatly with cell function, causing the cells to develop into widely different morphologies. The cells' division is impaired...

  6. Cell Division Synchronization

    Science.gov (United States)

    The report summarizes the progress in the design and construction of automatic equipment for synchronizing cell division in culture by periodic...Concurrent experiments in hypothermic synchronization of algal cell division are reported.

  7. Inhibition of Cell Division and DNA Replication Impair Mouse-Naïve Pluripotency Exit.

    Science.gov (United States)

    Waisman, Ariel; Vazquez Echegaray, Camila; Solari, Claudia; Cosentino, María Soledad; Martyn, Iain; Deglincerti, Alessia; Ozair, Mohammad Zeeshan; Ruzo, Albert; Barañao, Lino; Miriuka, Santiago; Brivanlou, Ali; Guberman, Alejandra

    2017-09-01

    The cell cycle has gained attention as a key determinant for cell fate decisions, but the contribution of DNA replication and mitosis in stem cell differentiation has not been extensively studied. To understand if these processes act as "windows of opportunity" for changes in cell identity, we established synchronized cultures of mouse embryonic stem cells as they exit the ground state of pluripotency. We show that initial transcriptional changes in this transition do not require passage through mitosis and that conversion to primed pluripotency is linked to lineage priming in the G1 phase. Importantly, we demonstrate that impairment of DNA replication severely blocks transcriptional switch to primed pluripotency, even in the absence of p53 activity induced by the DNA damage response. Our data suggest an important role for DNA replication during mouse embryonic stem cell differentiation, which could shed light on why pluripotent cells are only receptive to differentiation signals during G1, that is, before the S phase. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. The stem cell division theory of cancer.

    Science.gov (United States)

    López-Lázaro, Miguel

    2018-03-01

    All cancer registries constantly show striking differences in cancer incidence by age and among tissues. For example, lung cancer is diagnosed hundreds of times more often at age 70 than at age 20, and lung cancer in nonsmokers occurs thousands of times more frequently than heart cancer in smokers. An analysis of these differences using basic concepts in cell biology indicates that cancer is the end-result of the accumulation of cell divisions in stem cells. In other words, the main determinant of carcinogenesis is the number of cell divisions that the DNA of a stem cell has accumulated in any type of cell from the zygote. Cell division, process by which a cell copies and separates its cellular components to finally split into two cells, is necessary to produce the large number of cells required for living. However, cell division can lead to a variety of cancer-promoting errors, such as mutations and epigenetic mistakes occurring during DNA replication, chromosome aberrations arising during mitosis, errors in the distribution of cell-fate determinants between the daughter cells, and failures to restore physical interactions with other tissue components. Some of these errors are spontaneous, others are promoted by endogenous DNA damage occurring during quiescence, and others are influenced by pathological and environmental factors. The cell divisions required for carcinogenesis are primarily caused by multiple local and systemic physiological signals rather than by errors in the DNA of the cells. As carcinogenesis progresses, the accumulation of DNA errors promotes cell division and eventually triggers cell division under permissive extracellular environments. The accumulation of cell divisions in stem cells drives not only the accumulation of the DNA alterations required for carcinogenesis, but also the formation and growth of the abnormal cell populations that characterize the disease. This model of carcinogenesis provides a new framework for understanding the

  9. Cell growth and division cycle

    International Nuclear Information System (INIS)

    Darzynkiewicz, Z.

    1986-01-01

    The concept of the cell cycle in its present form was introduced more than three decades ago. Studying incorporation of DNA precursors by autoradiography, these authors observed that DNA synthesis in individual cells was discontinuous and occupied a discrete portion of the cell life (S phase). Mitotic division was seen to occur after a certain period of time following DNA replication. A distinct time interval between mitosis and DNA replication was also apparent. Thus, the cell cycle was subdivided into four consecutive phases, G/sub 1/, S, G/sub 2/, and M. The G/sub 1/ and G/sub 2/ phases represented the ''gaps'' between mitosis and the start of DNA replication, and between the end of DNA replication and the onset of mitosis, respectively. The cell cycle was defined as the interval between the midpoint of mitosis and the midpoint of the subsequent mitosis of the daughter cell(s). The authors' present knowledge on the cell cycle benefited mostly from the development of four different techniques: autoradiography, time-lapse cinematography, cell synchronization and flow cytometry. Of these, autoradiography has been the most extensively used, especially during the past two decades. By providing a means to analyse incorporation of precursors of DNA, RNA or proteins by individual cells and, in combination with various techniques of cell synchronization, autoradiography yielded most of the data fundamental to the current understanding of the cell cycle-related phenomena. Kinetics of cell progression through the cell cycle could be analysed in great detail after development of such sophisticated autoradiographic approaches as measurements of the fraction of labeled mitoses (''FLM curves'') or multiple sequential cell labelling with /sup 3/H- and /sup 14/C-TdR

  10. Hydroxychavicol, a key ingredient of Piper betle induces bacterial cell death by DNA damage and inhibition of cell division.

    Science.gov (United States)

    Singh, Deepti; Narayanamoorthy, Shwetha; Gamre, Sunita; Majumdar, Ananda Guha; Goswami, Manish; Gami, Umesh; Cherian, Susan; Subramanian, Mahesh

    2018-05-20

    Antibiotic resistance is a global problem and there is an urgent need to augment the arsenal against pathogenic bacteria. The emergence of different drug resistant bacteria is threatening human lives to be pushed towards the pre-antibiotic era. Botanical sources remain a vital source of diverse organic molecules that possess antibacterial property as well as augment existing antibacterial molecules. Piper betle, a climber, is widely used in south and south-east Asia whose leaves and nuts are consumed regularly. Hydroxychavicol (HC) isolated from Piper betle has been reported to possess antibacterial activity. It is currently not clear how the antibacterial activity of HC is manifested. In this investigation we show HC generates superoxide in E. coli cells. Antioxidants protected E. coli against HC induced cell death while gshA mutant was more sensitive to HC than wild type. DNA damage repair deficient mutants are hypersensitive to HC and HC induces the expression of DNA damage repair genes that repair oxidative DNA damage. HC treated E. coli cells are inhibited from growth and undergo DNA condensation. In vitro HC binds to DNA and cleaves it in presence of copper. Our data strongly indicates HC mediates bacterial cell death by ROS generation and DNA damage. Damage to iron sulfur proteins in the cells contribute to amplification of oxidative stress initiated by HC. Further HC is active against a number of Gram negative bacteria isolated from patients with a wide range of clinical symptoms and varied antibiotic resistance profiles. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Modes of overinitiation, dnaA gene expression, and inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia coli.

    Science.gov (United States)

    Fujimitsu, Kazuyuki; Su'etsugu, Masayuki; Yamaguchi, Yoko; Mazda, Kensaku; Fu, Nisi; Kawakami, Hironori; Katayama, Tsutomu

    2008-08-01

    The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.

  12. Modes of Overinitiation, dnaA Gene Expression, and Inhibition of Cell Division in a Novel Cold-Sensitive hda Mutant of Escherichia coli▿

    Science.gov (United States)

    Fujimitsu, Kazuyuki; Su'etsugu, Masayuki; Yamaguchi, Yoko; Mazda, Kensaku; Fu, Nisi; Kawakami, Hironori; Katayama, Tsutomu

    2008-01-01

    The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the β clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25°C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25°C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42°C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25°C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25°C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway. PMID:18502852

  13. A mechanism for ParB-dependent waves of ParA, a protein related to DNA segregation during cell division in prokaryotes

    DEFF Research Database (Denmark)

    Hunding, Axel; Gerdes, Kenn; Charbon, Gitte Ebersbach

    2003-01-01

    in an autocatalytic process. We discuss this mechanism in relation to recent models for MinDE oscillations in E.coli and to microtubule degradation in mitosis. The study points to an ancestral role for the presented pattern types in generating bipolarity in prokaryotes and eukaryotes.......Prokaryotic plasmids encode partitioning (par) loci involved in segregation of DNA to daughter cells at cell division. A functional fusion protein consisting of Walker-type ParA ATPase and green fluorescent protein (Gfp) oscillates back and forth within nucleoid regions with a wave period of about...

  14. Running on empty: does mitochondrial DNA mutation limit replicative lifespan in yeast?: Mutations that increase the division rate of cells lacking mitochondrial DNA also extend replicative lifespan in Saccharomyces cerevisiae.

    Science.gov (United States)

    Dunn, Cory D

    2011-10-01

    Mitochondrial DNA (mtDNA) mutations escalate with increasing age in higher organisms. However, it has so far been difficult to experimentally determine whether mtDNA mutation merely correlates with age or directly limits lifespan. A recent study shows that budding yeast can also lose functional mtDNA late in life. Interestingly, independent studies of replicative lifespan (RLS) and of mtDNA-deficient cells show that the same mutations can increase both RLS and the division rate of yeast lacking the mitochondrial genome. These exciting, parallel findings imply a potential causal relationship between mtDNA mutation and replicative senescence. Furthermore, these results suggest more efficient methods for discovering genes that determine lifespan. Copyright © 2011 WILEY Periodicals, Inc.

  15. Developmental control of cell division

    NARCIS (Netherlands)

    Boxem, M. (Mike)

    2002-01-01

    During development of multicellular organisms, cell divisions need to be coordinated with the developmental program of the entire organism. Although the mechanisms that drive cells through the division cycle are well understood, very little is known about the pathways that link extracellular signals

  16. Microbial mutagenesis and cell division

    International Nuclear Information System (INIS)

    Adler, H.I.; Carrasco, A.; Nagel, R.; Gill, J.S.; Crow, W.D.

    1982-01-01

    Our group has been pursuing three related objectives. The first of these is a study of a mechanism by which the bacterium Escherichia coli repairs radiation-induced damage. In particular, we have observed that cells of certain strains of this bacterium, mutant at the lon locus, can be restored to viability after exposure to ionizing radiation if they are incubated in a nutrient medium to which a preparation of partially purified bacterial membranes has been added. These preparations stimulate division by producing chemical alterations in the nutrient medium and simultaneously creating a highly anaerobic environment. A second objective of the group was to make use of lon mutants for a rapid, sensitive, and inexpensive assay for chemical mutagens. Cells of lon mutants form long multinucleate filaments if exposed to a variety of agents that react with DNA. These filaments can readily be observed microscopically 2 to 3 h after exposure to the suspect agent. A third objective of our group has been to make use of the oxygen reducing properties of bacterial membrane preparations to stimulate the growth of anaerobic bacteria. Our general goal is to develop basic microbiological techniques that will facilitate the application of genetic manipulation methods to important anaerobic species. To this end, we have developed a method, based on the use of membranes, that allows us to grow liquid cultures of Clostridium acetobutylicum from very small inocula to high titers without elaborate chemical or physical methods for excluding oxygen. We have also developed efficient methods for plating this bacterium that do not require the use of anaerobic incubators

  17. Geminin deploys multiple mechanisms to regulate Cdt1 before cell division thus ensuring the proper execution of DNA replication

    DEFF Research Database (Denmark)

    Ballabeni, Andrea; Zamponi, Raffaella; Moore, Jodene K

    2013-01-01

    the accumulation of Cdt1 in mitosis, because decreasing the Geminin levels prevents Cdt1 accumulation and impairs DNA replication. Geminin is known to inhibit Cdt1 function; its depletion during G2 leads to DNA rereplication and checkpoint activation. Here we show that, despite rapid Cdt1 protein turnover in G2...

  18. Ploidy-Dependent Unreductional Meiotic Cell Division in Polyploid Wheat

    Science.gov (United States)

    Meiosis includes one round of DNA replication and two successive nuclear divisions, i.e. meiosis I (reductional) and meiosis II (equational). This specialized cell division reduces chromosomes in half and generates haploid gametes in sexual reproduction of eukaryotes. It ensures faithful transmiss...

  19. Prokaryotic cell division: flexible and diverse

    NARCIS (Netherlands)

    den Blaauwen, T.

    2013-01-01

    Gram-negative rod-shaped bacteria have different approaches to position the cell division initiating Z-ring at the correct moment in their cell division cycle. The subsequent maturation into a functional division machine occurs in vastly different species in two steps with appreciable time in

  20. Heparan sulfate and cell division

    Directory of Open Access Journals (Sweden)

    Porcionatto M.A.

    1999-01-01

    Full Text Available Heparan sulfate is a component of vertebrate and invertebrate tissues which appears during the cytodifferentiation stage of embryonic development. Its structure varies according to the tissue and species of origin and is modified during neoplastic transformation. Several lines of experimental evidence suggest that heparan sulfate plays a role in cellular recognition, cellular adhesion and growth control. Heparan sulfate can participate in the process of cell division in two distinct ways, either as a positive or negative modulator of cellular proliferation, or as a response to a mitogenic stimulus.

  1. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2018-05-15

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  2. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  3. Chromosome replication, cell growth, division and shape: a personal perspective

    Directory of Open Access Journals (Sweden)

    Arieh eZaritsky

    2015-08-01

    Full Text Available The origins of Molecular Biology and Bacterial Physiology are reviewed, from our personal standpoints, emphasizing the coupling between bacterial growth, chromosome replication and cell division, dimensions and shape. Current knowledge is discussed with historical perspective, summarizing past and present achievements and enlightening ideas for future studies. An interactive simulation program of the Bacterial Cell Division Cycle (BCD, described as The Central Dogma in Bacteriology, is briefly represented. The coupled process of transcription/translation of genes encoding membrane proteins and insertion into the membrane (so-called transertion is invoked as the functional relationship between the only two unique macromolecules in the cell, DNA and peptidoglycan embodying the nucleoid and the sacculus respectively. We envision that nucleoid complexity, defined as the weighted-mean DNA content associated with the replication terminus, is directly related to cell shape through the transertion process. Accordingly, the primary signal for cell division transmitted by DNA dynamics (replication, transcription and segregation to the peptidoglycan biosynthetic machinery is of a physico-chemical nature, eg stress in the plasma membrane, relieving nucleoid occlusion in the cell's center hence enabling the divisome to assemble and function between segregated daughter nucleoids.

  4. Cell Division and Evolution of Biological Tissues

    Science.gov (United States)

    Rivier, Nicolas; Arcenegui-Siemens, Xavier; Schliecker, Gudrun

    A tissue is a geometrical, space-filling, random cellular network; it remains in this steady state while individual cells divide. Cell division (fragmentation) is a local, elementary topological transformation which establishes statistical equilibrium of the structure. Statistical equilibrium is characterized by observable relations (Lewis, Aboav) between cell shapes, sizes and those of their neighbours, obtained through maximum entropy and topological correlation extending to nearest neighbours only, i.e. maximal randomness. For a two-dimensional tissue (epithelium), the distribution of cell shapes and that of mother and daughter cells can be obtained from elementary geometrical and physical arguments, except for an exponential factor favouring division of larger cells, and exponential and combinatorial factors encouraging a most symmetric division. The resulting distributions are very narrow, and stationarity severely restricts the range of an adjustable structural parameter

  5. Nuclear size and cell division delay

    International Nuclear Information System (INIS)

    Bird, R.P.

    1986-01-01

    Radiation-induced division delay has been linked to damage at the nuclear envelope. Further, cells in G 2 phase are drastically arrested by high LET radiation such that single particles traversing cell nuclei may produce measurable division delay. A modest effort was initiated using two related cell lines of different size, near-diploid cells and near-tetraploid cells of Chinese hamster origin, to compare their sensitivity for radiation-induced division delay. If the nuclear surface is the critical target, then a larger nuclear cross-section presented to an alpha-particle beam should exhibit delay induced by a lesser particle fluence. Preliminary estimates of the extent of delay in asynchronous cultures following low doses of gamma-irradiation or of alpha-irradiation were made by in-situ observation of the time of onset of mitosis and by fixation and staining of cultures to determine the mitotic index as a function of time after irradiation. The basic approach to evaluating division delay will be to use Colecemid to accumulate mitotic cells over a period of time

  6. Genes involved in cell division in mycoplasmas

    Directory of Open Access Journals (Sweden)

    Frank Alarcón

    2007-01-01

    Full Text Available Bacterial cell division has been studied mainly in model systems such as Escherichia coli and Bacillus subtilis, where it is described as a complex process with the participation of a group of proteins which assemble into a multiprotein complex called the septal ring. Mycoplasmas are cell wall-less bacteria presenting a reduced genome. Thus, it was important to compare their genomes to analyze putative genes involved in cell division processes. The division and cell wall (dcw cluster, which in E. coli and B. subtilis is composed of 16 and 17 genes, respectively, is represented by only three to four genes in mycoplasmas. Even the most conserved protein, FtsZ, is not present in all mycoplasma genomes analyzed so far. A model for the FtsZ protein from Mycoplasma hyopneumoniae and Mycoplasma synoviae has been constructed. The conserved residues, essential for GTP/GDP binding, are present in FtsZ from both species. A strong conservation of hydrophobic amino acid patterns is observed, and is probably necessary for the structural stability of the protein when active. M. synoviae FtsZ presents an extended amino acid sequence at the C-terminal portion of the protein, which may participate in interactions with other still unknown proteins crucial for the cell division process.

  7. Mechanical Division of Cell-Sized Liposomes

    NARCIS (Netherlands)

    Deshpande, S.R.; Kerssemakers, J.W.J.; Dekker, C.

    2018-01-01

    Liposomes, self-assembled vesicles with a lipid-bilayer boundary similar to cell membranes, are extensively used in both fundamental and applied sciences. Manipulation of their physical properties, such as growth and division, may significantly expand their use as model systems in cellular and

  8. Cell cycle related /sup 125/IUDR-induced-division delay

    International Nuclear Information System (INIS)

    Scheniderman, M.H.; Hofer, K.G.

    1987-01-01

    A series of experiments were run to determine if /sup 125/I-decays, in /sup 125/IUdR labeled DNA, specifically accumulated at 1, 3, 5, 7 and 9 hours after plating labeled mitotic cells caused a change in the rate or time of cell entry into mitosis. To accomplish this, a pool of labeled mitotic cells was selected in mitosis and plated in replicate flasks. /sup 125/I decays were accumulated in groups of cells by cooling (4 0 C) for 2 hours starting at the designated times. After rewarding, colcemid was added to arrest cells in mitosis. The rate of cell progression into mitosis for each cell cycle time of accumulation was determined by scoring the mitotic index of cells sampled as a function of time after addition of the colcemid. The results are summarized: (1) Decays from /sup 125/I in /sup 125/I(UdR) labeled DNA reduced the rate of cell progression into mitosis and delayed the time of initiation of mitosis. (2) The reduced rate of progression and the delayed time of initiation of mitosis were independent of the cell cycle time that /sup 125/I-decays were accumulated. (3) The reduced rate of progression after cell cycle accumulation of /sup 125/I decay was statistically indistinguishable from the corresponding controls. (4) The delayed initiation of mitosis after specific cell cycle accumulation of /sup 125/I- decays was greater than the corresponding control. The relationship of these data to DNA and non-DNA division delay target(s) is emphasized

  9. Interdependence of bacterial cell division and genome segregation and its potential in drug development.

    Science.gov (United States)

    Misra, Hari S; Maurya, Ganesh K; Chaudhary, Reema; Misra, Chitra S

    2018-03-01

    Cell division and genome segregation are mutually interdependent processes, which are tightly linked with bacterial multiplication. Mechanisms underlying cell division and the cellular machinery involved are largely conserved across bacteria. Segregation of genome elements on the other hand, follows different pathways depending upon its type and the functional components encoded on these elements. Small molecules, that are known to inhibit cell division and/or resolution of intertwined circular chromosome and maintenace of DNA topology have earlier been tested as antibacterial agents. The utility of such drugs in controlling bacterial infections has witnessed only partial success, possibly due to functional redundancy associated with targeted components. However, in due course, literature has grown with newer information. This review has brought forth some recent findings on bacterial cell division with special emphasis on crosstalk between cell division and genome segregation that could be explored as novel targets in drug development. Copyright © 2018 Elsevier GmbH. All rights reserved.

  10. Control of cell division and radiation injury in mouse skin

    International Nuclear Information System (INIS)

    Yamaguchi, Takeo

    1974-01-01

    The method for determining the inhibitors of cell division (chalone-adrenalin system) in the irradiated epidermis and blood was developed using the epidermis of mouse ear conch during the cure of wounds (in vivo), and the epidermis cultured for a long period (in vitro). The whole body was irradiated with 200KV, 20 mA x-rays of 96 R/min filtered by 0.5 mmCu + 0.5 mmAl. Chalone, which is a physiologically intrinsic substance to control the proliferation, inhibits the DNA synthesis. From changes in cell division with time, chalone in the epidermis is considered to inhibit each process from G 2 to M, from G 2 to S, from G 1 to S. Adrenalin is indispensable when epidermal chalone acts the inhibition of cell division. Chalone activities in the epidermis irradiated with almost lethal doses were decreased. Factors to inhibit the proliferation of the epidermis by the potentiation of chalone and adrenalin are present in sera of animals irradiated to x-rays. (Serizawa, K.)

  11. Onset of cell division in maize germination: action of auxins

    International Nuclear Information System (INIS)

    de Jimenez, E.S.; Baiza, A.; Aguilar, R.

    1987-01-01

    Seed germination implies metabolic reactivation, synthesis of macromolecules and onset of cell division. During maize germination, meristematic tissues of embryos re-initiate cell division asynchronically. Since auxins are known to stimulate cell division, they asked how auxins might regulate cell cycle re-initiation. Embryonic tissues were incubated with and without auxins. A pulse of either 3 H-thymidine or 32 P-ortophosphate was given to the tissues. Mitotic indexes were determined and % of labeled mitotic cells recorded. Results indicated that meristematic cells re-initiate cell division either from G 1 or G 2 phases. Auxin stimulated differentially the cell division process of these cells. 32 P incorporation into cytoplasmic or nucleic histones was measured. Auxins stimulated this incorporation. Active turnover of histone phosphorylation occurred simultaneously to the cell division process. It is suggested that auxins might regulate the cell cycle by phosphorylation-dephosphorylation of histones

  12. Asymmetric cell division during T cell development controls downstream fate

    Science.gov (United States)

    Pham, Kim; Shimoni, Raz; Charnley, Mirren; Ludford-Menting, Mandy J.; Hawkins, Edwin D.; Ramsbottom, Kelly; Oliaro, Jane; Izon, David; Ting, Stephen B.; Reynolds, Joseph; Lythe, Grant; Molina-Paris, Carmen; Melichar, Heather; Robey, Ellen; Humbert, Patrick O.; Gu, Min

    2015-01-01

    During mammalian T cell development, the requirement for expansion of many individual T cell clones, rather than merely expansion of the entire T cell population, suggests a possible role for asymmetric cell division (ACD). We show that ACD of developing T cells controls cell fate through differential inheritance of cell fate determinants Numb and α-Adaptin. ACD occurs specifically during the β-selection stage of T cell development, and subsequent divisions are predominantly symmetric. ACD is controlled by interaction with stromal cells and chemokine receptor signaling and uses a conserved network of polarity regulators. The disruption of polarity by deletion of the polarity regulator, Scribble, or the altered inheritance of fate determinants impacts subsequent fate decisions to influence the numbers of DN4 cells arising after the β-selection checkpoint. These findings indicate that ACD enables the thymic microenvironment to orchestrate fate decisions related to differentiation and self-renewal. PMID:26370500

  13. Activation of cell divisions in legume nodulation

    DEFF Research Database (Denmark)

    Nadzieja, Marcin

    organogenesis. Coordination of these two interdependent processes results in formation of nodules - bacterial accommodating structures where fixation of atmospheric nitrogen takes place. Plant hormones such as auxin and cytokinin play important roles in nodulation. In some legumes the infection process...... of auxin transport inhibitors or cytokinin alone was shown to induce cortical cell divisions in the absence of rhizobia in certain legume species. While the roles of auxin and cytokinin in nodulation have been studied extensively, the precise timing, location and means of molecular crosstalk between...

  14. Inhibition of cell division in hupA hupB mutant bacteria lacking HU protein.

    OpenAIRE

    Dri, A M; Rouviere-Yaniv, J; Moreau, P L

    1991-01-01

    Escherichia coli hupA hypB double mutants that lack HU protein have severe cellular defects in cell division, DNA folding, and DNA partitioning. Here we show that the sfiA11 mutation, which alters the SfiA cell division inhibitor, reduces filamentation and production of anucleate cells in AB1157 hupA hupB strains. However, lexA3(Ind-) and sfiB(ftsZ)114 mutations, which normally counteract the effect of the SfiA inhibitor, could not restore a normal morphology to hupA hupB mutant bacteria. The...

  15. Radiomimetic effect of cisplatin on cucumber root development: the relationship between cell division and cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Dubrovsky, J. G. [Division of Experimental Biology, Center for Biological Research (CIB), PO Box 128, La Paz, BCS 23000 (Mexico)

    1993-07-01

    Cisplatin [DDP, cis-dichlorodiammine platinum (II)], a strong cytostatic and antineoplastic agent, was tested on seedlings of cucumber Cucumis sativus L. for its general effect on root development and its particular effects on root cell division and cell growth. DDP was characterized as a radiomimetic compound since both DDP (1·3 × 10{sup -5} M) and γ-irradiation (2·5-10 kGy) drastically and irreversibly stopped development of embryonic lateral root primordia (LRPs) in the radicle by inhibiting both mitotic activity and cell growth. In 20% of the LRPs of DDP-treated roots, cells did not divide at all. Dividing cells completed no more than two cell cycles. These effects were specific because when DDP was available to the roots only at the onset of cell division, cell proliferation and cell growth were similar to that produced by constant incubation. Neither DDP nor γ-irradiation affected non-meristematic cell elongation. It was concluded that cell growth of meristematic cells is closely related to cell division. However, non-meristematic cell growth is independent of DNA damage. This suggests DDP as a tool to reveal these autonomous processes in plants development and to detect tissue compartments in mature plant embryos which contain potentially non-meristematic cells. (author)

  16. Lipid Cell Biology: A Focus on Lipids in Cell Division.

    Science.gov (United States)

    Storck, Elisabeth M; Özbalci, Cagakan; Eggert, Ulrike S

    2018-06-20

    Cells depend on hugely diverse lipidomes for many functions. The actions and structural integrity of the plasma membrane and most organelles also critically depend on membranes and their lipid components. Despite the biological importance of lipids, our understanding of lipid engagement, especially the roles of lipid hydrophobic alkyl side chains, in key cellular processes is still developing. Emerging research has begun to dissect the importance of lipids in intricate events such as cell division. This review discusses how these structurally diverse biomolecules are spatially and temporally regulated during cell division, with a focus on cytokinesis. We analyze how lipids facilitate changes in cellular morphology during division and how they participate in key signaling events. We identify which cytokinesis proteins are associated with membranes, suggesting lipid interactions. More broadly, we highlight key unaddressed questions in lipid cell biology and techniques, including mass spectrometry, advanced imaging, and chemical biology, which will help us gain insights into the functional roles of lipids.

  17. Amoebiasis and its effect on cell division in the midgut of the African ...

    African Journals Online (AJOL)

    cells was noted in the nidi of the ventricular regions of locusts in- fected with parasites. ... migratoria and as these tissues undergo cell division the. R eprod u ced ..... repair or possibly could have completed DNA synthesis, divi- sion and ...

  18. Plant cortical microtubule dynamics and cell division plane orientation

    NARCIS (Netherlands)

    Chakrabortty, Bandan

    2017-01-01

    This thesis work aimed at a better understanding of the molecular basis of oriented cell division in plant cell. As, the efficiency of plant morphogenesis depends on oriented cell division, this work should contribute towards a fundamental understanding of the molecular basis of efficient plant

  19. Prophage induction and cell division in E. coli. Pt. 3

    International Nuclear Information System (INIS)

    George, J.; Castellazzi, M.; Buttin, G.

    1975-01-01

    In E. coli K12, cell filamentation promoted by tif is enhanced by the lon mutation; in contrast, prophage induction and repair of UV-irradiated phage lambda, also promoted by tif, are not affected by lon. From a tif lon double mutant, 'revertants' having recovered the ability to divide at 41 0 were isolated, among which most (95%) had also lost heir Lon filamentous phenotype after ultraviolet (UV) irradiation. From these 95% of revertants 94% are suppressed for the whole Tif phenotype, by additional mutations that render them deficient in DNA repair, as judged from their high UV sensitivity; some have been characterized as recA mutants. 1% have recovered a control on cell division at 41% or after UV irradiation by means of secondary mutations altering neither the other phenotypic properties of tif and lon, nor the repair and recombination ability of the cells: in particular, this class of 'revertants' remains thermoinducible upon lysogenisation; the mutations which specifically supress filamentation have been mapped at two loci, sfiA and sfiB, cotransducible respectively with pyrD and leu. In the remaining 5% of revertants that still exhibit an UV-induced filamentous growth, 3% can be tentatively classified as true tif + revertants; 2% behave as tif thermodependent revertants, showing suppression of Tif (and Lon) phenotype only at 41 0 : 2 recAts have been identified in this class. Non-lysogenic tif lon sfi and tif sfi strains remain viable during prolonged growth at 41 0 . Under these conditions, tif expresses mutator properties, which can be conveniently analyzed in this sfi background. The action of tif, lon and sfi mutations is tentatively interpreted on the basis of a negative control of cell division specifically associated with DNA repair. (orig.) [de

  20. Translational Control of Cell Division by Elongator

    Directory of Open Access Journals (Sweden)

    Fanelie Bauer

    2012-05-01

    Full Text Available Elongator is required for the synthesis of the mcm5s2 modification found on tRNAs recognizing AA-ending codons. In order to obtain a global picture of the role of Elongator in translation, we used reverse protein arrays to screen the fission yeast proteome for translation defects. Unexpectedly, this revealed that Elongator inactivation mainly affected three specific functional groups including proteins implicated in cell division. The absence of Elongator results in a delay in mitosis onset and cytokinesis defects. We demonstrate that the kinase Cdr2, which is a central regulator of mitosis and cytokinesis, is under translational control by Elongator due to the Lysine codon usage bias of the cdr2 coding sequence. These findings uncover a mechanism by which the codon usage, coupled to tRNA modifications, fundamentally contributes to gene expression and cellular functions.

  1. Quantitative regulation of B cell division destiny by signal strength.

    Science.gov (United States)

    Turner, Marian L; Hawkins, Edwin D; Hodgkin, Philip D

    2008-07-01

    Differentiation to Ab secreting and isotype-switched effector cells is tightly linked to cell division and therefore the degree of proliferation strongly influences the nature of the immune response. The maximum number of divisions reached, termed the population division destiny, is stochastically distributed in the population and is an important parameter in the quantitative outcome of lymphocyte responses. In this study, we further assessed the variables that regulate B cell division destiny in vitro in response to T cell- and TLR-dependent stimuli. Both the concentration and duration of stimulation were able to regulate the average maximum number of divisions undergone for each stimulus. Notably, a maximum division destiny was reached during provision of repeated saturating stimulation, revealing that an intrinsic limit to proliferation exists even under these conditions. This limit was linked directly to division number rather than time of exposure to stimulation and operated independently of the survival regulation of the cells. These results demonstrate that a B cell population's division destiny is regulable by the stimulatory conditions up to an inherent maximum value. Division destiny is a crucial parameter in regulating the extent of B cell responses and thereby also the nature of the immune response mounted.

  2. The role of genes controlling the replication and cell division in the repair of radiation damage in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Zhestyanikov, V D; Svetlova, M P; Tomilin, N V; Savel' eva, G E [AN SSSR, Leningrad. Inst. Tsitologii

    1975-01-01

    Mutations in genes controlling the replication (dnaEsup(ts), dnaBsup(ts), dnaGsup(ts) and cell division (lon) in Escherichia coli prevent the rejoining of the gamma radiation-induced single-strand breaks (dnaE in combination with polA1 mutation and dnaG at the restrictive temperature) and effective postreplication DNA repair in UV-irradiated cells (dnaG at the non-permissive temperature and lon mutation) and decrease the survival of UV- and gamma-irradiated bacteria.

  3. Are There Really Animals Like That? No Cell Division.

    Science.gov (United States)

    Blackwelder, R. E.; Garoian, G. S.

    1984-01-01

    Provides examples of animals in which growth occurs without cell division. Indicates that this phenomenon (called cell constancy or eutely) is an oddity of development that has arisen independently in several animal groups. (JN)

  4. Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme.

    Directory of Open Access Journals (Sweden)

    Anurag Kumar Sinha

    2017-10-01

    Full Text Available Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant.

  5. Modelling cell division and endoreduplication in tomato fruit pericarp

    NARCIS (Netherlands)

    Apri, M.; Kromdijk, J.; Visser, de P.H.B.; Gee, de M.; Molenaar, J.

    2014-01-01

    In many developing plant tissues and organs, differentiating cells switch from the classical cell cycle to an alternative partial cycle. This partial cycle bypasses mitosis and allows for multiple rounds of genome duplication without cell division, giving rise to cells with high ploidy numbers. This

  6. Cellular Clocks : Coupled Circadian Dispatch and Cell Division Cycles

    NARCIS (Netherlands)

    Merrow, Martha; Roenneberg, Till

    2004-01-01

    Gating of cell division by the circadian clock is well known, yet its mechanism is little understood. Genetically tractable model systems have led to new hypotheses and questions concerning the coupling of these two cellular cycles.

  7. Asymmetric cell division and its role in cell fate determination in the ...

    Indian Academy of Sciences (India)

    Supplementary figure 1. Light micrograph of an asymmetrically dividing T. indica cell at various time intervals. Progress over a 12 hr period, showing that the larger component does not undergo further division. (A) 0 h, cell division at an early stage. (B) 5 h, lower half of cell undergoing further division. (C) 12 h, differentiated ...

  8. Z ring as executor of bacterial cell division.

    Science.gov (United States)

    Dajkovic, Alex; Lutkenhaus, Joe

    2006-01-01

    It has become apparent that bacteria possess ancestors of the major eukaryotic cytoskeletal proteins. FtsZ, the ancestral homologue of tubulin, assembles into a cytoskeletal structure associated with cell division, designated the Z ring. Formation of the Z ring represents a major point of both spatial and temporal regulation of cell division. Here we discuss findings concerning the structure and the formation of the ring as well as its spatial and temporal regulation.

  9. Symmetric vs. asymmetric stem cell divisions: an adaptation against cancer?

    Directory of Open Access Journals (Sweden)

    Leili Shahriyari

    Full Text Available Traditionally, it has been held that a central characteristic of stem cells is their ability to divide asymmetrically. Recent advances in inducible genetic labeling provided ample evidence that symmetric stem cell divisions play an important role in adult mammalian homeostasis. It is well understood that the two types of cell divisions differ in terms of the stem cells' flexibility to expand when needed. On the contrary, the implications of symmetric and asymmetric divisions for mutation accumulation are still poorly understood. In this paper we study a stochastic model of a renewing tissue, and address the optimization problem of tissue architecture in the context of mutant production. Specifically, we study the process of tumor suppressor gene inactivation which usually takes place as a consequence of two "hits", and which is one of the most common patterns in carcinogenesis. We compare and contrast symmetric and asymmetric (and mixed stem cell divisions, and focus on the rate at which double-hit mutants are generated. It turns out that symmetrically-dividing cells generate such mutants at a rate which is significantly lower than that of asymmetrically-dividing cells. This result holds whether single-hit (intermediate mutants are disadvantageous, neutral, or advantageous. It is also independent on whether the carcinogenic double-hit mutants are produced only among the stem cells or also among more specialized cells. We argue that symmetric stem cell divisions in mammals could be an adaptation which helps delay the onset of cancers. We further investigate the question of the optimal fraction of stem cells in the tissue, and quantify the contribution of non-stem cells in mutant production. Our work provides a hypothesis to explain the observation that in mammalian cells, symmetric patterns of stem cell division seem to be very common.

  10. Transmission of persistent ionizing radiation-induced foci through cell division in human primary cells

    Energy Technology Data Exchange (ETDEWEB)

    Vaurijoux, Aurelie, E-mail: aurelie.vaurijoux@irsn.fr [Institut de Radioprotection et de Sureté Nucléaire (IRSN), Laboratoire de Dosimétrie Biologique, BP 17, 92262 Fontenay aux roses cedex (France); Voisin, Pascale; Freneau, Amelie [Institut de Radioprotection et de Sureté Nucléaire (IRSN), Laboratoire de Dosimétrie Biologique, BP 17, 92262 Fontenay aux roses cedex (France); Barquinero, Joan Francesc [Universitat Autònoma de Barcelona, Faculty of Biosciences, 08193 Cerdanyola del Vallès (Spain); Gruel, Gaetan [Institut de Radioprotection et de Sureté Nucléaire (IRSN), Laboratoire de Dosimétrie Biologique, BP 17, 92262 Fontenay aux roses cedex (France)

    2017-03-15

    Highlights: • Persistent IRIF do not permanently block cell proliferation. • Persistent IRIF are transmitted in part and sometimes asymmetrically to daughter cells. • IRIF differ in their nature before and after the first cell division. - Abstract: Unrepaired DNA double-strand breaks (DSBs) induced by ionizing radiation are associated with lethal effects and genomic instability. After the initial breaks and chromatin destabilization, a set of post-translational modifications of histones occurs, including phosphorylation of serine 139 of histone H2AX (γH2AX), which leads to the formation of ionizing radiation-induced foci (IRIF). DSB repair results in the disappearance of most IRIF within hours after exposure, although some remain 24 h after irradiation. Their relation to unrepaired DSBs is generally accepted but still controversial. This study evaluates the frequency and kinetics of persistent IRIF and analyzes their impact on cell proliferation. We observed persistent IRIF up to 7 days postirradiation, and more than 70% of cells exposed to 5 Gy had at least one of these persistent IRIF 24 h after exposure. Moreover we demonstrated that persistent IRIF did not block cell proliferation definitively. The frequency of IRIF was lower in daughter cells, due to asymmetric distribution of IRIF between some of them. We report a positive association between the presence of IRIF and the likelihood of DNA missegregation. Hence, the structure formed after the passage of a persistent IRI focus across the S and G2 phases may impede the correct segregation of the affected chromosome's sister chromatids. The ensuing abnormal resolution of anaphase might therefore cause the nature of IRIF in daughter-cell nuclei to differ before and after the first cell division. The resulting atypical chromosomal assembly may be lethal or result in a gene dosage imbalance and possibly enhanced genomic instability, in particular in the daughter cells.

  11. Transmission of persistent ionizing radiation-induced foci through cell division in human primary cells

    International Nuclear Information System (INIS)

    Vaurijoux, Aurelie; Voisin, Pascale; Freneau, Amelie; Barquinero, Joan Francesc; Gruel, Gaetan

    2017-01-01

    Highlights: • Persistent IRIF do not permanently block cell proliferation. • Persistent IRIF are transmitted in part and sometimes asymmetrically to daughter cells. • IRIF differ in their nature before and after the first cell division. - Abstract: Unrepaired DNA double-strand breaks (DSBs) induced by ionizing radiation are associated with lethal effects and genomic instability. After the initial breaks and chromatin destabilization, a set of post-translational modifications of histones occurs, including phosphorylation of serine 139 of histone H2AX (γH2AX), which leads to the formation of ionizing radiation-induced foci (IRIF). DSB repair results in the disappearance of most IRIF within hours after exposure, although some remain 24 h after irradiation. Their relation to unrepaired DSBs is generally accepted but still controversial. This study evaluates the frequency and kinetics of persistent IRIF and analyzes their impact on cell proliferation. We observed persistent IRIF up to 7 days postirradiation, and more than 70% of cells exposed to 5 Gy had at least one of these persistent IRIF 24 h after exposure. Moreover we demonstrated that persistent IRIF did not block cell proliferation definitively. The frequency of IRIF was lower in daughter cells, due to asymmetric distribution of IRIF between some of them. We report a positive association between the presence of IRIF and the likelihood of DNA missegregation. Hence, the structure formed after the passage of a persistent IRI focus across the S and G2 phases may impede the correct segregation of the affected chromosome's sister chromatids. The ensuing abnormal resolution of anaphase might therefore cause the nature of IRIF in daughter-cell nuclei to differ before and after the first cell division. The resulting atypical chromosomal assembly may be lethal or result in a gene dosage imbalance and possibly enhanced genomic instability, in particular in the daughter cells.

  12. Analytical model for macromolecular partitioning during yeast cell division

    International Nuclear Information System (INIS)

    Kinkhabwala, Ali; Khmelinskii, Anton; Knop, Michael

    2014-01-01

    Asymmetric cell division, whereby a parent cell generates two sibling cells with unequal content and thereby distinct fates, is central to cell differentiation, organism development and ageing. Unequal partitioning of the macromolecular content of the parent cell — which includes proteins, DNA, RNA, large proteinaceous assemblies and organelles — can be achieved by both passive (e.g. diffusion, localized retention sites) and active (e.g. motor-driven transport) processes operating in the presence of external polarity cues, internal asymmetries, spontaneous symmetry breaking, or stochastic effects. However, the quantitative contribution of different processes to the partitioning of macromolecular content is difficult to evaluate. Here we developed an analytical model that allows rapid quantitative assessment of partitioning as a function of various parameters in the budding yeast Saccharomyces cerevisiae. This model exposes quantitative degeneracies among the physical parameters that govern macromolecular partitioning, and reveals regions of the solution space where diffusion is sufficient to drive asymmetric partitioning and regions where asymmetric partitioning can only be achieved through additional processes such as motor-driven transport. Application of the model to different macromolecular assemblies suggests that partitioning of protein aggregates and episomes, but not prions, is diffusion-limited in yeast, consistent with previous reports. In contrast to computationally intensive stochastic simulations of particular scenarios, our analytical model provides an efficient and comprehensive overview of partitioning as a function of global and macromolecule-specific parameters. Identification of quantitative degeneracies among these parameters highlights the importance of their careful measurement for a given macromolecular species in order to understand the dominant processes responsible for its observed partitioning

  13. Stationary Size Distributions of Growing Cells with Binary and Multiple Cell Division

    Science.gov (United States)

    Rading, M. M.; Engel, T. A.; Lipowsky, R.; Valleriani, A.

    2011-10-01

    Populations of unicellular organisms that grow under constant environmental conditions are considered theoretically. The size distribution of these cells is calculated analytically, both for the usual process of binary division, in which one mother cell produces always two daughter cells, and for the more complex process of multiple division, in which one mother cell can produce 2 n daughter cells with n=1,2,3,… . The latter mode of division is inspired by the unicellular algae Chlamydomonas reinhardtii. The uniform response of the whole population to different environmental conditions is encoded in the individual rates of growth and division of the cells. The analytical treatment of the problem is based on size-dependent rules for cell growth and stochastic transition processes for cell division. The comparison between binary and multiple division shows that these different division processes lead to qualitatively different results for the size distribution and the population growth rates.

  14. Cell-Division Behavior in a Heterogeneous Swarm Environment.

    Science.gov (United States)

    Erskine, Adam; Herrmann, J Michael

    2015-01-01

    We present a system of virtual particles that interact using simple kinetic rules. It is known that heterogeneous mixtures of particles can produce particularly interesting behaviors. Here we present a two-species three-dimensional swarm in which a behavior emerges that resembles cell division. We show that the dividing behavior exists across a narrow but finite band of parameters and for a wide range of population sizes. When executed in a two-dimensional environment the swarm's characteristics and dynamism manifest differently. In further experiments we show that repeated divisions can occur if the system is extended by a biased equilibrium process to control the split of populations. We propose that this repeated division behavior provides a simple model for cell-division mechanisms and is of interest for the formation of morphological structure and to swarm robotics.

  15. A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells

    Science.gov (United States)

    Shimizu, Takashi; Eguchi, Kentaro; Nishida, Ikuo; Laukens, Kris; Witters, Erwin; van Onckelen, Harry; Nagata, Toshiyuki

    2006-06-01

    Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.

  16. Microtubule networks for plant cell division

    NARCIS (Netherlands)

    Keijzer, de Jeroen; Mulder, B.M.; Janson, M.E.

    2014-01-01

    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

  17. Novel Coiled-Coil Cell Division Factor ZapB Stimulates Z Ring Assembly and Cell Division

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Galli, Elizabeth; Møller-Jensen, Jakob

    2008-01-01

    Formation of the Z ring is the first known event in bacterial cell division. However, it is not yet known how the assembly and contraction of the Z ring is regulated. Here, we identify a novel cell division factor ZapB in Escherichia coli that simultaneously stimulates Z ring assembly and cell...... division. Deletion of zapB resulted in delayed cell division and the formation of ectopic Z rings and spirals whereas overexpression of ZapB resulted in nucleoid condensation and aberrant cell divisions. Localization of ZapB to the divisome depended on FtsZ but not FtsA, ZipA or FtsI and ZapB interacted...... with FtsZ in a bacterial two-hybrid analysis. The simultaneous inactivation of FtsA and ZipA prevented Z ring assembly and ZapB localization. Time lapse microscopy showed that ZapB-GFP is present at mid-cell in a pattern very similar to that of FtsZ. Cells carrying a zapB deletion and the ftsZ84ts allele...

  18. INO80 Chromatin Remodeling Coordinates Metabolic Homeostasis with Cell Division

    Directory of Open Access Journals (Sweden)

    Graeme J. Gowans

    2018-01-01

    Full Text Available Adaptive survival requires the coordination of nutrient availability with expenditure of cellular resources. For example, in nutrient-limited environments, 50% of all S. cerevisiae genes synchronize and exhibit periodic bursts of expression in coordination with respiration and cell division in the yeast metabolic cycle (YMC. Despite the importance of metabolic and proliferative synchrony, the majority of YMC regulators are currently unknown. Here, we demonstrate that the INO80 chromatin-remodeling complex is required to coordinate respiration and cell division with periodic gene expression. Specifically, INO80 mutants have severe defects in oxygen consumption and promiscuous cell division that is no longer coupled with metabolic status. In mutant cells, chromatin accessibility of periodic genes, including TORC1-responsive genes, is relatively static, concomitant with severely attenuated gene expression. Collectively, these results reveal that the INO80 complex mediates metabolic signaling to chromatin to restrict proliferation to metabolically optimal states.

  19. Primitive human hematopoietic cells give rise to differentially specified daughter cells upon their initial cell division.

    NARCIS (Netherlands)

    Giebel, B.; Zhang, T.; Beckmann, J.; Spanholtz, J.; Wernet, P.; Ho, A.; Punzel, M.

    2006-01-01

    It is often predicted that stem cells divide asymmetrically, creating a daughter cell that maintains the stem-cell capacity, and 1 daughter cell committed to differentiation. While asymmetric stem-cell divisions have been proven to occur in model organisms (eg, in Drosophila), it remains illusive

  20. Division of Labor in Biofilms: the Ecology of Cell Differentiation.

    Science.gov (United States)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    2015-04-01

    The dense aggregation of cells on a surface, as seen in biofilms, inevitably results in both environmental and cellular heterogeneity. For example, nutrient gradients can trigger cells to differentiate into various phenotypic states. Not only do cells adapt physiologically to the local environmental conditions, but they also differentiate into cell types that interact with each other. This allows for task differentiation and, hence, the division of labor. In this article, we focus on cell differentiation and the division of labor in three bacterial species: Myxococcus xanthus, Bacillus subtilis, and Pseudomonas aeruginosa. During biofilm formation each of these species differentiates into distinct cell types, in some cases leading to cooperative interactions. The division of labor and the cooperative interactions between cell types are assumed to yield an emergent ecological benefit. Yet in most cases the ecological benefits have yet to be elucidated. A notable exception is M. xanthus, in which cell differentiation within fruiting bodies facilitates the dispersal of spores. We argue that the ecological benefits of the division of labor might best be understood when we consider the dynamic nature of both biofilm formation and degradation.

  1. Asymmetric cell division requires specific mechanisms for adjusting global transcription.

    Science.gov (United States)

    Mena, Adriana; Medina, Daniel A; García-Martínez, José; Begley, Victoria; Singh, Abhyudai; Chávez, Sebastián; Muñoz-Centeno, Mari C; Pérez-Ortín, José E

    2017-12-01

    Most cells divide symmetrically into two approximately identical cells. There are many examples, however, of asymmetric cell division that can generate sibling cell size differences. Whereas physical asymmetric division mechanisms and cell fate consequences have been investigated, the specific problem caused by asymmetric division at the transcription level has not yet been addressed. In symmetrically dividing cells the nascent transcription rate increases in parallel to cell volume to compensate it by keeping the actual mRNA synthesis rate constant. This cannot apply to the yeast Saccharomyces cerevisiae, where this mechanism would provoke a never-ending increasing mRNA synthesis rate in smaller daughter cells. We show here that, contrarily to other eukaryotes with symmetric division, budding yeast keeps the nascent transcription rates of its RNA polymerases constant and increases mRNA stability. This control on RNA pol II-dependent transcription rate is obtained by controlling the cellular concentration of this enzyme. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. (1) The Relationship of Protein Expression and Cell Division, (2) 3D Imaging of Cells Using Digital Holography, and (3) General Chemistry Enrollment at University of Michigan

    Science.gov (United States)

    Matz, Rebecca L.

    2012-01-01

    Chapter 1: The role of cell division in protein expression is important to understand in order to guide the development of better nonviral gene delivery materials that can transport DNA to the nucleus with high efficiency for a variety of cell types, particularly when nondividing cells are targets of gene therapy. We evaluated the relationship…

  3. Modeling of Complex Life Cycle Prediction Based on Cell Division

    Directory of Open Access Journals (Sweden)

    Fucheng Zhang

    2017-01-01

    Full Text Available Effective fault diagnosis and reasonable life expectancy are of great significance and practical engineering value for the safety, reliability, and maintenance cost of equipment and working environment. At present, the life prediction methods of the equipment are equipment life prediction based on condition monitoring, combined forecasting model, and driven data. Most of them need to be based on a large amount of data to achieve the problem. For this issue, we propose learning from the mechanism of cell division in the organism. We have established a moderate complexity of life prediction model across studying the complex multifactor correlation life model. In this paper, we model the life prediction of cell division. Experiments show that our model can effectively simulate the state of cell division. Through the model of reference, we will use it for the equipment of the complex life prediction.

  4. Determination of cell division axes in the early embryogenesis of Caenorhabditis elegans

    OpenAIRE

    1987-01-01

    The establishment of cell division axes was examined in the early embryonic divisions of Caenorhabditis elegans. It has been shown previously that there are two different patterns of cleavage during early embryogenesis. In one set of cells, which undergo predominantly determinative divisions, the division axes are established successively in the same orientation, while division axes in the other set, which divide mainly proliferatively, have an orthogonal pattern of division. We have investig...

  5. Inhibition of cell division in hupA hupB mutant bacteria lacking HU protein.

    Science.gov (United States)

    Dri, A M; Rouviere-Yaniv, J; Moreau, P L

    1991-01-01

    Escherichia coli hupA hypB double mutants that lack HU protein have severe cellular defects in cell division, DNA folding, and DNA partitioning. Here we show that the sfiA11 mutation, which alters the SfiA cell division inhibitor, reduces filamentation and production of anucleate cells in AB1157 hupA hupB strains. However, lexA3(Ind-) and sfiB(ftsZ)114 mutations, which normally counteract the effect of the SfiA inhibitor, could not restore a normal morphology to hupA hupB mutant bacteria. The LexA repressor, which controls the expression of the sfiA gene, was present in hupA hupB mutant bacteria in concentrations half of those of the parent bacteria, but this decrease was independent of the specific cleavage of the LexA repressor by activated RecA protein. One possibility to account for the filamentous morphology of hupA hupB mutant bacteria is that the lack of HU protein alters the expression of specific genes, such as lexA and fts cell division genes. Images PMID:2019558

  6. DNA adducts in senescent cells

    International Nuclear Information System (INIS)

    Gaubatz, J.W.

    1987-01-01

    Perturbations in DNA repair and other metabolic processes during development and aging might affect the steady-state level of genomic damage. The persistence or accumulation of DNA lesions in postmitotic cells could have a significant impact on proper cellular function, interfering with gene regulation for example. To test the notion that DNA damage increases as a function of age in non-dividing cells, DNA was purified from heart tissue of C57BL/6Nia mice at different ages and analyzed by post labeling techniques to detect DNA adducts. In the present experiments, four-dimensional, thin-layer chromatography was used to isolate aromatic adducts that were labeled with carrier-free (γ- 32 P) ATP under DNA-P excess conditions. The complexity and frequency of aromatic adducts varied between DNA samples. Several adducts were present in all preparations and were clearly more abundant in nucleotide maps of mature and old heart DNA. However, a direct correlation with age was not observed. In contrast, experiments in which aromatic adducts were first isolated by phase-transfer to 1-butanol, then labeled with excess (γ- 32 P)ATP indicated that there was an age-related increase in these adducts. The results are consistent with their earlier studies that showed alkyl adducts increased during aging of mouse myocardium and suggest that a common repair pathway might be involved

  7. A crucial step in cell division identified | Center for Cancer Research

    Science.gov (United States)

    When cell division doesn’t go according to plan, the resulting daughter cells can become unstable or even cancerous. A team of CCR investigators has now discovered a crucial step required for normal cell division to occur. Read more...

  8. LexA Binds to Transcription Regulatory Site of Cell Division Gene ftsZ in Toxic Cyanobacterium Microcystis aeruginosa.

    Science.gov (United States)

    Honda, Takashi; Morimoto, Daichi; Sako, Yoshihiko; Yoshida, Takashi

    2018-05-17

    Previously, we showed that DNA replication and cell division in toxic cyanobacterium Microcystis aeruginosa are coordinated by transcriptional regulation of cell division gene ftsZ and that an unknown protein specifically bound upstream of ftsZ (BpFz; DNA-binding protein to an upstream site of ftsZ) during successful DNA replication and cell division. Here, we purified BpFz from M. aeruginosa strain NIES-298 using DNA-affinity chromatography and gel-slicing combined with gel electrophoresis mobility shift assay (EMSA). The N-terminal amino acid sequence of BpFz was identified as TNLESLTQ, which was identical to that of transcription repressor LexA from NIES-843. EMSA analysis using mutant probes showed that the sequence GTACTAN 3 GTGTTC was important in LexA binding. Comparison of the upstream regions of lexA in the genomes of closely related cyanobacteria suggested that the sequence TASTRNNNNTGTWC could be a putative LexA recognition sequence (LexA box). Searches for TASTRNNNNTGTWC as a transcriptional regulatory site (TRS) in the genome of M. aeruginosa NIES-843 showed that it was present in genes involved in cell division, photosynthesis, and extracellular polysaccharide biosynthesis. Considering that BpFz binds to the TRS of ftsZ during normal cell division, LexA may function as a transcriptional activator of genes related to cell reproduction in M. aeruginosa, including ftsZ. This may be an example of informality in the control of bacterial cell division.

  9. Control of sporulation-specific cell division in Streptomyces coelicolor

    NARCIS (Netherlands)

    Noens, Elke

    2007-01-01

    During developmental cell division in sporulation-committed aerial hyphae of streptomycetes, up to a hundred septa are simultaneously produced, in close harmony with synchromous chromosome condensation and segregation. Several unique protein families are involved in the control of this process,

  10. Bacterial Cell Wall Growth, Shape and Division

    NARCIS (Netherlands)

    Derouaux, A.; Terrak, M.; den Blaauwen, T.; Vollmer, W.; Remaut, H.; Fronzes, R.

    2014-01-01

    The shape of a bacterial cell is maintained by its peptidoglycan sacculus that completely surrounds the cytoplasmic membrane. During growth the sacculus is enlarged by peptidoglycan synthesis complexes that are controlled by components linked to the cytoskeleton and, in Gram-negative bacteria, by

  11. Spatial pattern of cell geometry and cell-division orientation in zebrafish lens epithelium

    Directory of Open Access Journals (Sweden)

    Toshiaki Mochizuki

    2014-09-01

    Full Text Available Cell proliferation is a key regulator of tissue morphogenesis. We examined cell proliferation and cell division in zebrafish lens epithelium by visualizing cell-cycle phases and nuclear positions, using fluorescent-labeled geminin and histone proteins. Proliferation was low in the anterior region of lens epithelium and higher in the marginal zone anterior to the equator, suggesting that the proliferation zone, called the germinative zone, is formed in zebrafish lens. Interestingly, cell-division orientation was biased longitudinally in the anterior region, shifted from longitudinal to circumferential along the anterior–posterior axis of lens sphere, and was biased circumferentially in the peripheral region. These data suggest that cell-division orientation is spatially regulated in zebrafish lens epithelium. The Hertwig rule indicates that cells tend to divide along their long axes. Orientation of long axes and cell division were biased similarly in zebrafish lens epithelium, suggesting that cell geometry correlates with cell-division orientation. A cell adhesion molecule, E-cadherin, is expressed in lens epithelium. In a zebrafish e-cadherin mutant, the long axes and cell-division orientation were shifted more longitudinally. These data suggest that E-cadherin is required for the spatial pattern of cell geometry and cell-division orientation in zebrafish lens epithelium.

  12. Dielectric modelling of cell division for budding and fission yeast

    International Nuclear Information System (INIS)

    Asami, Koji; Sekine, Katsuhisa

    2007-01-01

    The frequency dependence of complex permittivity or the dielectric spectrum of a system including a cell in cell division has been simulated by a numerical technique based on the three-dimensional finite difference method. Two different types of cell division characteristic of budding and fission yeast were examined. The yeast cells are both regarded as a body of rotation, and thus have anisotropic polarization, i.e. the effective permittivity of the cell depends on the orientation of the cell to the direction of an applied electric field. In the perpendicular orientation, where the rotational axis of the cell is perpendicular to the electric field direction, the dielectric spectra for both yeast cells included one dielectric relaxation and its intensity depended on the cell volume. In the parallel orientation, on the other hand, two dielectric relaxations appeared with bud growth for budding yeast and with septum formation for fission yeast. The low-frequency relaxation was shifted to a lower frequency region by narrowing the neck between the bud and the mother cell for budding yeast and by increasing the degree of septum formation for fission yeast. After cell separation, the low-frequency relaxation disappeared. The simulations well interpreted the oscillation of the relative permittivity of culture broth found for synchronous cell growth of budding yeast

  13. Asymmetric cell division of stem cells in the lung and other systems

    Directory of Open Access Journals (Sweden)

    Mohamed eBerika

    2014-07-01

    Full Text Available New insights have been added to identification, behavior and cellular properties of embryonic and tissue-specific stem cells over the last few years. The modes of stem cell division, asymmetric versus symmetric, are tightly regulated during development and regeneration. The proper choice of a stem cell to divide asymmetrically or symmetrically has great consequences for development and disease because inappropriate asymmetric division disrupts organ morphogenesis, whereas uncontrolled symmetric division induces tumorigenesis. Therefore, understanding the behavior of lung stem cells could identify innovative solutions for restoring normal morphogenesis and/or regeneration of different organs. In this concise review, we describe recent studies in our laboratory about the mode of division of lung epithelial stem cells. We also compare asymmetric cell division in the lung stem cells with other tissues in different organisms.

  14. Cell division orientation is coupled to cell-cell adhesion by the E-cadherin/LGN complex

    NARCIS (Netherlands)

    Gloerich, Martijn; Bianchini, Julie M.; Siemers, Kathleen A.; Cohen, Daniel J.; Nelson, W. James

    2017-01-01

    Both cell-cell adhesion and oriented cell division play prominent roles in establishing tissue architecture, but it is unclear how they might be coordinated. Here, we demonstrate that the cell-cell adhesion protein E-cadherin functions as an instructive cue for cell division orientation. This is

  15. DNA to DNA transcription might exist in eukaryotic cells

    OpenAIRE

    Li, Gao-De

    2016-01-01

    Till now, in biological sciences, the term, transcription, mainly refers to DNA to RNA transcription. But our recently published experimental findings obtained from Plasmodium falciparum strongly suggest the existence of DNA to DNA transcription in the genome of eukaryotic cells, which could shed some light on the functions of certain noncoding DNA in the human and other eukaryotic genomes.

  16. Abnormal number cell division of human thyroid anaplastic carcinoma cell line, SW 1736

    Directory of Open Access Journals (Sweden)

    Keiichi Ikeda

    2015-12-01

    Full Text Available Cell division, during which a mother cell usually divides into two daughter cells during one cell cycle, is the most important physiological event of cell biology. We observed one-to-four cell division during imaging of live SW1736 human thyroid anaplastic carcinoma cells transfected with a plasmid expressing the hybrid protein of green fluorescent protein and histone 2B (plasmid eGFP-H2B. Analysis of the images revealed a mother cell divided into four daughter cells. And one of the abnormally divided daughter cells subsequently formed a dinucleate cell.

  17. Formation of a cylindrical bridge in cell division

    Science.gov (United States)

    Citron, Daniel; Schmidt, Laura E.; Reichl, Elizabeth; Ren, Yixin; Robinson, Douglas; Zhang, Wendy W.

    2007-11-01

    In nature, the shape transition associated with the division of a mother cell into two daughter cells proceeds via a variety of routes. In the cylinder-thinning route, which has been observed in Dictyostelium and most animal cells, the mother cell first forms a broad bridge-like region, also known as a furrow, between two daughter cells. The furrow then rapidly evolves into a cylindrical bridge, which thins and eventually severs the mother cell into two. The fundamental mechanism underlying this division route is not understood. Recent experiments on Dictyostelium found that, while the cylinder-thinning route persists even when key actin cross-linking proteins are missing, it is disrupted by the removal of force-generating myosin-II proteins. Other measurements revealed that mutant cells lacking myosin-II have a much more uniform tension over the cell surface than wild-type cells. This suggests that tension variation may be important. Here we use a fluid model, previously shown to reproduce the thinning dynamics [Zhang & Robinson, PNAS 102, 7186 (2005)], to test this idea. Consistent with the experiments, the model shows that the cylinder formation process occurs regardless of the exact viscoelastic properties of the cell. In contrast to the experiments, a tension variation in the model hinders, rather then expedites, the cylinder formation.

  18. An Improved Model of Nonuniform Coleochaete Cell Division.

    Science.gov (United States)

    Wang, Yuandi; Cong, Jinyu

    2016-08-01

    Cell division is a key biological process in which cells divide forming new daughter cells. In the present study, we investigate continuously how a Coleochaete cell divides by introducing a modified differential equation model in parametric equation form. We discuss both the influence of "dead" cells and the effects of various end-points on the formation of the new cells' boundaries. We find that the boundary condition on the free end-point is different from that on the fixed end-point; the former has a direction perpendicular to the surface. It is also shown that the outer boundaries of new cells are arc-shaped. The numerical experiments and theoretical analyses for this model to construct the outer boundary are given.

  19. Cell division control by the Chromosomal Passenger Complex

    Energy Technology Data Exchange (ETDEWEB)

    Waal, Maike S. van der; Hengeveld, Rutger C.C.; Horst, Armando van der; Lens, Susanne M.A., E-mail: s.m.a.lens@umcutrecht.nl

    2012-07-15

    The Chromosomal Passenger Complex (CPC) consisting of Aurora B kinase, INCENP, Survivin and Borealin, is essential for genomic stability by controlling multiple processes during both nuclear and cytoplasmic division. In mitosis it ensures accurate segregation of the duplicated chromosomes by regulating the mitotic checkpoint, destabilizing incorrectly attached spindle microtubules and by promoting the axial shortening of chromosomal arms in anaphase. During cytokinesis the CPC most likely prevents chromosome damage by imposing an abscission delay when a chromosome bridge connects the two daughter cells. Moreover, by controlling proper cytoplasmic division, the CPC averts tetraploidization. This review describes recent insights on how the CPC is capable of conducting its various functions in the dividing cell to ensure chromosomal stability.

  20. Asymmetries in Cell Division, Cell Size, and Furrowing in the Xenopus laevis Embryo.

    Science.gov (United States)

    Tassan, Jean-Pierre; Wühr, Martin; Hatte, Guillaume; Kubiak, Jacek

    2017-01-01

    Asymmetric cell divisions produce two daughter cells with distinct fate. During embryogenesis, this mechanism is fundamental to build tissues and organs because it generates cell diversity. In adults, it remains crucial to maintain stem cells. The enthusiasm for asymmetric cell division is not only motivated by the beauty of the mechanism and the fundamental questions it raises, but has also very pragmatic reasons. Indeed, misregulation of asymmetric cell divisions is believed to have dramatic consequences potentially leading to pathogenesis such as cancers. In diverse model organisms, asymmetric cell divisions result in two daughter cells, which differ not only by their fate but also in size. This is the case for the early Xenopus laevis embryo, in which the two first embryonic divisions are perpendicular to each other and generate two pairs of blastomeres, which usually differ in size: one pair of blastomeres is smaller than the other. Small blastomeres will produce embryonic dorsal structures, whereas the larger pair will evolve into ventral structures. Here, we present a speculative model on the origin of the asymmetry of this cell division in the Xenopus embryo. We also discuss the apparently coincident asymmetric distribution of cell fate determinants and cell-size asymmetry of the 4-cell stage embryo. Finally, we discuss the asymmetric furrowing during epithelial cell cytokinesis occurring later during Xenopus laevis embryo development.

  1. An archaebacterial homologue of the essential eubacterial cell division protein FtsZ.

    Science.gov (United States)

    Baumann, P; Jackson, S P

    1996-06-25

    Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes,. respectively). Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria. Currently, little is known about the archaeal cell division apparatus. In Bacteria, a crucial component of the cell division machinery is FtsZ, a GTPase that localizes to a ring at the site of septation. Interestingly, FtsZ is distantly related in sequence to eukaryotic tubulins, which also interact with GTP and are components of the eukaryotic cell cytoskeleton. By screening for the ability to bind radiolabeled nucleotides, we have identified a protein of the hyperthermophilic archaeon Pyrococcus woesei that interacts tightly and specifically with GTP. Furthermore, through screening an expression library of P. woesei genomic DNA, we have cloned the gene encoding this protein. Sequence comparisons reveal that the P. woesei GTP-binding protein is strikingly related in sequence to eubacterial FtsZ and is marginally more similar to eukaryotic tubulins than are bacterial FtsZ proteins. Phylogenetic analyses reinforce the notion that there is an evolutionary linkage between FtsZ and tubulins. These findings suggest that the archaeal cell division apparatus may be fundamentally similar to that of Bacteria and lead us to consider the evolutionary relationships between Archaea, Bacteria, and Eucarya.

  2. Correlation between cationic lipid-based transfection and cell division

    Energy Technology Data Exchange (ETDEWEB)

    Kirchenbuechler, Inka; Kirchenbuechler, David; Elbaum, Michael, E-mail: michael@elbaum.ac.il

    2016-07-01

    We evaluate the temporal relation between protein expression by cationic lipid-mediated transfection and cell division using time lapse fluorescence microscopy. Detailed image analysis provides new insights on the single cell level while simultaneously achieving appropriate statistics. Earlier evidence by less direct methods such as flow cytometry indicates a primary route for transfection involving nuclear envelope breakdown, but also suggests the existence of a pathway independent of mitosis. We confirm and quantify both mechanisms. We found the timing for successful transfection to be unexpectedly flexible, contrary to assertions of a narrow time window. Specifically, cells dividing more than 24 h after exposure to the transfection medium express the probed protein at a comparable level to cells in a mitotic state during or shortly after transfection. This finding can have a profound impact on the guidance and development of non-viral gene delivery materials. - Highlights: • Cationic lipid-based transfection supports protein expression without cell division. • Protein expression is unrelated to cell cycle status at the time of transfection. • Time-lapse imaging provides direct evaluation without statistical averaging. • Lipoplex dissociation is a likely target for improvement of transfection efficiency.

  3. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    Science.gov (United States)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  4. Primary radiation damage and disturbance in cell divisions

    International Nuclear Information System (INIS)

    Kim, Jin Kyu; Lee, Yun-Jong; Kim, Jae-Hun; Petin, Vladislav G.; Nili, Mohammad

    2008-01-01

    Survived cells from a homogeneous population exposed to ionizing radiation form various colonies of different sizes and morphology on a solid nutrient medium, which appear at different time intervals after irradiation. Such a phenomenon agrees well with the modern theory of microdosimetry and classical hit-and-target models of radiobiology. According to the hit-principle, individual cells exposed to the same dose of radiation are damaged in different manners. It means that the survived cells can differ in the content of sublethal damage (hits) produced by the energy absorbed into the cell and which is not enough to give rise to effective radiation damage which is responsible for cell killing or inactivation. In diploid yeast cells, the growth rate of cells from 250 colonies of various sizes appeared at different time intervals after irradiation with 600 Gy of gamma radiation from a 60 Co isotopic source was analyzed. The survival rate after irradiation was 20%. Based on the analyses results, it was possible to categorize the clones grown from irradiated cells according to the number of sub-lesions from 1 to 4. The clones with various numbers of sub-lesions were shown to be different in their viability, radiosensitivity, sensitivity to environmental conditions, and the frequency of recombination and respiratory deficient mutations. Cells from unstable clones exhibited an enhanced radiosensitivity, and an increased portion of morphologically changed cells, nonviable cells and respiration mutants, as well. The degree of expression of the foregoing effects was higher if the number of primary sublethal lesions was greater in the originally irradiated cell. Disturbance in cell division can be characterized by cell inactivation or incorrect distribution of mitochondria between daughter cells. Thus, the suggested methodology of identification of cells with a definite number of primary sublethal lesions will promote further elucidation of the nature of primary radiation

  5. Temporal controls of the asymmetric cell division cycle in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    Shenghua Li

    2009-08-01

    Full Text Available The asymmetric cell division cycle of Caulobacter crescentus is orchestrated by an elaborate gene-protein regulatory network, centered on three major control proteins, DnaA, GcrA and CtrA. The regulatory network is cast into a quantitative computational model to investigate in a systematic fashion how these three proteins control the relevant genetic, biochemical and physiological properties of proliferating bacteria. Different controls for both swarmer and stalked cell cycles are represented in the mathematical scheme. The model is validated against observed phenotypes of wild-type cells and relevant mutants, and it predicts the phenotypes of novel mutants and of known mutants under novel experimental conditions. Because the cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, the model we are proposing here may be applicable to other genera of importance to agriculture and medicine (e.g., Rhizobium, Brucella.

  6. Temporal controls of the asymmetric cell division cycle in Caulobacter crescentus.

    Science.gov (United States)

    Li, Shenghua; Brazhnik, Paul; Sobral, Bruno; Tyson, John J

    2009-08-01

    The asymmetric cell division cycle of Caulobacter crescentus is orchestrated by an elaborate gene-protein regulatory network, centered on three major control proteins, DnaA, GcrA and CtrA. The regulatory network is cast into a quantitative computational model to investigate in a systematic fashion how these three proteins control the relevant genetic, biochemical and physiological properties of proliferating bacteria. Different controls for both swarmer and stalked cell cycles are represented in the mathematical scheme. The model is validated against observed phenotypes of wild-type cells and relevant mutants, and it predicts the phenotypes of novel mutants and of known mutants under novel experimental conditions. Because the cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, the model we are proposing here may be applicable to other genera of importance to agriculture and medicine (e.g., Rhizobium, Brucella).

  7. DNA repair , cell repair and radiosensitivity

    International Nuclear Information System (INIS)

    Zhestyanikov, V.D.

    1983-01-01

    Data obtained in laboratory of radiation cytology and literature data testifying to a considerable role of DNA repair in cell sensitivity to radiation and chemical DNA-tropic agents have been considered. Data pointing to the probability of contribution of inducible repair of DNA into plant cells sensitivity to X-rays are obtained. Certain violations of DNA repair do not result in the increase of radiosensitivity. It is assumed that in the cases unknown mechanisms of DNA repair operate

  8. Visualization of DNA double-strand break repair: From molecules to cells

    NARCIS (Netherlands)

    Krawczyk, Przemek M.; Stap, Jan; Aten, Jacob A.

    2008-01-01

    DNA double-strand break (DSB) signaling and repair processes are positioned at the crossroad of nuclear pathways that regulate DNA replication, cell division, senescence and apoptosis. Importantly, errors in DSB repair may lead to lethal or potentially tumorigenic chromosome rearrangements.

  9. Control of the meiotic cell division program in plants

    NARCIS (Netherlands)

    Wijnker, T.G.; Schnittger, A.

    2013-01-01

    While the question of why organisms reproduce sexually is still a matter of controversy, it is clear that the foundation of sexual reproduction is the formation of gametes with half the genomic DNA content of a somatic cell. This reduction in genomic content is accomplished through meiosis that, in

  10. Mechanisms of DNA uptake by cells

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1977-01-01

    Three categories of cellular uptake of DNA can be distinguished. First, in the highly transformable bacteria, such as Diplococcus pneumoniae, Haemophilus influenzae and Bacillus subtilis, elaborate mechanisms of DNA transport have evolved, presumably for the purpose of genetic exchange. These mechanisms can introduce substantial amounts of DNA into the cell. Second, methods have been devised for the forced introduction of DNA by manipulation of bacterial cells under nonphysiological conditions. By such means small but significant amounts of DNA have been introduced into various bacteria, including Escherichia coli. Third, mammalian cells are able to take up biologically active DNA. This has been most clearly demonstrated with viral DNA, although the mechanism of uptake is not well understood. The intention, here, is to survey current understanding of the various mechanisms of DNA uptake. A review of experience with the bacterial systems may throw some light on the mammalian system and lead to suggestions for enhancing DNA uptake by mammalian cells.

  11. Dido3 PHD Modulates Cell Differentiation and Division

    Directory of Open Access Journals (Sweden)

    Jovylyn Gatchalian

    2013-07-01

    Full Text Available Death Inducer Obliterator 3 (Dido3 is implicated in the maintenance of stem cell genomic stability and tumorigenesis. Here, we show that Dido3 regulates the expression of stemness genes in embryonic stem cells through its plant homeodomain (PHD finger. Binding of Dido3 PHD to histone H3K4me3 is disrupted by threonine phosphorylation that triggers Dido3 translocation from chromatin to the mitotic spindle. The crystal structure of Dido3 PHD in complex with H3K4me3 reveals an atypical aromatic-cage-like binding site that contains a histidine residue. Biochemical, structural, and mutational analyses of the binding mechanism identified the determinants of specificity and affinity and explained the inability of homologous PHF3 to bind H3K4me3. Together, our findings reveal a link between the transcriptional control in embryonic development and regulation of cell division.

  12. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

    1982-01-01

    The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

  13. Cell Division, a new open access online forum for and from the cell cycle community

    Directory of Open Access Journals (Sweden)

    Kaldis Philipp

    2006-04-01

    Full Text Available Abstract Cell Division is a new, open access, peer-reviewed online journal that publishes cutting-edge articles, commentaries and reviews on all exciting aspects of cell cycle control in eukaryotes. A major goal of this new journal is to publish timely and significant studies on the aberrations of the cell cycle network that occur in cancer and other diseases.

  14. Colonic stem cell data are consistent with the immortal model of stem cell division under non-random strand segregation.

    Science.gov (United States)

    Walters, K

    2009-06-01

    Colonic stem cells are thought to reside towards the base of crypts of the colon, but their numbers and proliferation mechanisms are not well characterized. A defining property of stem cells is that they are able to divide asymmetrically, but it is not known whether they always divide asymmetrically (immortal model) or whether there are occasional symmetrical divisions (stochastic model). By measuring diversity of methylation patterns in colon crypt samples, a recent study found evidence in favour of the stochastic model, assuming random segregation of stem cell DNA strands during cell division. Here, the effect of preferential segregation of the template strand is considered to be consistent with the 'immortal strand hypothesis', and explore the effect on conclusions of previously published results. For a sample of crypts, it is shown how, under the immortal model, to calculate mean and variance of the number of unique methylation patterns allowing for non-random strand segregation and compare them with those observed. The calculated mean and variance are consistent with an immortal model that incorporates non-random strand segregation for a range of stem cell numbers and levels of preferential strand segregation. Allowing for preferential strand segregation considerably alters previously published conclusions relating to stem cell numbers and turnover mechanisms. Evidence in favour of the stochastic model may not be as strong as previously thought.

  15. Chromosomal DNA replication of Vicia faba cells

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1976-01-01

    The chromosomal DNA replication of higher plant cells has been investigated by DNA fiber autoradiography. The nuclear DNA fibers of Vicia root meristematic cells are organized into many tandem arrays of replication units or replicons which exist as clusters with respect to replication. DNA is replicated bidirectionally from the initiation points at the average rate of 0.15 μm/min at 20 0 C, and the average interinitiation interval is about 16 μm. The manner of chromosomal DNA replication in this higher plant is similar to that found in other eukaryotic cells at a subchromosomal level. (auth.)

  16. Polyploid tumour cells elicit paradiploid progeny through depolyploidizing divisions and regulated autophagic degradation.

    Science.gov (United States)

    Erenpreisa, Jekaterina; Salmina, Kristine; Huna, Anda; Kosmacek, Elizabeth A; Cragg, Mark S; Ianzini, Fiorenza; Anisimov, Alim P

    2011-07-01

    'Neosis' describes the process whereby p53 function-deficient tumour cells undergo self-renewal after genotoxic damage apparently via senescing ETCs (endopolyploid tumour cells). We previously reported that autophagic digestion and extrusion of DNA occurs in ETC and subsequently revealed that self-renewal transcription factors are also activated under these conditions. Here, we further studied this phenomenon in a range of cell lines after genotoxic damage induced by gamma irradiation, ETO (etoposide) or PXT (paclitaxel) treatment. These experiments revealed that chromatin degradation by autophagy was compatible with continuing mitotic activity in ETC. While the actively polyploidizing primary ETC produced early after genotoxic insult activated self-renewal factors throughout the polygenome, the secondary ETC restored after failed multipolar mitosis underwent subnuclei differentiation. As such, only a subset of subnuclei continued to express OCT4 and NANOG, while those lacking these factors stopped DNA replication and underwent degradation and elimination through autophagy. The surviving subnuclei sequestered nascent cytoplasm to form subcells, while being retained within the confines of the old ETC. Finally, the preformed paradiploid subcells became released from their linking chromosome bridges through autophagy and subsequently began cell divisions. These data show that 'neotic' ETC resulting from genotoxically damaged p53 function-deficient tumour cells develop through a heteronuclear system differentiating the polyploid genome into rejuvenated 'viable' subcells (which provide mitotically propagating paradiploid descendents) and subnuclei, which become degraded and eliminated by autophagy. The whole process reduces aneuploidy in descendants of ETC.

  17. DNA repair in human cells

    International Nuclear Information System (INIS)

    Regan, J.D.; Carrier, W.L.; Kusano, I.; Furuno-Fukushi, I.; Dunn, W.C. Jr.; Francis, A.A.; Lee, W.H.

    1982-01-01

    Our primary objective is to elucidate the molecular events in human cells when cellular macromolecules such as DNA are damaged by radiation or chemical agents. We study and characterize (i) the sequence of DNA repair events, (ii) the various modalities of repair, (iii) the genetic inhibition of repair due to mutation, (iv) the physiological inhibition of repair due to mutation, (v) the physiological inhibition of repair due to biochemical inhibitors, and (vi) the genetic basis of repair. Our ultimate goals are to (i) isolate and analyze the repair component of the mutagenic and/or carcinogenic event in human cells, and (ii) elucidate the magnitude and significance of this repair component as it impinges on the practical problems of human irradiation or exposure to actual or potential chemical mutagens and carcinogens. The significance of these studies lies in (i) the ubiquitousness of repair (most organisms, including man, have several complex repair systems), (ii) the belief that mutagenic and carcinogenic events may arise only from residual (nonrepaired) lesions or that error-prone repair systems may be the major induction mechanisms of the mutagenic or carcinogenic event, and (iii) the clear association of repair defects and highly carcinogenic disease states in man [xeroderma pigmentosum (XP)

  18. Single-cell analysis of growth and cell division of the anaerobe Desulfovibrio vulgaris Hildenborough

    Directory of Open Access Journals (Sweden)

    Anouchka eFievet

    2015-12-01

    Full Text Available Recent years have seen significant progress in understanding basic bacterial cell cycle properties such as cell growth and cell division. While characterization and regulation of bacterial cell cycle is quite well documented in the case of fast growing aerobic model organisms, no data has been so far reported for anaerobic bacteria. This lack of information in anaerobic microorganisms can mainly be explained by the absence of molecular and cellular tools such as single cell microscopy and fluorescent probes usable for anaerobes and essential to study cellular events and/or subcellular localization of the actors involved in cell cycle.In this study, single-cell microscopy has been adapted to study for the first time, in real time, the cell cycle of a bacterial anaerobe, Desulfovibrio vulgaris Hildenborough (DvH. This single-cell analysis provides mechanistic insights into the cell division cycle of DvH, which seems to be governed by the recently discussed so-called incremental model that generates remarkably homogeneous cell sizes. Furthermore, cell division was reversibly blocked during oxygen exposure. This may constitute a strategy for anaerobic cells to cope with transient exposure to oxygen that they may encounter in their natural environment, thereby contributing to their aerotolerance. This study lays the foundation for the first molecular, single-cell assay that will address factors that cannot otherwise be resolved in bulk assays and that will allow visualization of a wide range of molecular mechanisms within living anaerobic cells.

  19. From HeLa cell division to infectious diarrhoea

    International Nuclear Information System (INIS)

    Stephen, J.; Osborne, M.P.; Spencer, A.J.; Warley, A.

    1990-01-01

    Hela S3 cells were grown in suspension both randomly and, synchronously using hydroxyurea which blocks cells at the G1/S interface. Cryosections were prepared, freeze-dried and analyzed by X-ray microanalysis. As cells moved into S and through M phases [Na] and [Cl] increased; both returned to normal levels upon re-entering G1 phase. The Na/K ratio was 1:1 in G1 phase. Infection of HeLa S3 cells in G1 phase with vaccinia virus resulted in no change in intracellular [Na]. Infection of neonatal mice with murine rotavirus was localized to villus tip enterocytes and gave rise to diarrhoea which was maximal at 72h post-infection (p.i.). Diarrhoea was preceded by ischemia of villi (18-42h p.i.) and villus shortening (maximal at 42h p.i.), and was also coincident with a dramatic regrowth of villi. At 48h p.i. a proliferative zone of electron lucent cells was observed in villus base regions. Cryosections of infected gut, taken before, during, and after infection, together with corresponding age-matched controls, were freeze-dried and analysed by X-ray microanalysis. At 48h p.i. electron lucent villus base cells were shown to be more hydrated, and, to contain higher levels of both Na and Cl and lower levels of P, S, K and Mg than corresponding control cells. These studies increase confidence in the use of X-ray microanalysis in studying biological systems, provide some insight into the process of cell division, and constitute the basis of a new concept of diarrhoeal secretion.27 references

  20. From HeLa cell division to infectious diarrhoea

    Energy Technology Data Exchange (ETDEWEB)

    Stephen, J.; Osborne, M.P.; Spencer, A.J.; Warley, A. (Univ. of Birmingham (England))

    1990-09-01

    Hela S3 cells were grown in suspension both randomly and, synchronously using hydroxyurea which blocks cells at the G1/S interface. Cryosections were prepared, freeze-dried and analyzed by X-ray microanalysis. As cells moved into S and through M phases (Na) and (Cl) increased; both returned to normal levels upon re-entering G1 phase. The Na/K ratio was 1:1 in G1 phase. Infection of HeLa S3 cells in G1 phase with vaccinia virus resulted in no change in intracellular (Na). Infection of neonatal mice with murine rotavirus was localized to villus tip enterocytes and gave rise to diarrhoea which was maximal at 72h post-infection (p.i.). Diarrhoea was preceded by ischemia of villi (18-42h p.i.) and villus shortening (maximal at 42h p.i.), and was also coincident with a dramatic regrowth of villi. At 48h p.i. a proliferative zone of electron lucent cells was observed in villus base regions. Cryosections of infected gut, taken before, during, and after infection, together with corresponding age-matched controls, were freeze-dried and analysed by X-ray microanalysis. At 48h p.i. electron lucent villus base cells were shown to be more hydrated, and, to contain higher levels of both Na and Cl and lower levels of P, S, K and Mg than corresponding control cells. These studies increase confidence in the use of X-ray microanalysis in studying biological systems, provide some insight into the process of cell division, and constitute the basis of a new concept of diarrhoeal secretion.27 references.

  1. Loss of CDKC;2 increases both cell division and drought tolerance in Arabidopsis thaliana.

    Science.gov (United States)

    Zhao, Lina; Li, Yaqiong; Xie, Qi; Wu, Yaorong

    2017-09-01

    Drought stress is one of the abiotic stresses that limit plant growth and agricultural productivity. To further understand the mechanism of drought tolerance and identify the genes involved in this process, a genetic screen for altered drought response was conducted in Arabidopsis. One mutant with enhanced drought tolerance was isolated and named Arabidopsis drought tolerance mutant 1 (atdtm1), which has larger lateral organs, prolonged growth duration, increased relative water content and a reduced leaf stomatal density compared with the wild type. The loss of AtDTM1 increases cell division during leaf development. The phenotype is caused by the loss of a T-DNA tagged gene encoding CYCLIN-DEPENDENT KINASE C;2 (CDKC;2), which functions in the regulation of transcription by influencing the phosphorylation status of RNA polymerase II (Pol II). Here, we show that CDKC;2 affects the transcription of downstream genes such as cell cycle genes and genes involved in stomatal development, resulting in altered plant organ size as well as drought tolerance of the plant. These results reveal the crucial role of CDKC;2 in modulating both cell division and the drought response in Arabidopsis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  2. Live birth potential of good morphology and vitrified blastocysts presenting abnormal cell divisions

    DEFF Research Database (Denmark)

    Azzarello, Antonino; Høst, Thomas; Hay-Schmidt, Anders

    2017-01-01

    a lower live birth rate (17.0%) than blastocyst with solely regular cell divisions (29.3%). ACDs could occur at more than one cell division in the same good morphology blastocyst. Reported as independent events, we observed ACDs occurring more frequently at the later cell cycles (1st: 1.3%; 2nd: 8.0%; 3rd...

  3. THE MECHANISM OF 5-AMINOURACIL-INDUCED SYNCHRONY OF CELL DIVISION IN VI CIA FABA ROOT MERISTEMS

    Science.gov (United States)

    Prensky, Wolf; Smith, Harold H.

    1965-01-01

    Cessation of mitosis was brought about in Vicia faba roots incubated for 24 hours in the thymine analogue, 5-aminouracil. Recovery of mitotic activity began 8 hours after removal from 5-aminouracil and reached a peak at 15 hours. If colchicine was added 4 hours before the peak of mitoses, up to 80 per cent of all cells accumulated in mitotic division stages. By use of single and double labeling techniques, it was shown that synchrony of cell divisions resulted from depression in the rate of DNA synthesis by 5-aminouracil, which brought about an accumulation of cells in the S phase of the cell cycle. Treatment with 5-aminouracil may have also caused a delay in the rate of exit of cells from the G2 period. It appeared to have no effect on the duration of the G1 period. When roots were removed from 5-aminouracil, DNA synthesis resumed in all cells in the S phase. Although thymidine antagonized the effects of 5-aminouracil, an exogenous supply of it was not necessary for the resumption of DNA synthesis, as shown by incorporation studies with tritiated deoxycytidine. PMID:19866644

  4. Division of Labor

    KAUST Repository

    Oke, Muse; Zaher, Manal S.; Hamdan, Samir

    2014-01-01

    The first assignment of DNA polymerases at the eukaryotic replication fork was possible after the in vitro reconstitution of the simian virus 40 (SV40) replication system. In this system, DNA polymerase α (Pol α) provides both leading and lagging strands with RNA-DNA primers that are extended by DNA polymerase δ (Pol δ). Extrapolating the architecture of the replication fork from the SV40 model system to an actual eukaryotic cell has been challenged by the discovery of a third DNA polymerase in Saccharomyces cerevisiae, DNA polymerase ε (Pol ε). A division of labor has been proposed for the eukaryotic replication fork whereby Pol ε replicates the leading strand and Pol δ replicates the lagging strand. However, an alternative model of unequal division of labor in which Pol δ can still participate in leading-strand synthesis is plausible.

  5. Division of Labor

    KAUST Repository

    Oke, Muse

    2014-09-12

    The first assignment of DNA polymerases at the eukaryotic replication fork was possible after the in vitro reconstitution of the simian virus 40 (SV40) replication system. In this system, DNA polymerase α (Pol α) provides both leading and lagging strands with RNA-DNA primers that are extended by DNA polymerase δ (Pol δ). Extrapolating the architecture of the replication fork from the SV40 model system to an actual eukaryotic cell has been challenged by the discovery of a third DNA polymerase in Saccharomyces cerevisiae, DNA polymerase ε (Pol ε). A division of labor has been proposed for the eukaryotic replication fork whereby Pol ε replicates the leading strand and Pol δ replicates the lagging strand. However, an alternative model of unequal division of labor in which Pol δ can still participate in leading-strand synthesis is plausible.

  6. Stochastic modeling of cell growth with symmetric or asymmetric division

    Science.gov (United States)

    Marantan, Andrew; Amir, Ariel

    2016-07-01

    We consider a class of biologically motivated stochastic processes in which a unicellular organism divides its resources (volume or damaged proteins, in particular) symmetrically or asymmetrically between its progeny. Assuming the final amount of the resource is controlled by a growth policy and subject to additive and multiplicative noise, we derive the recursive integral equation describing the evolution of the resource distribution over subsequent generations and use it to study the properties of stable resource distributions. We find conditions under which a unique stable resource distribution exists and calculate its moments for the class of affine linear growth policies. Moreover, we apply an asymptotic analysis to elucidate the conditions under which the stable distribution (when it exists) has a power-law tail. Finally, we use the results of this asymptotic analysis along with the moment equations to draw a stability phase diagram for the system that reveals the counterintuitive result that asymmetry serves to increase stability while at the same time widening the stable distribution. We also briefly discuss how cells can divide damaged proteins asymmetrically between their progeny as a form of damage control. In the appendixes, motivated by the asymmetric division of cell volume in Saccharomyces cerevisiae, we extend our results to the case wherein mother and daughter cells follow different growth policies.

  7. Planar cell polarity signaling coordinates oriented cell division and cell rearrangement in clonally expanding growth plate cartilage

    OpenAIRE

    Li, Yuwei; Li, Ang; Junge, Jason; Bronner, Marianne

    2017-01-01

    Both oriented cell divisions and cell rearrangements are critical for proper embryogenesis and organogenesis. However, little is known about how these two cellular events are integrated. Here we examine the linkage between these processes in chick limb cartilage. By combining retroviral-based multicolor clonal analysis with live imaging, the results show that single chondrocyte precursors can generate both single-column and multi-column clones through oriented division followed by cell rearra...

  8. Cell division cycle 20 overexpression predicts poor prognosis for patients with lung adenocarcinoma.

    Science.gov (United States)

    Shi, Run; Sun, Qi; Sun, Jing; Wang, Xin; Xia, Wenjie; Dong, Gaochao; Wang, Anpeng; Jiang, Feng; Xu, Lin

    2017-03-01

    The cell division cycle 20, a key component of spindle assembly checkpoint, is an essential activator of the anaphase-promoting complex. Aberrant expression of cell division cycle 20 has been detected in various human cancers. However, its clinical significance has never been deeply investigated in non-small-cell lung cancer. By analyzing The Cancer Genome Atlas database and using some certain online databases, we validated overexpression of cell division cycle 20 in both messenger RNA and protein levels, explored its clinical significance, and evaluated the prognostic role of cell division cycle 20 in non-small-cell lung cancer. Cell division cycle 20 expression was significantly correlated with sex (p = 0.003), histological classification (p overexpression of cell division cycle 20 was significantly associated with bigger primary tumor size (p = 0.0023), higher MKI67 level (r = 0.7618, p Overexpression of cell division cycle 20 is associated with poor prognosis in lung adenocarcinoma patients, and its overexpression can also be used to identify high-risk groups. In conclusion, cell division cycle 20 might serve as a potential biomarker for lung adenocarcinoma patients.

  9. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

    Energy Technology Data Exchange (ETDEWEB)

    Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

    2014-11-01

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

  10. Uncovering the link between malfunctions in Drosophila neuroblast asymmetric cell division and tumorigenesis

    Directory of Open Access Journals (Sweden)

    Kelsom Corey

    2012-11-01

    Full Text Available Abstract Asymmetric cell division is a developmental process utilized by several organisms. On the most basic level, an asymmetric division produces two daughter cells, each possessing a different identity or fate. Drosophila melanogaster progenitor cells, referred to as neuroblasts, undergo asymmetric division to produce a daughter neuroblast and another cell known as a ganglion mother cell (GMC. There are several features of asymmetric division in Drosophila that make it a very complex process, and these aspects will be discussed at length. The cell fate determinants that play a role in specifying daughter cell fate, as well as the mechanisms behind setting up cortical polarity within neuroblasts, have proved to be essential to ensuring that neurogenesis occurs properly. The role that mitotic spindle orientation plays in coordinating asymmetric division, as well as how cell cycle regulators influence asymmetric division machinery, will also be addressed. Most significantly, malfunctions during asymmetric cell division have shown to be causally linked with neoplastic growth and tumor formation. Therefore, it is imperative that the developmental repercussions as a result of asymmetric cell division gone awry be understood.

  11. Regulation of the Min Cell Division Inhibition Complex by the Rcs Phosphorelay in Proteus mirabilis.

    Science.gov (United States)

    Howery, Kristen E; Clemmer, Katy M; Şimşek, Emrah; Kim, Minsu; Rather, Philip N

    2015-08-01

    A key regulator of swarming in Proteus mirabilis is the Rcs phosphorelay, which represses flhDC, encoding the master flagellar regulator FlhD4C2. Mutants in rcsB, the response regulator in the Rcs phosphorelay, hyperswarm on solid agar and differentiate into swarmer cells in liquid, demonstrating that this system also influences the expression of genes central to differentiation. To gain a further understanding of RcsB-regulated genes involved in swarmer cell differentiation, transcriptome sequencing (RNA-Seq) was used to examine the RcsB regulon. Among the 133 genes identified, minC and minD, encoding cell division inhibitors, were identified as RcsB-activated genes. A third gene, minE, was shown to be part of an operon with minCD. To examine minCDE regulation, the min promoter was identified by 5' rapid amplification of cDNA ends (5'-RACE), and both transcriptional lacZ fusions and quantitative real-time reverse transcriptase (qRT) PCR were used to confirm that the minCDE operon was RcsB activated. Purified RcsB was capable of directly binding the minC promoter region. To determine the role of RcsB-mediated activation of minCDE in swarmer cell differentiation, a polar minC mutation was constructed. This mutant formed minicells during growth in liquid, produced shortened swarmer cells during differentiation, and exhibited decreased swarming motility. This work describes the regulation and role of the MinCDE cell division system in P. mirabilis swarming and swarmer cell elongation. Prior to this study, the mechanisms that inhibit cell division and allow swarmer cell elongation were unknown. In addition, this work outlines for the first time the RcsB regulon in P. mirabilis. Taken together, the data presented in this study begin to address how P. mirabilis elongates upon contact with a solid surface. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Explanatory chapter: introducing exogenous DNA into cells.

    Science.gov (United States)

    Koontz, Laura

    2013-01-01

    The ability to efficiently introduce DNA into cells is essential for many experiments in biology. This is an explanatory chapter providing an overview of the various methods for introducing DNA into bacteria, yeast, and mammalian cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Cell division requirement for activation of murine leukemia virus in cell culture by irradiation

    International Nuclear Information System (INIS)

    Otten, J.A.; Quarles, J.M.; Tennant, R.W.

    1976-01-01

    Actively dividing cultures of AKR mouse cells were exposed to relatively low dose-rates of γ radiation and tested for activation of endogenous leukemia viruses. Efficient and reproducible induction of virus was obtained with actively dividing cells, but cultures deprived of serum to inhibit cell division before and during γ irradiation were not activated, even when medium with serum was added immediately after irradiation. These results show that cell division was required for virus induction but that a stable intermediate similar to the state induced by halogenated pyrimidines was not formed. In actively dividing AKR cell cultures, virus activation appeared to be proportional to the dose of γ radiation; the estimated frequency of activation was 1-8 x 10 - 5 per exposed cell and the efficiency of activation was approximately 0.012 inductions per cell per rad. Other normal primary and established mouse cell cultures tested were not activated by γ radiation. The requirement of cell division for radiation and chemical activation may reflect some common mechanism for initiation of virus expression

  14. Mechanical Regulation in Cell Division and in Neurotransmitter Release

    Science.gov (United States)

    Thiyagarajan, Sathish

    During their lifecycle, cells must produce forces which play important roles in several subcellular processes. Force-producing components are organized into macromolecular assemblies of proteins that are often dynamic, and are constructed or disassembled in response to various signals. The forces themselves may directly be involved in subcellular mechanics, or they may influence mechanosensing proteins either within or outside these structures. These proteins play different roles: they may ensure the stability of the force-producing structure, or they may send signals to a coupled process. The generation and sensing of subcellular forces is an active research topic, and this thesis focusses on the roles of these forces in two key areas: cell division and neurotransmitter release. The first part of the thesis deals with the effect of force on cell wall growth regulation during division in the fission yeast Schizosaccharomyces pombe, a cigar-shaped, unicellular organism. During cytokinesis, the last stage of cell division in which the cell physically divides into two, a tense cytokinetic ring anchored to the cellular membrane assembles and constricts, accompanied by the inward centripetal growth of new cell wall, called septum, in the wake of the inward-moving membrane. The contour of the septum hole maintains its circularity as it reduces in size--an indication of regulated growth. To characterize the cell wall growth process, we performed image analysis on contours of the leading edge of the septum obtained via fluorescence microscopy in the labs of our collaborators. We quantified the deviations from circularity using the edge roughness. The roughness was spatially correlated, suggestive of regulated growth. We hypothesized that the cell wall growers are mechanosensitive and respond to the force exerted by the ring. A mathematical model based on this hypothesis then showed that this leads to corrections of roughness in a curvature-dependent fashion. Thus, one of

  15. DNA cytoskeleton for stabilizing artificial cells.

    Science.gov (United States)

    Kurokawa, Chikako; Fujiwara, Kei; Morita, Masamune; Kawamata, Ibuki; Kawagishi, Yui; Sakai, Atsushi; Murayama, Yoshihiro; Nomura, Shin-Ichiro M; Murata, Satoshi; Takinoue, Masahiro; Yanagisawa, Miho

    2017-07-11

    Cell-sized liposomes and droplets coated with lipid layers have been used as platforms for understanding live cells, constructing artificial cells, and implementing functional biomedical tools such as biosensing platforms and drug delivery systems. However, these systems are very fragile, which results from the absence of cytoskeletons in these systems. Here, we construct an artificial cytoskeleton using DNA nanostructures. The designed DNA oligomers form a Y-shaped nanostructure and connect to each other with their complementary sticky ends to form networks. To undercoat lipid membranes with this DNA network, we used cationic lipids that attract negatively charged DNA. By encapsulating the DNA into the droplets, we successfully created a DNA shell underneath the membrane. The DNA shells increased interfacial tension, elastic modulus, and shear modulus of the droplet surface, consequently stabilizing the lipid droplets. Such drastic changes in stability were detected only when the DNA shell was in the gel phase. Furthermore, we demonstrate that liposomes with the DNA gel shell are substantially tolerant against outer osmotic shock. These results clearly show the DNA gel shell is a stabilizer of the lipid membrane akin to the cytoskeleton in live cells.

  16. Uhrf1 controls the self-renewal versus differentiation of hematopoietic stem cells by epigenetically regulating the cell-division modes.

    Science.gov (United States)

    Zhao, Jingyao; Chen, Xufeng; Song, Guangrong; Zhang, Jiali; Liu, Haifeng; Liu, Xiaolong

    2017-01-10

    Hematopoietic stem cells (HSCs) are able to both self-renew and differentiate. However, how individual HSC makes the decision between self-renewal and differentiation remains largely unknown. Here we report that ablation of the key epigenetic regulator Uhrf1 in the hematopoietic system depletes the HSC pool, leading to hematopoietic failure and lethality. Uhrf1-deficient HSCs display normal survival and proliferation, yet undergo erythroid-biased differentiation at the expense of self-renewal capacity. Notably, Uhrf1 is required for the establishment of DNA methylation patterns of erythroid-specific genes during HSC division. The expression of these genes is enhanced in the absence of Uhrf1, which disrupts the HSC-division modes by promoting the symmetric differentiation and suppressing the symmetric self-renewal. Moreover, overexpression of one of the up-regulated genes, Gata1, in HSCs is sufficient to phenocopy Uhrf1-deficient HSCs, which show impaired HSC symmetric self-renewal and increased differentiation commitment. Taken together, our findings suggest that Uhrf1 controls the self-renewal versus differentiation of HSC through epigenetically regulating the cell-division modes, thus providing unique insights into the relationship among Uhrf1-mediated DNA methylation, cell-division mode, and HSC fate decision.

  17. BioClips of symmetric and asymmetric cell division.

    Science.gov (United States)

    Lu, Fong-Mei; Eliceiri, Kevin W; White, John G

    2007-05-01

    Animations have long been used as tools to illustrate complex processes in such diverse fields as mechanical engineering, astronomy, bacteriology and physics. Animations in biology hold particular educational promise for depicting complex dynamic processes, such as photosynthesis, motility, viral replication and cellular respiration, which cannot be easily explained using static two-dimensional images. However, these animations have often been restrictive in scope, having been created for a specific classroom or research audience. In recent years, a new type of animation has emerged called the BioClip (http://www.bioclips.com) that strives to present science in an interactive multimedia format, which is, at once, informative and entertaining, by combining animations, text descriptions and music in one portable cross-platform document. In the present article, we illustrate the educational value of this new electronic resource by reviewing in depth two BioClips our group has created which describe the processes of symmetric and asymmetric cell division (http://www.wormclassroom.org/cb/bioclip).

  18. Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism.

    Science.gov (United States)

    Steinhauser, Matthew L; Bailey, Andrew P; Senyo, Samuel E; Guillermier, Christelle; Perlstein, Todd S; Gould, Alex P; Lee, Richard T; Lechene, Claude P

    2012-01-15

    Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter, but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with submicrometre resolution. Here we apply MIMS to diverse organisms, including Drosophila, mice and humans. We test the 'immortal strand hypothesis', which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labelling mice with (15)N-thymidine from gestation until post-natal week 8, we find no (15)N label retention by dividing small intestinal crypt cells after a four-week chase. In adult mice administered (15)N-thymidine pulse-chase, we find that proliferating crypt cells dilute the (15)N label, consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human haematopoietic system. These studies show that MIMS provides high-resolution quantification of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.

  19. An Equatorial Contractile Mechanism Drives Cell Elongation but not Cell Division

    Science.gov (United States)

    Denker, Elsa; Bhattachan, Punit; Deng, Wei; Mathiesen, Birthe T.; Jiang, Di

    2014-01-01

    Cell shape changes and proliferation are two fundamental strategies for morphogenesis in animal development. During embryogenesis of the simple chordate Ciona intestinalis, elongation of individual notochord cells constitutes a crucial stage of notochord growth, which contributes to the establishment of the larval body plan. The mechanism of cell elongation is elusive. Here we show that although notochord cells do not divide, they use a cytokinesis-like actomyosin mechanism to drive cell elongation. The actomyosin network forming at the equator of each notochord cell includes phosphorylated myosin regulatory light chain, α-actinin, cofilin, tropomyosin, and talin. We demonstrate that cofilin and α-actinin are two crucial components for cell elongation. Cortical flow contributes to the assembly of the actomyosin ring. Similar to cytokinetic cells, membrane blebs that cause local contractions form at the basal cortex next to the equator and participate in force generation. We present a model in which the cooperation of equatorial actomyosin ring-based constriction and bleb-associated contractions at the basal cortex promotes cell elongation. Our results demonstrate that a cytokinesis-like contractile mechanism is co-opted in a completely different developmental scenario to achieve cell shape change instead of cell division. We discuss the occurrences of actomyosin rings aside from cell division, suggesting that circumferential contraction is an evolutionally conserved mechanism to drive cell or tissue elongation. PMID:24503569

  20. DNA replication and repair in Tilapia cells

    International Nuclear Information System (INIS)

    Yew, F.H.; Chang, L.M.

    1984-01-01

    The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also efficient in excision repair, which in other fish cell lines has been found to be at a low level or absent. Their response to the inhibitors hydroxyurea and 1-β-D-arabinofuranosylcytosine is less sensitive than that of other cell lines, yet the cells seem to have very small pools of DNA precursor. (author)

  1. DNA repair in murine embryonic stem cells and differentiated cells

    International Nuclear Information System (INIS)

    Tichy, Elisia D.; Stambrook, Peter J.

    2008-01-01

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells

  2. Asymmetric cell division and its role in cell fate determination in the green alga Tetraselmis indica

    Digital Repository Service at National Institute of Oceanography (India)

    Arora, M.; Anil, A.C.; Burgess, K.; Delany, J.E.; Mesbahi, E.

    is a mechanism to ensure survival upon exposure to stress. Int. J. Food Microbiol. 78 19-30 De Smet I and Beeckman T 2011 Asymmetric cell division in land plants and algae: the driving force for differentiation. Nature Rev. Mol. Cell Biol. 12 177... of Prasinophytes, but are as evolved as any other green alga or land plant. These organisms share several ultrastructural features with the other core Chlorophytes (Trebouxiophyceae, Ulvophyceae and Chlorophyceae). However, the role of Chlorodendrophycean algae...

  3. Growth-arrest-specific protein 2 inhibits cell division in Xenopus embryos.

    Directory of Open Access Journals (Sweden)

    Tong Zhang

    Full Text Available Growth-arrest-specific 2 gene was originally identified in murine fibroblasts under growth arrest conditions. Furthermore, serum stimulation of quiescent, non-dividing cells leads to the down-regulation of gas2 and results in re-entry into the cell cycle. Cytoskeleton rearrangements are critical for cell cycle progression and cell division and the Gas2 protein has been shown to co-localize with actin and microtubules in interphase mammalian cells. Despite these findings, direct evidence supporting a role for Gas2 in the mechanism of cell division has not been reported.To determine whether the Gas2 protein plays a role in cell division, we over-expressed the full-length Gas2 protein and Gas2 truncations containing either the actin-binding CH domain or the tubulin-binding Gas2 domain in Xenopus laevis embryos. We found that both the full-length Gas2 protein and the Gas2 domain, but not the CH domain, inhibited cell division and resulted in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that Gas2 function is mediated by microtubule binding. Gas2 co-localized with microtubules at the cell cortex of Gas2-injected Xenopus embryos using cryo-confocal microscopy and co-sedimented with microtubules in cytoskeleton co-sedimentation assays. To investigate the mechanism of Gas2-induced cell division arrest, we showed, using a wound-induced contractile array assay, that Gas2 stabilized microtubules. Finally, electron microscopy studies demonstrated that Gas2 bundled microtubules into higher-order structures.Our experiments show that Gas2 inhibits cell division in Xenopus embryos. We propose that Gas2 function is mediated by binding and bundling microtubules, leading to cell division arrest.

  4. Phenotypic plasticity and effects of selection on cell division symmetry in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Uttara N Lele

    Full Text Available Aging has been demonstrated in unicellular organisms and is presumably due to asymmetric distribution of damaged proteins and other components during cell division. Whether the asymmetry-induced aging is inevitable or an adaptive and adaptable response is debated. Although asymmetric division leads to aging and death of some cells, it increases the effective growth rate of the population as shown by theoretical and empirical studies. Mathematical models predict on the other hand, that if the cells divide symmetrically, cellular aging may be delayed or absent, growth rate will be reduced but growth yield will increase at optimum repair rates. Therefore in nutritionally dilute (oligotrophic environments, where growth yield may be more critical for survival, symmetric division may get selected. These predictions have not been empirically tested so far. We report here that Escherichia coli grown in oligotrophic environments had greater morphological and functional symmetry in cell division. Both phenotypic plasticity and genetic selection appeared to shape cell division time asymmetry but plasticity was lost on prolonged selection. Lineages selected on high nutrient concentration showed greater frequency of presumably old or dead cells. Further, there was a negative correlation between cell division time asymmetry and growth yield but there was no significant correlation between asymmetry and growth rate. The results suggest that cellular aging driven by asymmetric division may not be hardwired but shows substantial plasticity as well as evolvability in response to the nutritional environment.

  5. Plant Cell Division Analyzed by Transient Agrobacterium-Mediated Transformation of Tobacco BY-2 Cells.

    Science.gov (United States)

    Buschmann, Henrik

    2016-01-01

    The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.

  6. Thermosensitive mutant of Bacillus subtilis deficient in uracil and cell division

    Energy Technology Data Exchange (ETDEWEB)

    Nagai, K; Some, H; Tamura, G

    1976-01-01

    Thermonsensitive division mutants were derived from Bacillus subtilis Marburg 168 thy trp/sub 2/ by means of membrane filtration after nitrosoguanidine mutagenesis. Among them, ts42 requiring uracil for normal growth at 48/sup 0/C was investigated. In the absence of uracil, the mutant cells grew normally at 37/sup 0/C and stopped dividing after temperature shift to 48/sup 0/C resulting in filaments of two to four times length of normal rods. The total cell number after the temperature shift increased two to three fold in 90 min and remained constant thereafter. The viable count after the temperature shift to 48/sup 0/C, increased 1.5 to 2 fold in initial 60 min and then decreased exponentially. A rapid restoration of colony forming ability was shown when the mutant cells were shifted back to the permissive temperature after 120 to 180 min of incubation at 48/sup 0/C or when uracil was introduced to the culture at 48/sup 0/C. This recovery of viability was partly observed even in the presence of chloramphenicol. The synthesis of RNA of this mutant was shown to decline 20 min after the temperature shift to 48/sup 0/C whereas the syntheses of DNA and protein proceeded for more than 80 min at that temperature. No newly isolated uracil requiring mutants formed filaments in the medium lacking uracil or showed growth pattern like ts42.

  7. ESCRT-III mediated cell division in Sulfolobus acidocaldarius –A reconstitution perspective

    Directory of Open Access Journals (Sweden)

    Tobias eHärtel

    2014-06-01

    Full Text Available In the framework of Synthetic Biology, it has become an intriguing question what would be the minimal representation of cell division machinery. Thus, it seems appropriate to compare how cell division is realized in different microorganisms. In particular, the cell division system of Crenarchaeota lacks certain proteins found in most bacteria and Euryarchaeota, such as FtsZ, MreB or the Min system. The Sulfolobaceae family encodes functional homologs of the eukaryotic proteins Vps4 and ESCRT-III. ESCRT-III is essential for several eukaryotic pathways, e.g. budding of intralumenal vesicles (ILVs, or cytokinesis, whereas Vps4 dissociates the ESCRT-III complex from the membrane. CdvA (Cell Division A is required for the recruitment of crenarchaeal ESCRT-III proteins to the membrane at mid-cell. The proteins polymerize and form a smaller structure during constriction. Thus, ESCRT-III mediated cell division in S. acidocaldarius shows functional analogies to the Z ring observed in prokaryotes like E. coli, which has recently begun to be reconstituted in vitro. In this short perspective, we discuss the possibility of building such an in vitro cell division system on basis of archaeal ESCRT-III.

  8. Tumor-Initiating Label-Retaining Cancer Cells in Human Gastrointestinal Cancers Undergo Asymmetric Cell Division

    Science.gov (United States)

    Xin, Hong-Wu; Hari, Danielle M.; Mullinax, John E.; Ambe, Chenwi M.; Koizumi, Tomotake; Ray, Satyajit; Anderson, Andrew J.; Wiegand, Gordon W.; Garfield, Susan H.; Thorgeirsson, Snorri S.; Avital, Itzhak

    2012-01-01

    Label-retaining cells (LRCs) have been proposed to represent adult tissue stem cells. LRCs are hypothesized to result from either slow cycling or asymmetric cell division (ACD). However, the stem cell nature and whether LRC undergo ACD remain controversial. Here, we demonstrate label-retaining cancer cells (LRCCs) in several gastrointestinal (GI) cancers including fresh surgical specimens. Using a novel method for isolation of live LRCC, we demonstrate that a subpopulation of LRCC is actively dividing and exhibits stem cells and pluripotency gene expression profiles. Using real-time confocal microscopic cinematography, we show live LRCC undergoing asymmetric nonrandom chromosomal cosegregation LRC division. Importantly, LRCCs have greater tumor-initiating capacity than non-LRCCs. Based on our data and that cancers develop in tissues that harbor normal-LRC, we propose that LRCC might represent a novel population of GI stem-like cancer cells. LRCC may provide novel mechanistic insights into the biology of cancer and regenerative medicine and present novel targets for cancer treatment. PMID:22331764

  9. ParA and ParB coordinate chromosome segregation with cell elongation and division during Streptomyces sporulation

    Science.gov (United States)

    Donczew, Magdalena; Mackiewicz, Paweł; Wróbel, Agnieszka; Flärdh, Klas; Zakrzewska-Czerwińska, Jolanta

    2016-01-01

    In unicellular bacteria, the ParA and ParB proteins segregate chromosomes and coordinate this process with cell division and chromosome replication. During sporulation of mycelial Streptomyces, ParA and ParB uniformly distribute multiple chromosomes along the filamentous sporogenic hyphal compartment, which then differentiates into a chain of unigenomic spores. However, chromosome segregation must be coordinated with cell elongation and multiple divisions. Here, we addressed the question of whether ParA and ParB are involved in the synchronization of cell-cycle processes during sporulation in Streptomyces. To answer this question, we used time-lapse microscopy, which allows the monitoring of growth and division of single sporogenic hyphae. We showed that sporogenic hyphae stop extending at the time of ParA accumulation and Z-ring formation. We demonstrated that both ParA and ParB affect the rate of hyphal extension. Additionally, we showed that ParA promotes the formation of massive nucleoprotein complexes by ParB. We also showed that FtsZ ring assembly is affected by the ParB protein and/or unsegregated DNA. Our results indicate the existence of a checkpoint between the extension and septation of sporogenic hyphae that involves the ParA and ParB proteins. PMID:27248800

  10. Movement of beta-irradiated epidermal basal cells to the spinous-granular layers in the absence of cell division

    International Nuclear Information System (INIS)

    Etoh, H.; Taguchi, Y.H.; Tabachnick, J.

    1975-01-01

    Guinea-pig epidermis was irradiated with 3000 rad of beta rays 1 hr after two injections of [ 3 H]thymidine 5 hr apart (labeled cells in S phase and G 2 phase) or 18 hr after injection (labeled early G 1 cells). In nonirradiated epidermis labeled basal cells divided within 24 hr with daughter cells remaining in the basal layer, and approximately 50 percent of the labeled cells moved into the spinal layer by the 3rd day. Cell division in nonirradiated epidermis diluted the number of silver grains/nucleus, and lightly labeled cells were found in the granular layer by day 7. Beta irradiation inhibited cell division but it did not slow the rate of transit (ca 8 days) of irradiated labeled cells from basal to granular layer, some of these remaining heavily labeled. Although cell division may play some role in upward movement of basal cells in normal epidermis detachment of a basal cell from the basement membrane and its transit to the granular layer is unimpaired in the absence of cell division. These findings suggest that some radioresistant metabolic function(s), not cell division, is responsible for upward movement of basal cells. (auth)

  11. Somatic mosaicism in families with hemophilia B: 11% of germline mutations originate within a few cell divisions post-fertilization

    Energy Technology Data Exchange (ETDEWEB)

    Knoell, A.; Ketterling, R.P.; Vielhaber, E. [Mayo Clinic/Foundation, Rochester, MN (United States)] [and others

    1994-09-01

    Previous molecular estimates of mosaicism in the dystrophin and other genes generally have focused on the transmission of the mutated allele to two or more children by an individual without the mutation in leukocyte DNA. We have analyzed 414 families with hemophilia B by direct genomic sequencing and haplotype analysis, and have deduced the origin of mutation in 56 families. There was no origin individual who transmitted a mutant allele to more than one child. However, somatic mosaicism was detected by sequence analysis of four origin individuals (3{female} and 1{male}). The sensitivity of this analysis is typically one part in ten. In one additional female who had close to a 50:50 ratio of mutant to normal alleles, three of four noncarrier daughters inherited the haplotype associated with the mutant allele. This highlights a caveat in molecular analysis: a presumptive carrier in a family with sporadic disease does not necessarily have a 50% probability of transmitting the mutant allele to her offspring. After eliminating those families in which mosaicism could not be detected because of a total gene deletion or absence of DNA from a deduced origin individual, 5 of 43 origin individuals exhibited somatic mosaicism at a level that reflects a mutation within the first few cell divisions after fertilization. In one patient, analysis of cervical scrapings and buccal mucosa confirm the generalized distribution of somatic mutation. Are the first few cell divisions post-fertilization highly mutagenic, or do mutations at later divisions also give rise to somatic mosaicism? To address this question, DNA from origin individuals are being analyzed to detect somatic mosaicism at a sensitivity of 1:1000. Single nucleotide primer extension (SNuPE) has been utilized in eight families to date and no mosaicism has been detected. When the remaining 30 samples are analyzed, it will be possible to compare the frequency of somatic mosaicism at 0.1-10% with that of {ge}10%.

  12. Control of cell division and the spatial localization of assembled gene products in Caulobacter crescentus

    International Nuclear Information System (INIS)

    Nathan, P.D.

    1988-01-01

    Experiments are described that examine the role of penicillin-binding proteins (PBPs) in the regulation of cell division in Caulobacter crescentus; and the spatial localization of methyl-accepting chemotaxis proteins (MCPs) in C. crescentus swarmer and predivisional cells. In the analysis of PBP function, in vivo and in vitro assays are used to directly label C. crescentus PBPs with [ 3 H] penicillin G in wild type strain CB15, in a series of conditional cell division mutants and in new temperature sensitive cephalosporin C resistant mutants PC8002 and PC8003. 14 PBPs are characterized and a high molecular weight PBP (PBP 1B) that is required for cell division is identified. PBP 1B competes for β-lactams that induce filament formation and may be a high affinity binding protein. A second high molecular weight PBP (PBP 1C) is also associated with defective cell division. The examination of PBP patterns in synchronous swarmer cells reveals that the in vivo activity of PBP 1B and PBP 1C increases at the time that the cell division pathway is initiated. None of the PBPs, however, appear to be differentially localized in the C. crescentus cell. In the analysis of MCP localization, in vivo and in vitro assays are used to directly label C. crescentus MCPs with methyl- 3 H. MCPs are examined in flagellated and non-flagellated vesicles prepared from cells by immunoaffinity chromatography

  13. Using stochastic cell division and death to probe minimal units of cellular replication

    Science.gov (United States)

    Chib, Savita; Das, Suman; Venkatesan, Soumya; Sai Narain Seshasayee, Aswin; Thattai, Mukund

    2018-03-01

    The invariant cell initiation mass measured in bacterial growth experiments has been interpreted as a minimal unit of cellular replication. Here we argue that the existence of such minimal units induces a coupling between the rates of stochastic cell division and death. To probe this coupling we tracked live and dead cells in Escherichia coli populations treated with a ribosome-targeting antibiotic. We find that the growth exponent from macroscopic cell growth or decay measurements can be represented as the difference of microscopic first-order cell division and death rates. The boundary between cell growth and decay, at which the number of live cells remains constant over time, occurs at the minimal inhibitory concentration (MIC) of the antibiotic. This state appears macroscopically static but is microscopically dynamic: division and death rates exactly cancel at MIC but each is remarkably high, reaching 60% of the antibiotic-free division rate. A stochastic model of cells as collections of minimal replicating units we term ‘widgets’ reproduces both steady-state and transient features of our experiments. Sub-cellular fluctuations of widget numbers stochastically drive each new daughter cell to one of two alternate fates, division or death. First-order division or death rates emerge as eigenvalues of a stationary Markov process, and can be expressed in terms of the widget’s molecular properties. High division and death rates at MIC arise due to low mean and high relative fluctuations of widget number. Isolating cells at the threshold of irreversible death might allow molecular characterization of this minimal replication unit.

  14. Periplasmic Acid Stress Increases Cell Division Asymmetry (Polar Aging of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Michelle W Clark

    Full Text Available Under certain kinds of cytoplasmic stress, Escherichia coli selectively reproduce by distributing the newer cytoplasmic components to new-pole cells while sequestering older, damaged components in cells inheriting the old pole. This phenomenon is termed polar aging or cell division asymmetry. It is unknown whether cell division asymmetry can arise from a periplasmic stress, such as the stress of extracellular acid, which is mediated by the periplasm. We tested the effect of periplasmic acid stress on growth and division of adherent single cells. We tracked individual cell lineages over five or more generations, using fluorescence microscopy with ratiometric pHluorin to measure cytoplasmic pH. Adherent colonies were perfused continually with LBK medium buffered at pH 6.00 or at pH 7.50; the external pH determines periplasmic pH. In each experiment, cell lineages were mapped to correlate division time, pole age and cell generation number. In colonies perfused at pH 6.0, the cells inheriting the oldest pole divided significantly more slowly than the cells inheriting the newest pole. In colonies perfused at pH 7.50 (near or above cytoplasmic pH, no significant cell division asymmetry was observed. Under both conditions (periplasmic pH 6.0 or pH 7.5 the cells maintained cytoplasmic pH values at 7.2-7.3. No evidence of cytoplasmic protein aggregation was seen. Thus, periplasmic acid stress leads to cell division asymmetry with minimal cytoplasmic stress.

  15. From cell differentiation to cell collectives: Bacillus subtilis uses division of labor to migrate.

    Science.gov (United States)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    2015-04-01

    The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called "van Gogh bundles") of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.

  16. From cell differentiation to cell collectives: Bacillus subtilis uses division of labor to migrate.

    Directory of Open Access Journals (Sweden)

    Jordi van Gestel

    2015-04-01

    Full Text Available The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called "van Gogh bundles" of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.

  17. Investigation of roles for LRR-RLKs PNL1 and PNL2 in asymmetric cell division in Arabidopsis thaliana

    OpenAIRE

    Rodriguez, Maiti Celina

    2008-01-01

    Asymmetric cell division is a vital component of plant development. It enables cell differentiation and cell diversity. A key component of asymmetric cell division is cell signaling. Signals are believed to control polarization and orientation of asymmetric divisions during stomatal development. The findings of this report suggest that PNL1 and PNL2, two LRR-RLKs found in Arabidopsis and closely related to maize PAN1 LRR-RLK, are possibly involved in the signaling events occurring during the ...

  18. DNA Based Electrochromic and Photovoltaic Cells

    Science.gov (United States)

    2012-01-01

    using deoxyribonucleic acid complex as an electron blocking layer App. Phys. Lett. 88 (2006) 171109. 23. F.H.C. Crick , J.D. Watson . The complementary...9550-09-1-0647 final 01-09-2009 ; 30-11-2011 DNA Based Electrochromic and Photovoltaic Cells FA 9550-09-1-0647 Pawlicka, Agnieszka, J. Instituto de...Available. DNA is an abundant natural product with very good biodegradation properties and can be used to obtain gel polymer electrolytes (GPEs) with high

  19. DNA repair studies in mammalian germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.; Owens, J.G.

    1984-01-01

    In submammalian test systems, nitrosocarbamates (NEC) are 100-fold more mutagenic than are their corresponding nitrosourea homologues. To learn more about its interaction with germ-cell DNA in the mouse testis, male mice were given i.p. injections of NEC. Testicular injections of [ 3 H]dThd were given along with the NEC. Sixteen days after treatment, sperm were recovered from the caudal epididymides and assayed for an unscheduled-DNA-synthesis

  20. Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells.

    Science.gov (United States)

    Burke, Russell T; Marcus, Joshua M; Orth, James D

    2017-06-13

    Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos. Previous work has shown strong cell cycle effects and drug-induced cell death across many different cancer-derived cell lines. Here, we report strong cell cycle-associated DNA double-stranded break formation upon the treatment of cancer cells with SINE. In multiple cell models, selinexor treatment results in the formation of clustered DNA damage foci in 30-40% of cells within 8 hours that is dependent upon cysteine-528. DNA damage strongly correlates with G1/S-phase and decreased DNA replication. Live cell microscopy reveals an association between DNA damage and cell fate. Cells that form damage in G1-phase more often die or arrest, while those damaged in S/G2-phase frequently progress to cell division. Up to half of all treated cells form damage foci, and most cells that die after being damaged, were damaged in G1-phase. By comparison, non-transformed cell lines show strong cell cycle effects but little DNA damage and less death than cancer cells. Significant drug combination effects occur when selinexor is paired with different classes of agents that either cause DNA damage or that diminish DNA damage repair. These data present a novel effect of exportin-1 inhibition and provide a strong rationale for multiple combination treatments of selinexor with agents that are currently in use for the treatment of different solid cancers.

  1. Characterization of harpy/Rca1/emi1 mutants: patterning in the absence of cell division.

    Science.gov (United States)

    Riley, Bruce B; Sweet, Elly M; Heck, Rebecca; Evans, Adrienne; McFarland, Karen N; Warga, Rachel M; Kane, Donald A

    2010-03-01

    We have characterized mutations in the early arrest gene, harpy (hrp), and show that they introduce premature stops in the coding region of early mitotic inhibitor1 (Rca1/emi1). In harpy mutants, cells stop dividing during early gastrulation. Lineage analysis confirms that there is little change in cell number after approximately cycle-14. Gross patterning occurs relatively normally, and many organ primordia are produced on time but with smaller numbers of cells. Despite the lack of cell division, some organ systems continue to increase in cell number, suggesting recruitment from surrounding areas. Analysis of bromodeoxyuridine incorporation shows that endoreduplication continues in many cells well past the first day of development, but cells cease endoreduplication once they begin to differentiate and express cell-type markers. Despite relatively normal gross patterning, harpy mutants show several defects in morphogenesis, cell migration and differentiation resulting directly or indirectly from the arrest of cell division. Copyright (c) 2010 Wiley-Liss, Inc.

  2. Stem cells propagate their DNA by random segregation in the flatworm Macrostomum lignano.

    Directory of Open Access Journals (Sweden)

    Freija Verdoodt

    Full Text Available Adult stem cells are proposed to have acquired special features to prevent an accumulation of DNA-replication errors. Two such mechanisms, frequently suggested to serve this goal are cellular quiescence, and non-random segregation of DNA strands during stem cell division, a theory designated as the immortal strand hypothesis. To date, it has been difficult to test the in vivo relevance of both mechanisms in stem cell systems. It has been shown that in the flatworm Macrostomum lignano pluripotent stem cells (neoblasts are present in adult animals. We sought to address by which means M. lignano neoblasts protect themselves against the accumulation of genomic errors, by studying the exact mode of DNA-segregation during their division. In this study, we demonstrated four lines of in vivo evidence in favor of cellular quiescence. Firstly, performing BrdU pulse-chase experiments, we localized 'Label-Retaining Cells' (LRCs. Secondly, EDU pulse-chase combined with Vasa labeling demonstrated the presence of neoblasts among the LRCs, while the majority of LRCs were differentiated cells. We showed that stem cells lose their label at a slow rate, indicating cellular quiescence. Thirdly, CldU/IdU- double labeling studies confirmed that label-retaining stem cells showed low proliferative activity. Finally, the use of the actin inhibitor, cytochalasin D, unequivocally demonstrated random segregation of DNA-strands in LRCs. Altogether, our data unambiguously demonstrated that the majority of neoblasts in M. lignano distribute their DNA randomly during cell division, and that label-retention is a direct result of cellular quiescence, rather than a sign of co-segregation of labeled strands.

  3. Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus

    DEFF Research Database (Denmark)

    Jensen, Rasmus Bugge

    2006-01-01

    Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication...

  4. Ciprofloxacin Derivatives Affect Parasite Cell Division and Increase the Survival of Mice Infected with Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Erica S Martins-Duarte

    Full Text Available Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a worldwide disease whose clinical manifestations include encephalitis and congenital malformations in newborns. Previously, we described the synthesis of new ethyl-ester derivatives of the antibiotic ciprofloxacin with ~40-fold increased activity against T. gondii in vitro, compared with the original compound. Cipro derivatives are expected to target the parasite's DNA gyrase complex in the apicoplast. The activity of these compounds in vivo, as well as their mode of action, remained thus far uncharacterized. Here, we examined the activity of the Cipro derivatives in vivo, in a model of acute murine toxoplasmosis. In addition, we investigated the cellular effects T. gondii tachyzoites in vitro, by immunofluorescence and transmission electron microscopy (TEM. When compared with Cipro treatment, 7-day treatments with Cipro derivatives increased mouse survival significantly, with 13-25% of mice surviving for up to 60 days post-infection (vs. complete lethality 10 days post-infection, with Cipro treatment. Light microscopy examination early (6 and 24h post-infection revealed that 6-h treatments with Cipro derivatives inhibited the initial event of parasite cell division inside host cells, in an irreversible manner. By TEM and immunofluorescence, the main cellular effects observed after treatment with Cipro derivatives and Cipro were cell scission inhibition--with the appearance of 'tethered' parasites--malformation of the inner membrane complex, and apicoplast enlargement and missegregation. Interestingly, tethered daughter cells resulting from Cipro derivatives, and also Cipro, treatment did not show MORN1 cap or centrocone localization. The biological activity of Cipro derivatives against C. parvum, an apicomplexan species that lacks the apicoplast, is, approximately, 50 fold lower than that in T. gondii tachyzoites, supporting that these compounds targets the apicoplast. Our results

  5. DNA fork displacement rates in human cells

    International Nuclear Information System (INIS)

    Kapp, L.N.; Painter, R.B.

    1981-01-01

    DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 μm/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions. (Auth.)

  6. DNA fork displacement rates in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Kapp, L.N.; Painter, R.B. (California Univ., San Francisco (USA). Lab. of Radiobiology)

    1981-11-27

    DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 ..mu..m/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions.

  7. Planar cell polarity signaling coordinates oriented cell division and cell rearrangement in clonally expanding growth plate cartilage.

    Science.gov (United States)

    Li, Yuwei; Li, Ang; Junge, Jason; Bronner, Marianne

    2017-10-10

    Both oriented cell divisions and cell rearrangements are critical for proper embryogenesis and organogenesis. However, little is known about how these two cellular events are integrated. Here we examine the linkage between these processes in chick limb cartilage. By combining retroviral-based multicolor clonal analysis with live imaging, the results show that single chondrocyte precursors can generate both single-column and multi-column clones through oriented division followed by cell rearrangements. Focusing on single column formation, we show that this stereotypical tissue architecture is established by a pivot-like process between sister cells. After mediolateral cell division, N-cadherin is enriched in the post-cleavage furrow; then one cell pivots around the other, resulting in stacking into a column. Perturbation analyses demonstrate that planar cell polarity signaling enables cells to pivot in the direction of limb elongation via this N-cadherin-mediated coupling. Our work provides new insights into the mechanisms generating appropriate tissue architecture of limb skeleton.

  8. Patterns of DNA methylation in development, division of labor and hybridization in an ant with genetic caste determination.

    Directory of Open Access Journals (Sweden)

    Chris R Smith

    Full Text Available BACKGROUND: DNA methylation is a common regulator of gene expression, including acting as a regulator of developmental events and behavioral changes in adults. Using the unique system of genetic caste determination in Pogonomyrmex barbatus, we were able to document changes in DNA methylation during development, and also across both ancient and contemporary hybridization events. METHODOLOGY/PRINCIPAL FINDINGS: Sodium bisulfite sequencing demonstrated in vivo methylation of symmetric CG dinucleotides in P. barbatus. We also found methylation of non-CpG sequences. This validated two bioinformatics methods for predicting gene methylation, the bias in observed to expected ratio of CpG dinucleotides and the density of CpG/TpG single nucleotide polymorphisms (SNP. Frequencies of genomic DNA methylation were determined for different developmental stages and castes using ms-AFLP assays. The genetic caste determination system (GCD is probably the product of an ancestral hybridization event between P. barbatus and P. rugosus. Two lineages obligately co-occur within a GCD population, and queens are derived from intra-lineage matings whereas workers are produced from inter-lineage matings. Relative DNA methylation levels of queens and workers from GCD lineages (contemporary hybrids were not significantly different until adulthood. Virgin queens had significantly higher relative levels of DNA methylation compared to workers. Worker DNA methylation did not vary among developmental stages within each lineage, but was significantly different between the currently hybridizing lineages. Finally, workers of the two genetic caste determination lineages had half as many methylated cytosines as workers from the putative parental species, which have environmental caste determination. CONCLUSIONS/SIGNIFICANCE: These results suggest that DNA methylation may be a conserved regulatory mechanism moderating division of labor in both bees and ants. Current and historic

  9. DNA synthesis in irradiated mammalian cells

    International Nuclear Information System (INIS)

    Painter, R.B.; California Univ., San Francisco; Young, B.R.

    1987-01-01

    One of the first responses observed in S phase mammalian cells that have suffered DNA damage is the inhibition of initiation of DNA replicons. In cells exposed to ionizing radiation, a single-strand break appears to be the stimulus for this effect, whereby the initiation of many adjacent replicons (a replicon cluster) is blocked by a single-strand break in any one of them. In cells exposed to ultraviolet light (u.v.), replicon initiation is blocked at fluences that induce about one pyrimidine dimer per replicon. The inhibition of replicon initiation by u.v. in Chinese hamster cells that are incapable of excising pyrimidine dimers from their DNA is virtually the same as in cells that are proficient in dimer excision. Therefore, a single-strand break formed during excision repair of pyrimidine dimers is not the stimulus for inhibition of replicon initiation in u.v.-irradiated cells. Considering this fact, as well as the comparative insensitivity of human ataxia telangiectasia cells to u.v.-induced inhibition of replicon initiation, we propose that a relatively rare lesion is the stimulus for u.v. -induced inhibition of replicon initiation. (author

  10. Exposure to Sub-lethal 2,4-Dichlorophenoxyacetic Acid Arrests Cell Division and Alters Cell Surface Properties in Escherichia coli

    Science.gov (United States)

    Bhat, Supriya V.; Kamencic, Belma; Körnig, André; Shahina, Zinnat; Dahms, Tanya E. S.

    2018-01-01

    Escherichia coli is a robust, easily adaptable and culturable bacterium in vitro, and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force – laser scanning confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in E. coli WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests E. coli cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage. PMID:29472899

  11. Prenatal Cell-Free DNA Screening

    Science.gov (United States)

    ... poses no physical risks for you or your baby. While prenatal cell-free DNA screening might cause anxiety, it might help you avoid the need for more invasive tests, treatment or monitoring during your pregnancy. Keep in mind, however, that ...

  12. Colocalization and interaction between elongasome and divisome during a preparative cell division phase in Escherichia coli

    NARCIS (Netherlands)

    Ploeg, van der R.; Verheul, J.; Vischer, N.O.E.; Alexeeva, S.V.; Hoogendoorn, E.; Postma, M.; Banzhaf, M.; Vollmer, W.; Blaauwen, den T.

    2013-01-01

    The rod-shaped bacterium Escherichia coli grows by insertion of peptidoglycan into the lateral wall during cell elongation and synthesis of new poles during cell division. The monofunctional transpeptidases PBP2 and PBP3 are part of specialized protein complexes called elongasome and divisome,

  13. A Bistable Circuit Involving SCARECROW-RETINOBLASTOMA Integrates Cues to Inform Asymmetric Stem Cell Division

    Science.gov (United States)

    Cruz-Ramírez, Alfredo; Díaz-Triviño, Sara; Blilou, Ikram; Grieneisen, Verônica A.; Sozzani, Rosangela; Zamioudis, Christos; Miskolczi, Pál; Nieuwland, Jeroen; Benjamins, René; Dhonukshe, Pankaj; Caballero-Pérez, Juan; Horvath, Beatrix; Long, Yuchen; Mähönen, Ari Pekka; Zhang, Hongtao; Xu, Jian; Murray, James A.H.; Benfey, Philip N.; Bako, Laszlo; Marée, Athanasius F.M.; Scheres, Ben

    2012-01-01

    SUMMARY In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOMA-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a “flip flop” that constrains asymmetric cell division to the stem cell region. PMID:22921914

  14. The progression of the intra-erythrocytic cell cycle of Plasmodium falciparum and the role of the centriolar plaques in asynchronous mitotic division during schizogony

    DEFF Research Database (Denmark)

    Arnot, David E; Ronander, Elena; Bengtsson, Dominique C

    2011-01-01

    The cell division cycle and mitosis of intra-erythrocytic (IE) Plasmodium falciparum are poorly understood aspects of parasite development which affect malaria molecular pathogenesis. Specifically, the timing of the multiple gap (G), DNA synthesis (S) and chromosome separation (M) phases of paras......The cell division cycle and mitosis of intra-erythrocytic (IE) Plasmodium falciparum are poorly understood aspects of parasite development which affect malaria molecular pathogenesis. Specifically, the timing of the multiple gap (G), DNA synthesis (S) and chromosome separation (M) phases...... of parasite mitosis are not well defined, nor whether genome divisions are immediately followed by cleavage of the nuclear envelope. Curiously, daughter merozoite numbers do not follow the geometric expansion expected from equal numbers of binary divisions, an outcome difficult to explain using the standard...

  15. Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions, Exploiting Sites of Cell Division and Senescent Cell Extrusion.

    Directory of Open Access Journals (Sweden)

    Guillaume Golovkine

    2016-01-01

    Full Text Available To achieve systemic infection, bacterial pathogens must overcome the critical and challenging step of transmigration across epithelial barriers. This is particularly true for opportunistic pathogens such as Pseudomonas aeruginosa, an agent which causes nosocomial infections. Despite extensive study, details on the mechanisms used by this bacterium to transmigrate across epithelial tissues, as well as the entry sites it uses, remain speculative. Here, using real-time microscopy and a model epithelial barrier, we show that P. aeruginosa employs a paracellular transmigration route, taking advantage of altered cell-cell junctions at sites of cell division or when senescent cells are expelled from the cell layer. Once a bacterium transmigrates, it is followed by a cohort of bacteria using the same entry point. The basal compartment is then invaded radially from the initial penetration site. Effective transmigration and propagation require type 4 pili, the type 3 secretion system (T3SS and a flagellum, although flagellum-deficient bacteria can occasionally invade the basal compartment from wounded areas. In the basal compartment, the bacteria inject the T3SS toxins into host cells, disrupting the cytoskeleton and focal contacts to allow their progression under the cells. Thus, P. aeruginosa exploits intrinsic host cell processes to breach the epithelium and invade the subcellular compartment.

  16. Roles of Mitochondrial DNA Mutations in Stem Cell Ageing

    Directory of Open Access Journals (Sweden)

    Tianhong Su

    2018-03-01

    Full Text Available Mitochondrial DNA (mtDNA mutations accumulate in somatic stem cells during ageing and cause mitochondrial dysfunction. In this review, we summarize the studies that link mtDNA mutations to stem cell ageing. We discuss the age-related behaviours of the somatic mtDNA mutations in stem cell populations and how they potentially contribute to stem cell ageing by altering mitochondrial properties in humans and in mtDNA-mutator mice. We also draw attention to the diverse fates of the mtDNA mutations with different origins during ageing, with potential selective pressures on the germline inherited but not the somatic mtDNA mutations.

  17. Division of labor in biofilms : The ecology of cell differentiation

    NARCIS (Netherlands)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    The dense aggregation of cells on a surface, as seen in biofilms, inevitably results in both environmental and cellular heterogeneity. For example, nutrient gradients can trigger cells to differentiate into various phenotypic states. Not only do cells adapt physiologically to the local environmental

  18. Drosophila Sulf1 is required for the termination of intestinal stem cell division during regeneration.

    Science.gov (United States)

    Takemura, Masahiko; Nakato, Hiroshi

    2017-01-15

    Stem cell division is activated to trigger regeneration in response to tissue damage. The molecular mechanisms by which this stem cell mitotic activity is properly repressed at the end of regeneration are poorly understood. Here, we show that a specific modification of heparan sulfate is crucial for regulating Drosophila intestinal stem cell (ISC) division during normal midgut homeostasis and regeneration. Loss of the extracellular heparan sulfate endosulfatase Sulf1 resulted in increased ISC division during normal homeostasis, which was caused by upregulation of mitogenic signaling including the JAK-STAT, EGFR and Hedgehog pathways. Using a regeneration model, we found that ISCs failed to properly halt division at the termination stage in Sulf1 mutants, showing that Sulf1 is required for terminating ISC division at the end of regeneration. We propose that post-transcriptional regulation of mitogen signaling by heparan sulfate structural modifications provides a new regulatory step for precise temporal control of stem cell activity during regeneration. © 2017. Published by The Company of Biologists Ltd.

  19. Mechanisms of dealing with DNA damage in terminally differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Fortini, P. [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy); Dogliotti, E., E-mail: eugenia.dogliotti@iss.it [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy)

    2010-03-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  20. Mechanisms of dealing with DNA damage in terminally differentiated cells

    International Nuclear Information System (INIS)

    Fortini, P.; Dogliotti, E.

    2010-01-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  1. A plant U-box protein, PUB4, regulates asymmetric cell division and cell proliferation in the root meristem

    NARCIS (Netherlands)

    Kinoshita, A.; Hove, ten C.A.; Tabata, R.; Yamada, M.; Shimizu, N.; Ishida, T.; Yamaguchi, K.; Shigenobu, S.; Takebayashi, Y.; Luchies, J.; Kobayashi, M.; Kurata, T.; Wada, T.; Seo, M.; Hasebe, M.; Blilou, I.; Fukuda, H.; Scheres, B.; Heidstra, R.; Kamiya, Y.; Sawa, S.

    2015-01-01

    The root meristem (RM) is a fundamental structure that is responsible for postembryonic root growth. The RM contains the quiescent center (QC), stem cells and frequently dividing meristematic cells, in which the timing and the frequency of cell division are tightly regulated. In Arabidopsis

  2. Regulation of the number of cell division rounds by tissue-specific transcription factors and Cdk inhibitor during ascidian embryogenesis.

    Directory of Open Access Journals (Sweden)

    Mami Kuwajima

    Full Text Available Mechanisms that regulate the number of cell division rounds during embryogenesis have remained largely elusive. To investigate this issue, we used the ascidian, which develops into a tadpole larva with a small number of cells. The embryonic cells divide 11.45 times on average from fertilization to hatching. The number of cell division rounds varies depending on embryonic lineages. Notochord and muscle consist of large postmitotic cells and stop dividing early in developing embryos. Here we show that conversion of mesenchyme to muscle cell fates by inhibition of inductive FGF signaling or mis-expression of a muscle-specific key transcription factor for muscle differentiation, Tbx6, changed the number of cell divisions in accordance with the altered fate. Tbx6 likely activates a putative mechanism to halt cell division at a specific stage. However, precocious expression of Tbx6 has no effect on progression of the developmental clock itself. Zygotic expression of a cyclin-dependent kinase inhibitor, CKI-b, is initiated in muscle and then in notochord precursors. CKI-b is possibly downstream of tissue-specific key transcription factors of notochord and muscle. In the two distinct muscle lineages, postmitotic muscle cells are generated after 9 and 8 rounds of cell division depending on lineage, but the final cell divisions occur at a similar developmental stage. CKI-b gene expression starts simultaneously in both muscle lineages at the 110-cell stage, suggesting that CKI-b protein accumulation halts cell division at a similar stage. The difference in the number of cell divisions would be due to the cumulative difference in cell cycle length. These results suggest that muscle cells do not count the number of cell division rounds, and that accumulation of CKI-b protein triggered by tissue-specific key transcription factors after cell fate determination might act as a kind of timer that measures elapsed time before cell division termination.

  3. DNA asymmetry in stem cells - immortal or mortal?

    Science.gov (United States)

    Yadlapalli, Swathi; Yamashita, Yukiko M

    2013-09-15

    The immortal strand hypothesis proposes that stem cells retain a template copy of genomic DNA (i.e. an 'immortal strand') to avoid replication-induced mutations. An alternative hypothesis suggests that certain cells segregate sister chromatids non-randomly to transmit distinct epigenetic information. However, this area of research has been highly controversial, with conflicting data even from the same cell types. Moreover, historically, the same term of 'non-random sister chromatid segregation' or 'biased sister chromatid segregation' has been used to indicate distinct biological processes, generating a confusion in the biological significance and potential mechanism of each phenomenon. Here, we discuss the models of non-random sister chromatid segregation, and we explore the strengths and limitations of the various techniques and experimental model systems used to study this question. We also describe our recent study on Drosophila male germline stem cells, where sister chromatids of X and Y chromosomes are segregated non-randomly during cell division. We aim to integrate the existing evidence to speculate on the underlying mechanisms and biological relevance of this long-standing observation on non-random sister chromatid segregation.

  4. Polarity, cell division, and out-of-equilibrium dynamics control the growth of epithelial structures

    Science.gov (United States)

    Cerruti, Benedetta; Puliafito, Alberto; Shewan, Annette M.; Yu, Wei; Combes, Alexander N.; Little, Melissa H.; Chianale, Federica; Primo, Luca; Serini, Guido; Mostov, Keith E.; Celani, Antonio

    2013-01-01

    The growth of a well-formed epithelial structure is governed by mechanical constraints, cellular apico-basal polarity, and spatially controlled cell division. Here we compared the predictions of a mathematical model of epithelial growth with the morphological analysis of 3D epithelial structures. In both in vitro cyst models and in developing epithelial structures in vivo, epithelial growth could take place close to or far from mechanical equilibrium, and was determined by the hierarchy of time-scales of cell division, cell–cell rearrangements, and lumen dynamics. Equilibrium properties could be inferred by the analysis of cell–cell contact topologies, and the nonequilibrium phenotype was altered by inhibiting ROCK activity. The occurrence of an aberrant multilumen phenotype was linked to fast nonequilibrium growth, even when geometric control of cell division was correctly enforced. We predicted and verified experimentally that slowing down cell division partially rescued a multilumen phenotype induced by altered polarity. These results improve our understanding of the development of epithelial organs and, ultimately, of carcinogenesis. PMID:24145168

  5. Resveratrol protects mouse embryonic stem cells from ionizing radiation by accelerating recovery from DNA strand breakage.

    Science.gov (United States)

    Denissova, Natalia G; Nasello, Cara M; Yeung, Percy L; Tischfield, Jay A; Brenneman, Mark A

    2012-01-01

    Resveratrol has elicited many provocative anticancer effects in laboratory animals and cultured cells, including reduced levels of oxidative DNA damage, inhibition of tumor initiation and progression and induction of apoptosis in tumor cells. Use of resveratrol as a cancer-preventive agent in humans will require that its anticancer effects not be accompanied by damage to normal tissue stem or progenitor cells. In mouse embryonic stem cells (mESC) or early mouse embryos exposed to ethanol, resveratrol has been shown to suppress apoptosis and promote survival. However, in cells exposed to genotoxic stress, survival may come at the expense of genome stability. To learn whether resveratrol can protect stem cells from DNA damage and to study its effects on genomic integrity, we exposed mESC pretreated with resveratrol to ionizing radiation (IR). Forty-eight hours pretreatment with a comparatively low concentration of resveratrol (10 μM) improved survival of mESC >2-fold after exposure to 5 Gy of X-rays. Cells pretreated with resveratrol sustained the same levels of reactive oxygen species and DNA strand breakage after IR as mock-treated controls, but repaired DNA damage more rapidly and resumed cell division sooner. Frequencies of IR-induced mutation at a chromosomal reporter locus were not increased in cells pretreated with resveratrol as compared with controls, indicating that resveratrol can improve viability in mESC after DNA damage without compromising genomic integrity.

  6. Molecular Programs Underlying Asymmetric Stem Cell Division and Their Disruption in Malignancy.

    Science.gov (United States)

    Mukherjee, Subhas; Brat, Daniel J

    2017-01-01

    Asymmetric division of stem cells is a highly conserved and tightly regulated process by which a single stem cell produces two unequal daughter cells. One retains its stem cell identity while the other becomes specialized through a differentiation program and loses stem cell properties. Coordinating these events requires control over numerous intra- and extracellular biological processes and signaling networks. In the initial stages, critical events include the compartmentalization of fate determining proteins within the mother cell and their subsequent passage to the appropriate daughter cell in order to direct their destiny. Disturbance of these events results in an altered dynamic of self-renewing and differentiation within the cell population, which is highly relevant to the growth and progression of cancer. Other critical events include proper asymmetric spindle assembly, extrinsic regulation through micro-environmental cues, and non-canonical signaling networks that impact cell division and fate determination. In this review, we discuss mechanisms that maintain the delicate balance of asymmetric cell division in normal tissues and describe the current understanding how some of these mechanisms are deregulated in cancer.

  7. Induced DNA repair pathway in mammalian cells

    International Nuclear Information System (INIS)

    Overberg, R.

    1985-01-01

    The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 μM cycloheximide subsequent to ultraviolet irradiation is lower than that of cells incubated without cycloheximide. The drop in survival is considerably larger than that produced by incubation of unirradiated cells with cycloheximide. The phenomenon was also observed when PtK-2 cells were incubated with emetine, another protein synthesis inhibitor, or with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, a RNA synthesis inhibitor. PtK cells which received a preliminary UV treatment followed by an incubation period without cycloheximide and then a second irradiation and 24 hour incubation with cycloheximide, survived the effects of the second irradiation better than cells which were incubated in the presence of cycloheximide after the first and second UV irradiation. The application of cycloheximide for 24 hours after UV irradiation of PtK cells resulted in one-half as many 6-thioguanine resistant cells as compared to the number of 6-thioguanine resistant cells found when cycloheximide was not used. These experiments indicate that a UV-inducible cycloheximide-sensitive DNA repair pathway is present in PtK and xeroderma pigmentosum cells, which is error-prone in PtK cells

  8. An experimental and computational framework to build a dynamic protein atlas of human cell division

    OpenAIRE

    Kavur, Marina; Kavur, Marina; Kavur, Marina; Ellenberg, Jan; Peters, Jan-Michael; Ladurner, Rene; Martinic, Marina; Kueblbeck, Moritz; Nijmeijer, Bianca; Wachsmuth, Malte; Koch, Birgit; Walther, Nike; Politi, Antonio; Heriche, Jean-Karim; Hossain, M.

    2017-01-01

    Essential biological functions of human cells, such as division, require the tight coordination of the activity of hundreds of proteins in space and time. While live cell imaging is a powerful tool to study the distribution and dynamics of individual proteins after fluorescence tagging, it has not yet been used to map protein networks due to the lack of systematic and quantitative experimental and computational approaches. Using the cell and nuclear boundaries as landmarks, we generated a 4D ...

  9. Repetitious nature of repaired DNA in mammalian cells

    International Nuclear Information System (INIS)

    1978-01-01

    The report consists of three appendices, as follows: summary of preliminary studies of the comparative DNA repair in normal lymphoblastoid and Burkitt's lymphoma cell lines; nonuniform reassociation of human lymphoblastoid cell DNA repair replicated following methyl methane sulfonate treatment; and preliminary DNA single-strand breakage studies in the L5178Y cell line

  10. Increased DNA-repair in spleen cells of M. Hodgkin

    International Nuclear Information System (INIS)

    Frischauf, H.; Neumann, E.; Howanietz, L.; Dolejs, I.; Tuschl, H.; Altmann, H.

    1974-11-01

    In spleen cells of control patients and cells of Morbus Hodgkin, DNA-repair after gamma- and UV-irradiation was determined measuring the incorporated 3H-thymidine activity in the DNA. Additionally, the ratio of labeled cells compared to non-labeled cells and the grains per cell were evaluated by autoradiographic investigations. DNA-content per cell was measured using pulsecytophotometry. A significant increase of DNA-repair capacity after gamma-irradiation was found by density gradient centrifugation in alkaline sucrose. The same trend could be shown by investigations of unscheduled DNA-synthesis using autoradiographic method. (author)

  11. Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.

    Science.gov (United States)

    Berezuk, Alison M; Goodyear, Mara; Khursigara, Cezar M

    2014-08-22

    In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Engineered cell-cell communication via DNA messaging

    Directory of Open Access Journals (Sweden)

    Ortiz Monica E

    2012-09-01

    Full Text Available Abstract Background Evolution has selected for organisms that benefit from genetically encoded cell-cell communication. Engineers have begun to repurpose elements of natural communication systems to realize programmed pattern formation and coordinate other population-level behaviors. However, existing engineered systems rely on system-specific small molecules to send molecular messages among cells. Thus, the information transmission capacity of current engineered biological communication systems is physically limited by specific biomolecules that are capable of sending only a single message, typically “regulate transcription.” Results We have engineered a cell-cell communication platform using bacteriophage M13 gene products to autonomously package and deliver heterologous DNA messages of varying lengths and encoded functions. We demonstrate the decoupling of messages from a common communication channel via the autonomous transmission of various arbitrary genetic messages. Further, we increase the range of engineered DNA messaging across semisolid media by linking message transmission or receipt to active cellular chemotaxis. Conclusions We demonstrate decoupling of a communication channel from message transmission within engineered biological systems via the autonomous targeted transduction of user-specified heterologous DNA messages. We also demonstrate that bacteriophage M13 particle production and message transduction occurs among chemotactic bacteria. We use chemotaxis to improve the range of DNA messaging, increasing both transmission distance and communication bit rates relative to existing small molecule-based communication systems. We postulate that integration of different engineered cell-cell communication platforms will allow for more complex spatial programming of dynamic cellular consortia.

  13. Overly long centrioles and defective cell division upon excess of the SAS-4-related protein CPAP.

    Science.gov (United States)

    Kohlmaier, Gregor; Loncarek, Jadranka; Meng, Xing; McEwen, Bruce F; Mogensen, Mette M; Spektor, Alexander; Dynlacht, Brian D; Khodjakov, Alexey; Gönczy, Pierre

    2009-06-23

    The centrosome is the principal microtubule organizing center (MTOC) of animal cells. Accurate centrosome duplication is fundamental for genome integrity and entails the formation of one procentriole next to each existing centriole, once per cell cycle. The procentriole then elongates to eventually reach the same size as the centriole. The mechanisms that govern elongation of the centriolar cylinder and their potential relevance for cell division are not known. Here, we show that the SAS-4-related protein CPAP is required for centrosome duplication in cycling human cells. Furthermore, we demonstrate that CPAP overexpression results in the formation of abnormally long centrioles. This also promotes formation of more than one procentriole in the vicinity of such overly long centrioles, eventually resulting in the presence of supernumerary MTOCs. This in turn leads to multipolar spindle assembly and cytokinesis defects. Overall, our findings suggest that centriole length must be carefully regulated to restrict procentriole number and thus ensure accurate cell division.

  14. Sporulation-specific cell division defects in ylmE mutants of Streptomyces coelicolor are rescued by additional deletion of ylmD.

    Science.gov (United States)

    Zhang, Le; Willemse, Joost; Hoskisson, Paul A; van Wezel, Gilles P

    2018-05-09

    Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that deletion of ylmD (SCO2081) or ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, has major consequences for sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with low viability and high heat sensitivity. FtsZ-ring formation was also highly disturbed in ylmE mutants. Deletion of ylmD had a far less severe effect on sporulation. Interestingly, the additional deletion of ylmD restored sporulation to the ylmE null mutant. YlmD and YlmE are not part of the divisome, but instead localize diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work reveals a function for YlmD and YlmE in the control of sporulation-specific cell division in S. coelicolor, whereby the presence of YlmD alone results in major developmental defects.

  15. Effect of anolyte on growth and division of Chinese hamster cancerous cells

    Directory of Open Access Journals (Sweden)

    saeed Mohammadzadeh

    2009-04-01

    Full Text Available Background: At present, cancer can be controlled by chemotherapy, but unfortunately, this method has strong side effects and scientist try to reduce them using different substances. 2 kinds of activated water called anolyte and catholyte have electrochemical property and antibacterial and oxidative properties respectively. The aim of this research is to study the effect of anolyte on growth and division of cancerous cells. Materials and Methods: In this research, different concentration of anolyte, 1 . 7, 2, 5,8.3 and 10 percent of anolyte and control with 2 and 5 percent of serum physiologic were added on converted cell of Chinese hamster (line b11dii-FAF28 clone 237 in 12 plastic and 15 glass flasks. After adding, converted cell was counted with the help of hoemocytometer and microscope. Data of experiment analyzed and results compared by t test, as well as using Excell software their diagrams were drawn. Results: The results indicated that anolyte had significant effect on cancer cells. In concentration of 1.7% cell division was decreased but in concentration of 8.3 %, division of cancerous cells was blocked and cells were fixed. Conclusion: Considering the low amount of sodium chloride in anolyte, it seems that, this solution (Anolyte hasn’t side effects and advers effect on the cells body.

  16. SEPT9_v1 Functions in Breast Cancer Cell Division

    Science.gov (United States)

    2012-01-01

    in PBS containing 3% PFA and stained with antibodies to SEPT2, gp135/podocalyxin (3F2/D8 mouse hybridoma supernatant), and Na/K-ATPase ( chicken ...Science 317(5836):372–376. Rohatgi R, Snell WJ. 2010. The ciliary membrane. Curr Opin Cell Biol 22(4):541–546. Romio L, Fry AM, Winyard PJD, Malcolm...Making sense of cilia and flag- ella. J Cell Biol 179(4):575–582. Snell WJ, Pan J, Wang Q. 2004. Cilia and flagella revealed: From flagellar assembly in

  17. Structural and functional characterizations of SsgB, a conserved activator of developmental cell division in morphologically complex actinomycetes.

    Science.gov (United States)

    Xu, Qingping; Traag, Bjørn A; Willemse, Joost; McMullan, Daniel; Miller, Mitchell D; Elsliger, Marc-André; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Bakolitsa, Constantina; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chruszcz, Maksymilian; Clayton, Thomas; Das, Debanu; Deller, Marc C; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Grzechnik, Slawomir K; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Krishna, S Sri; Kumar, Abhinav; Marciano, David; Minor, Wladek; Mommaas, A Mieke; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L; Sefcovic, Natasha; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Wang, Shuren; Weekes, Dana; Hodgson, Keith O; Wooley, John; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A; van Wezel, Gilles P

    2009-09-11

    SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 A resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic "whirly" single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners.

  18. Structural and Functional Characterizations of SsgB, a Conserved Activator of Developmental Cell Division in Morphologically Complex Actinomycetes*

    Science.gov (United States)

    Xu, Qingping; Traag, Bjørn A.; Willemse, Joost; McMullan, Daniel; Miller, Mitchell D.; Elsliger, Marc-André; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chruszcz, Maksymilian; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Grzechnik, Slawomir K.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; Minor, Wladek; Mommaas, A. Mieke; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; van den Bedem, Henry; Wang, Shuren; Weekes, Dana; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.; van Wezel, Gilles P.

    2009-01-01

    SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 Å resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic “whirly” single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners. PMID:19567872

  19. Exploring Middle School Students' Conceptions of the Relationship between Genetic Inheritance and Cell Division

    Science.gov (United States)

    Williams, Michelle; DeBarger, Angela Haydel; Montgomery, Beronda L.; Zhou, Xuechun; Tate, Erika

    2012-01-01

    This study examines students' understanding of the normative connections between key concepts of cell division, including both mitosis and meiosis, and underlying biological principles that are critical for an in-depth understanding of genetic inheritance. Using a structural equation modeling method, we examine middle school students'…

  20. Microgravity effects during fertilization, cell division, development, and calcium metabolism in sea urchins

    Science.gov (United States)

    Schatten, Heide

    1996-01-01

    The overall objectives of this project are to explore the role of microgravity during fertilization, early development, cytoskeletal organization, and skeletal calcium deposition in a model development system: the sea urchin eggs and embryos. While pursuing these objectives, we have also helped to develop, test, and fly the Aquatic Research Facility (ARF) system. Cells were fixed at preselected time points to preserve the structures and organelles of interest with regards to cell biology events during development. The protocols used for the analysis of the results had been developed during the earlier part of this research and were applied for post-flight analysis using light and (immuno)fluorescence microscopy, scanning electron microscopy, and transmission electron microscopy. The structures of interest are: microtubules during fertilization, cell division, and cilia movement; microfilaments during cell surface restructuring and cell division; centrosomes and centrioles during cell division, cell differentiation, and cilia formation and movement; membranes, Golgi, endoplasmic reticulum, mitochondria, and chromosomes at all stages of development; and calcium deposits during spicule formation in late-stage embryos. In addition to further explore aspects important or living in space, several aspects of this research are also aimed at understanding diseases that affect humans on Earth which may be accelerated in space.

  1. Building the perfect parasite: cell division in apicomplexa.

    Directory of Open Access Journals (Sweden)

    Boris Striepen

    2007-06-01

    Full Text Available Apicomplexans are pathogens responsible for malaria, toxoplasmosis, and crytposporidiosis in humans, and a wide range of livestock diseases. These unicellular eukaryotes are stealthy invaders, sheltering from the immune response in the cells of their hosts, while at the same time tapping into these cells as source of nutrients. The complexity and beauty of the structures formed during their intracellular development have made apicomplexans the darling of electron microscopists. Dramatic technological progress over the last decade has transformed apicomplexans into respectable genetic model organisms. Extensive genomic resources are now available for many apicomplexan species. At the same time, parasite transfection has enabled researchers to test the function of specific genes through reverse and forward genetic approaches with increasing sophistication. Transfection also introduced the use of fluorescent reporters, opening the field to dynamic real time microscopic observation. Parasite cell biologists have used these tools to take a fresh look at a classic problem: how do apicomplexans build the perfect invasion machine, the zoite, and how is this process fine-tuned to fit the specific niche of each pathogen in this ancient and very diverse group? This work has unearthed a treasure trove of novel structures and mechanisms that are the focus of this review.

  2. Attempt to stimulate cell division in Saccharomyces cerevisiae with weak ultraviolet light

    International Nuclear Information System (INIS)

    Quickenden, T.I.; Matich, A.J.; Pung, S.H.; Tilbury, R.N.

    1989-01-01

    Liquid cultures of the yeast Saccharomyces cerevisiae were irradiated with weak light having irradiances ranging from ca. 1 X 10(2) to 5 X 10(9) photons cm-2 s-1 and at wavelengths ranging from 200 to 700 nm. When particular care was taken to control the temperature of the cultures and the flow rate of oxygen, no evidence was obtained for stimulation of either yeast growth or division by the incident light. These results do not support the claims of early workers that very low intensity uv light can stimulate cell division in living organisms

  3. Xanthomonas citri MinC Oscillates from Pole to Pole to Ensure Proper Cell Division and Shape

    NARCIS (Netherlands)

    Soibelmann Glock Lorenzoni, André; Dantas, Giordanni; Bergsma, Tessa; Ferreira, Henrique; Scheffers, Dirk

    2017-01-01

    Xanthomonas citri (Xac) is the causal agent of citrus canker, a disease that affects citrus crops and causes economic impact worldwide. To further characterize cell division in this plant pathogen, we investigated the role of the protein MinC in cell division, chromosome segregation, and

  4. From centriole biogenesis to cellular function: centrioles are essential for cell division at critical developmental stages.

    Science.gov (United States)

    Rodrigues-Martins, Ana; Riparbelli, Maria; Callaini, Giuliano; Glover, David M; Bettencourt-Dias, Monica

    2008-01-01

    Centrioles are essential for the formation of cilia, flagella and centrosome organization. Abnormalities in centrosome structure and number in many cancers can be associated with aberrant cell division and genomic instability.(1,2) Canonical centriole duplication occurs in coordination with the cell division cycle, such that a single new "daughter" centriole arises next to each "mother" centriole. If destroyed, or eliminated during development, centrioles can form de novo.(3-5) Here we discuss our recent data demonstrating a molecular pathway that operates in both de novo and canonical centriole biogenesis involving SAK/PLK4, SAS-6 and SAS-4.(6) We showed that centriole biogenesis is a self-assembly process locally triggered by high SAK/PLK4 activity that may or not be associated with an existing centriole. SAS-6 acts downstream of SAK/PLK4 to organize nine precentriolar units, which we call here enatosomes, fitting together laterally and longitudinally, specifying a tube-like centriole precursor.(7,8) The identification of mutants impaired in centriole biogenesis has permitted the study of the physiological consequences of their absence in the whole organism. In Drosophila, centrioles are not necessary for somatic cell divisions.(9,10) However, we show here that mitotic abnormalities arise in syncytial SAK/PLK4-derived mutant embryos resulting in lethality. Moreover male meiosis fails in both SAK/PLK4 and DSAS-4 mutant spermatids that have no centrioles. These results show diversity in the need for centrioles in cell division. This suggests that tissue specific constraints selected for different contributions of centrosome-independent and dependent mechanisms in spindle function. This heterogeneity should be taken into account both in reaching an understanding of spindle function and when designing drugs that target cell division.

  5. FtsZ-less prokaryotic cell division as well as FtsZ- and dynamin-less chloroplast and non-photosynthetic plastid division

    Directory of Open Access Journals (Sweden)

    Shin-Ya eMiyagishima

    2014-09-01

    Full Text Available The chloroplast division machinery is a mixture of a stromal FtsZ-based complex descended from a cyanobacterial ancestor of chloroplasts and a cytosolic dynamin-related protein (DRP 5B-based complex derived from the eukaryotic host. Molecular genetic studies have shown that each component of the division machinery is normally essential for normal chloroplast division. However, several exceptions have been found. In the absence of the FtsZ ring, nonphotosynthetic plastids are able to proliferate, likely by elongation and budding. Depletion of DRP5B impairs, but does not stop chloroplast division. Chloroplasts in glaucophytes, which possesses a peptidoglycan (PG layer, divide without DRP5B. Certain parasitic eukaryotes possess nonphotosynthetic plastids of secondary endosymbiotic origin, but neither FtsZ nor DRP5B is encoded in their genomes. Elucidation of the FtsZ- and/or DRP5B-less chloroplast division mechanism will lead to a better understanding of the function and evolution of the chloroplast division machinery and the finding of the as-yet-unknown mechanism that is likely involved in chloroplast division. Recent studies have shown that FtsZ was lost from a variety of prokaryotes, many of which lost PG by regressive evolution. In addition, even some of the FtsZ-bearing bacteria are able to divide when FtsZ and PG are depleted experimentally. In some cases, alternative mechanisms for cell division, such as budding by an increase of the cell surface-to-volume ratio, are proposed. Although PG is believed to have been lost from chloroplasts other than in glaucophytes, there is some indirect evidence for the existence of PG in chloroplasts. Such information is also useful for understanding how nonphotosynthetic plastids are able to divide in FtsZ-depleted cells and the reason for the retention of FtsZ in chloroplast division. Here we summarize information to facilitate analyses of FtsZ- and/or DRP5B-less chloroplast and nonphotosynthetic plastid

  6. An Aminopropyl Carbazole Derivative Induces Neurogenesis by Increasing Final Cell Division in Neural Stem Cells.

    Science.gov (United States)

    Shin, Jae-Yeon; Kong, Sun-Young; Yoon, Hye Jin; Ann, Jihyae; Lee, Jeewoo; Kim, Hyun-Jung

    2015-07-01

    P7C3 and its derivatives, 1-(3,6-dibromo-9H-carbazol-9-yl)-3-(p-tolylamino)propan-2-ol (1) and N-(3-(3,6-dibromo-9H-carbazol-9-yl)-2-hydroxypropyl)-N-(3-methoxyphenyl)-4-methylbenzenesulfonamide (2), were previously reported to increase neurogenesis in rat neural stem cells (NSCs). Although P7C3 is known to increase neurogenesis by protecting newborn neurons, it is not known whether its derivatives also have protective effects to increase neurogenesis. In the current study, we examined how 1 induces neurogenesis. The treatment of 1 in NSCs increased numbers of cells in the absence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), while not affecting those in the presence of growth factors. Compound 1 did not induce astrocytogenesis during NSC differentiation. 5-Bromo-2'-deoxyuridine (BrdU) pulsing experiments showed that 1 significantly enhanced BrdU-positive neurons. Taken together, our data suggest that 1 promotes neurogenesis by the induction of final cell division during NSC differentiation.

  7. Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

    Science.gov (United States)

    Petojevic, Tatjana; Pesavento, James J.; Costa, Alessandro; Liang, Jingdan; Wang, Zhijun; Berger, James M.; Botchan, Michael R.

    2015-01-01

    DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2–7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2–7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2–7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel. PMID:25561522

  8. An archaebacterial homologue of the essential eubacterial cell division protein FtsZ.

    OpenAIRE

    Baumann, P; Jackson, S P

    1996-01-01

    Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes,. respectively). Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria. Currently, little is known about the archaeal cell division apparatus. In Bacteria, a crucial component of the cell d...

  9. Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells

    International Nuclear Information System (INIS)

    Akimoto, Miho; Niikura, Mamoru; Ichikawa, Masami; Yonekawa, Hiromichi; Nakada, Kazuto; Honma, Yoshio; Hayashi, Jun-Ichi

    2005-01-01

    Recent studies showed high frequencies of homoplasmic mtDNA mutations in various human tumor types, suggesting that the mutated mtDNA haplotypes somehow contribute to expression of tumor phenotypes. We directly addressed this issue by isolating mouse mtDNA-less (ρ 0 ) cells for complete mtDNA replacement between normal cells and their carcinogen-induced transformants, and examined the effect of the mtDNA replacement on expression of tumorigenicity, a phenotype forming tumors in nude mice. The results showed that genome chimera cells carrying nuclear DNA from tumor cells and mtDNA from normal cells expressed tumorigenicity, whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells did not. These observations provided direct evidence that nuclear DNA, but not mtDNA, is responsible for carcinogen-induced malignant transformation, although it remains possible that mtDNA mutations and resultant respiration defects may influence the degree of malignancy, such as invasive or metastatic properties

  10. Cell division in Escherichia coli BS-12 is hypersensitive to deoxyribonucleic acid damage by ultraviolet light

    International Nuclear Information System (INIS)

    Bridges, B.A.; Mottershead, R.P.; Green, M.H.

    1977-01-01

    Escherichia coli BS-12 uvrA lon is hypersensitive to ultraviolet light. On minimal agar plates at densities in excess of about 10(7) bacteria per plate, as few as one or two photoreversible pyrimidine dimers in the entire genome are sufficient to cause inhibition of cell division. Most of the resulting filaments are unable to divide or form a viable colony. Inhibition of cell division appears to be a rapid consequence of replication of deoxyribonucleic acid containing a pyrimidine dimer. Photoreversibility of the inhibition of cell division persists indefinitely, indicating that the continued presence of the pyrimidine dimers (or the continued generation of daughter strand gaps) is necessary to maintain the division-inhibited state. In view of the kinetics for the production of filamentation by ultraviolet light and the extremely low average inducing fluence (0.03 J/m2), it is concluded that the initiating signal is not the same as that causing other inducible phenomena such as prophage induction or Weigle reactivation

  11. Regulation of DNA repair processes in mammalian cell

    International Nuclear Information System (INIS)

    Bil'din, V.N.; Sergina, T.B.; Zhestyanikov, V.D.

    1992-01-01

    A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase α and β (aphidicolin) and DNA polymerase β (2', 3'-dideoxythjymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gy) or by γ-ray irradiation (150 Gy) is more sensitive to aphidicolin and mitogen-simulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase α

  12. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1990-01-01

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60 Co γ-rays, or to KUR radiation in the presence or absence of 10 B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10 B(n,α) 7 Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  13. Fission yeast cells undergo nuclear division in the absence of spindle microtubules.

    Directory of Open Access Journals (Sweden)

    Stefania Castagnetti

    2010-10-01

    Full Text Available Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.

  14. Involvement of DNA methylation in the control of cell growth during heat stress in tobacco BY-2 cells.

    Science.gov (United States)

    Centomani, Isabella; Sgobba, Alessandra; D'Addabbo, Pietro; Dipierro, Nunzio; Paradiso, Annalisa; De Gara, Laura; Dipierro, Silvio; Viggiano, Luigi; de Pinto, Maria Concetta

    2015-11-01

    The alteration of growth patterns, through the adjustment of cell division and expansion, is a characteristic response of plants to environmental stress. In order to study this response in more depth, the effect of heat stress on growth was investigated in tobacco BY-2 cells. The results indicate that heat stress inhibited cell division, by slowing cell cycle progression. Cells were stopped in the pre-mitotic phases, as shown by the increased expression of CycD3-1 and by the decrease in the NtCycA13, NtCyc29 and CDKB1-1 transcripts. The decrease in cell length and the reduced expression of Nt-EXPA5 indicated that cell expansion was also inhibited. Since DNA methylation plays a key role in controlling gene expression, the possibility that the altered expression of genes involved in the control of cell growth, observed during heat stress, could be due to changes in the methylation state of their promoters was investigated. The results show that the altered expression of CycD3-1 and Nt-EXPA5 was consistent with changes in the methylation state of the upstream region of these genes. These results suggest that DNA methylation, controlling the expression of genes involved in plant development, contributes to growth alteration occurring in response to environmental changes.

  15. Saturation of DNA repair in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, F E; Setlow, R B

    1979-01-01

    Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20 J/m/sup 2/. We measured excision repair in normal human fibroblasts up to 80 J/m/sup 2/. The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus, and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24h following two 20 J/m/sup 2/ doses separated by 24h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the rate limiting steps in repair.

  16. The C. elegans engrailed homolog ceh-16 regulates the self-renewal expansion division of stem cell-like seam cells.

    Science.gov (United States)

    Huang, Xinxin; Tian, E; Xu, Yanhua; Zhang, Hong

    2009-09-15

    Stem cells undergo symmetric and asymmetric division to maintain the dynamic equilibrium of the stem cell pool and also to generate a variety of differentiated cells. The homeostatic mechanism controlling the choice between self-renewal and differentiation of stem cells is poorly understood. We show here that ceh-16, encoding the C. elegans ortholog of the transcription factor Engrailed, controls symmetric and asymmetric division of stem cell-like seam cells. Loss of function of ceh-16 causes certain seam cells, which normally undergo symmetric self-renewal expansion division with both daughters adopting the seam cell fate, to divide asymmetrically with only one daughter retaining the seam cell fate. The human engrailed homolog En2 functionally substitutes the role of ceh-16 in promoting self-renewal expansion division of seam cells. Loss of function of apr-1, encoding the C. elegans homolog of the Wnt signaling component APC, results in transformation of self-renewal maintenance seam cell division to self-renewal expansion division, leading to seam cell hyperplasia. The apr-1 mutation suppresses the seam cell division defect in ceh-16 mutants. Our study reveals that ceh-16 interacts with the Wnt signaling pathway to control the choice between self-renewal expansion and maintenance division and also demonstrates an evolutionarily conserved function of engrailed in promoting cell proliferation.

  17. DNA-repair synthesis in ataxia telangiectasia lymphoblastoid cells

    Energy Technology Data Exchange (ETDEWEB)

    Ford, M.D.; Houldsworth, J.; Lavin, M.F. (Queensland Univ., Brisbane (Australia). Dept. of Biochemistry)

    1981-12-01

    The ability of a number of Epstein-Barr virus-transformed lymphoblastoid cells from ataxia telangiectasia (AT) patients to repair ..gamma..-radiation damage to DNA was determined. All of these AT cells were previously shown to be hypersensitive to ..gamma..-radiation. Two methods were used to determine DNA-repair synthesis: isopycnic gradient analysis and a method employing hydroxyurea to inhibit semiconservative DNA synthesis. Control, AT heterozygote and AT homozygote cells were demonstrated to have similar capacities for repair of radiation damage to DNA. In addition at high radiation doses (10-40 krad) the extent of inhibition of DNA synthesis was similar in the different cell types.

  18. A local maximum in gibberellin levels regulates maize leaf growth by spatial control of cell division.

    Science.gov (United States)

    Nelissen, Hilde; Rymen, Bart; Jikumaru, Yusuke; Demuynck, Kirin; Van Lijsebettens, Mieke; Kamiya, Yuji; Inzé, Dirk; Beemster, Gerrit T S

    2012-07-10

    Plant growth rate is largely determined by the transition between the successive phases of cell division and expansion. A key role for hormone signaling in determining this transition was inferred from genetic approaches and transcriptome analysis in the Arabidopsis root tip. We used the developmental gradient at the maize leaf base as a model to study this transition, because it allows a direct comparison between endogenous hormone concentrations and the transitions between dividing, expanding, and mature tissue. Concentrations of auxin and cytokinins are highest in dividing tissues, whereas bioactive gibberellins (GAs) show a peak at the transition zone between the division and expansion zone. Combined metabolic and transcriptomic profiling revealed that this GA maximum is established by GA biosynthesis in the division zone (DZ) and active GA catabolism at the onset of the expansion zone. Mutants defective in GA synthesis and signaling, and transgenic plants overproducing GAs, demonstrate that altering GA levels specifically affects the size of the DZ, resulting in proportional changes in organ growth rates. This work thereby provides a novel molecular mechanism for the regulation of the transition from cell division to expansion that controls organ growth and size. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Termination of T cell priming relies on a phase of unresponsiveness promoting disengagement from APCs and T cell division.

    Science.gov (United States)

    Bohineust, Armelle; Garcia, Zacarias; Beuneu, Hélène; Lemaître, Fabrice; Bousso, Philippe

    2018-05-07

    T cells are primed in secondary lymphoid organs by establishing stable interactions with antigen-presenting cells (APCs). However, the cellular mechanisms underlying the termination of T cell priming and the initiation of clonal expansion remain largely unknown. Using intravital imaging, we observed that T cells typically divide without being associated to APCs. Supporting these findings, we demonstrate that recently activated T cells have an intrinsic defect in establishing stable contacts with APCs, a feature that was reflected by a blunted capacity to stop upon T cell receptor (TCR) engagement. T cell unresponsiveness was caused, in part, by a general block in extracellular calcium entry. Forcing TCR signals in activated T cells antagonized cell division, suggesting that T cell hyporesponsiveness acts as a safeguard mechanism against signals detrimental to mitosis. We propose that transient unresponsiveness represents an essential phase of T cell priming that promotes T cell disengagement from APCs and favors effective clonal expansion. © 2018 Bohineust et al.

  20. DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

    Directory of Open Access Journals (Sweden)

    Katherine S Lawrence

    2015-04-01

    Full Text Available Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR, which monitors DNA integrity, and the spindle assembly checkpoint (SAC, which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

  1. [Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development].

    Science.gov (United States)

    Bellé, Robert; Le Bouffant, Ronan; Morales, Julia; Cosson, Bertrand; Cormier, Patrick; Mulner-Lorillon, Odile

    2007-01-01

    Cell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology

  2. Live Cell Characterization of DNA Aggregation Delivered through Lipofection.

    Science.gov (United States)

    Mieruszynski, Stephen; Briggs, Candida; Digman, Michelle A; Gratton, Enrico; Jones, Mark R

    2015-05-27

    DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.

  3. Individuality and universality in the growth-division laws of single E. coli cells

    Science.gov (United States)

    Kennard, Andrew S.; Osella, Matteo; Javer, Avelino; Grilli, Jacopo; Nghe, Philippe; Tans, Sander J.; Cicuta, Pietro; Cosentino Lagomarsino, Marco

    2016-01-01

    The mean size of exponentially dividing Escherichia coli cells in different nutrient conditions is known to depend on the mean growth rate only. However, the joint fluctuations relating cell size, doubling time, and individual growth rate are only starting to be characterized. Recent studies in bacteria reported a universal trend where the spread in both size and doubling times is a linear function of the population means of these variables. Here we combine experiments and theory and use scaling concepts to elucidate the constraints posed by the second observation on the division control mechanism and on the joint fluctuations of sizes and doubling times. We found that scaling relations based on the means collapse both size and doubling-time distributions across different conditions and explain how the shape of their joint fluctuations deviates from the means. Our data on these joint fluctuations highlight the importance of cell individuality: Single cells do not follow the dependence observed for the means between size and either growth rate or inverse doubling time. Our calculations show that these results emerge from a broad class of division control mechanisms requiring a certain scaling form of the "division hazard rate function," which defines the probability rate of dividing as a function of measurable parameters. This "model free" approach gives a rationale for the universal body-size distributions observed in microbial ecosystems across many microbial species, presumably dividing with multiple mechanisms. Additionally, our experiments show a crossover between fast and slow growth in the relation between individual-cell growth rate and division time, which can be understood in terms of different regimes of genome replication control.

  4. Raman spectroscopy for DNA quantification in cell nucleus.

    Science.gov (United States)

    Okotrub, K A; Surovtsev, N V; Semeshin, V F; Omelyanchuk, L V

    2015-01-01

    Here we demonstrate the feasibility of a novel approach to quantify DNA in cell nuclei. This approach is based on spectroscopy analysis of Raman light scattering, and avoids the problem of nonstoichiometric binding of dyes to DNA, as it directly measures the signal from DNA. Quantitative analysis of nuclear DNA contribution to Raman spectrum could be reliably performed using intensity of a phosphate mode at 1096 cm(-1) . When compared to the known DNA standards from cells of different animals, our results matched those values at error of 10%. We therefore suggest that this approach will be useful to expand the list of DNA standards, to properly adjust the duration of hydrolysis in Feulgen staining, to assay the applicability of fuchsines for DNA quantification, as well as to measure DNA content in cells with complex hydrolysis patterns, when Feulgen densitometry is inappropriate. © 2014 International Society for Advancement of Cytometry.

  5. Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

    Science.gov (United States)

    Pu, Xinzhu; Wang, Zemin; Klaunig, James E

    2015-08-06

    Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

  6. Cell division genes promote asymmetric interaction between Numb and Notch in the Drosophila CNS.

    Science.gov (United States)

    Wai, P; Truong, B; Bhat, K M

    1999-06-01

    Cell intrinsic and cell extrinsic factors mediate asymmetric cell divisions during neurogenesis in the Drosophila embryo. In the NB4-2->GMC-1->RP2/sib lineage, one of the well-studied neuronal lineages in the ventral nerve cord, the Notch (N) signaling interacts with the asymmetrically localized Numb (Nb) to specify sibling neuronal fates to daughter cells of GMC-1. In this current study, we have investigated asymmetric cell fate specifications by N and Nb in the context of cell cycle. We have used loss-of-function mutations in N and nb, cell division mutants cyclinA (cycA), regulator of cyclin A1 (rca1) and string/cdc25 phosphatase (stg), and the microtubule destabilizing agent, nocodazole, to investigate this issue. We report that the loss of cycA, rca1 or stg leads to a block in the division of GMC-1, however, this GMC-1 exclusively adopts an RP2 identity. While the loss of N leads to the specification of RP2 fates to both progeny of GMC-1 and loss of nb results in the specification of sib fates to these daughter cells, the GMC-1 in the double mutant between nb and cycA assumes a sib fate. These epistasis results indicate that both N and nb function downstream of cell division genes and that progression through cell cycle is required for the asymmetric localization of Nb. In the absence of entry to metaphase, the Nb protein prevents the N signaling from specifying sib fate to the RP2/sib precursor. These results are also consistent with our finding that the sib cell is specified as RP2 in N; nb double mutants. Finally, our results show that nocodazole-arrested GMC-1 in wild-type embryos randomly assumes either an RP2 fate or a sib fate. This suggests that microtubules are involved in mediating the antagonistic interaction between Nb and N during RP2 and sib fate specification.

  7. Cytosolic DNA Sensor Upregulation Accompanies DNA Electrotransfer in B16.F10 Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Katarina Znidar

    2016-01-01

    Full Text Available In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNβ mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI, DEAD (Asp-Glu-Ala-Asp box polypeptide 60 (DDX60, and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo.

  8. A Resistance-Nodulation-Cell Division Family Xenobiotic Efflux Pump in an Obligate Anaerobe, Porphyromonas gingivalis

    OpenAIRE

    Ikeda, Takeshi; Yoshimura, Fuminobu

    2002-01-01

    Porphyromonas gingivalis, a gram-negative obligate anaerobe, contains two homologs of an Escherichia coli resistance-nodulation-cell division-type multidrug exporter gene, acrB, in putative operons, together with homologs of membrane fusion protein gene acrA and outer membrane channel gene tolC. MIC determination and accumulation assays with mutants with disruptions of one or more genes showed that one cluster, named xepCAB, pumped out multiple agents including rifampin, puromycin, and ethidi...

  9. Characterization of substances that restore impaired cell division of UV-irradiated E. coli B

    International Nuclear Information System (INIS)

    Yoshiyama, Y.; Shimoii, H.; Tamura, G.

    1981-01-01

    Substances which restore impaired cell division in UV-irradiated E. coli B were surveyed among various bacteria. The active substance was found only in several genera of Gram-negative bacteria, i.e., Escherichia, Enterobacter, Salmonella and some species of Pseudomonas. The activity in the dialyzed cell extract of E. coli B/r was observed in the presence of β-NAD and was enhanced by Mg 2+ and Mn 2+ . The active substance was very labile, but the activity was protected by 1 mM dithiothreitol in the process of purification. The activity of a fraction recovered through DEAE-cellulose column chromatography was stimulated by the presence of membrane fraction. Upon treatment with lipid-degrading enzymes and proteases, the division-stimulating activity was lost or reduced. It appears that the inactivation by lipase and phospholipase A2 was due to the formation of lysophospholipids and that a proteinous substance participated in the recovery of impaired cell division of UV-irradiated E. coli B

  10. Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions.

    Directory of Open Access Journals (Sweden)

    Nan Li

    Full Text Available Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG. In this study we used mouse embryonic stem (MES and mouse embryonic fibroblast (MEF cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS. Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR defects.

  11. Timing of initiation of macronuclear DNA synthesis is set during the preceding cell cycle in Paramecium tetraurelia: analysis of the effects of abrupt changes in nutrient level

    International Nuclear Information System (INIS)

    Ching, A.S.L.; Berger, J.D.

    1986-01-01

    In many eukaryotic organisms, initiation of DNA synthesis is associated with a major control point within the cell cycle and reflects the commitment of the cell to the DNA replication-division portion of the cell cycle. In paramecium, the timing of DNA synthesis initiation is established prior to fission during the preceding cell cycle. DNA synthesis normally starts at 0.25 in the cell cycle. When dividing cells are subjected to abrupt nutrient shift-up by transfer from a chemostat culture to medium with excess food, or shift-down from a well-fed culture to exhausted medium, DNA synthesis initiation in the post-shift cell cycle occurs at 0.25 of the parental cell cycle and not at either 0.25 in the post-shift cell cycle or at 0.25 in the equilibrium cell cycle produced under the post-shift conditions. The long delay prior to initiation of DNA synthesis following nutritional shift-up is not a consequence of continued slow growth because the rate of protein synthesis increases rapidly to the normal level after shift-up. Analysis of the relation between increase in cell mass and initiation of DNA synthesis following nutritional shifts indicates that increase in cell mass, per se, is neither a necessary nor a sufficient condition for initiation of DNA synthesis, in spite of the strong association between accumulation of cell mass and initiation of DNA synthesis in cells growing under steady-state conditions

  12. A millifluidic study of cell-to-cell heterogeneity in growth-rate and cell-division capability in populations of isogenic cells of Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Shima P Damodaran

    Full Text Available To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers and a significant subpopulation of slowly dividing cells (slow-growers. These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

  13. LocZ Is a New Cell Division Protein Involved in Proper Septum Placement in Streptococcus pneumoniae

    Science.gov (United States)

    Holečková, Nela; Molle, Virginie; Buriánková, Karolína; Benada, Oldřich; Kofroňová, Olga; Ulrych, Aleš; Branny, Pavel

    2014-01-01

    ABSTRACT How bacteria control proper septum placement at midcell, to guarantee the generation of identical daughter cells, is still largely unknown. Although different systems involved in the selection of the division site have been described in selected species, these do not appear to be widely conserved. Here, we report that LocZ (Spr0334), a newly identified cell division protein, is involved in proper septum placement in Streptococcus pneumoniae. We show that locZ is not essential but that its deletion results in cell division defects and shape deformation, causing cells to divide asymmetrically and generate unequally sized, occasionally anucleated, daughter cells. LocZ has a unique localization profile. It arrives early at midcell, before FtsZ and FtsA, and leaves the septum early, apparently moving along with the equatorial rings that mark the future division sites. Consistently, cells lacking LocZ also show misplacement of the Z-ring, suggesting that it could act as a positive regulator to determine septum placement. LocZ was identified as a substrate of the Ser/Thr protein kinase StkP, which regulates cell division in S. pneumoniae. Interestingly, homologues of LocZ are found only in streptococci, lactococci, and enterococci, indicating that this close phylogenetically related group of bacteria evolved a specific solution to spatially regulate cell division. PMID:25550321

  14. Chlamydia co-opts the rod shape-determining proteins MreB and Pbp2 for cell division.

    Science.gov (United States)

    Ouellette, Scot P; Karimova, Gouzel; Subtil, Agathe; Ladant, Daniel

    2012-07-01

    Chlamydiae are obligate intracellular bacterial pathogens that have extensively reduced their genome in adapting to the intracellular environment. The chlamydial genome contains only three annotated cell division genes and lacks ftsZ. How this obligate intracellular pathogen divides is uncharacterized. Chlamydiae contain two high-molecular-weight (HMW) penicillin binding proteins (Pbp) implicated in peptidoglycan synthesis, Pbp2 and Pbp3/FtsI. We show here, using HMW Pbp-specific penicillin derivatives, that both Pbp2 and Pbp3 are essential for chlamydial cell division. Ultrastructural analyses of antibiotic-treated cultures revealed distinct phenotypes: Pbp2 inhibition induced internal cell bodies within a single outer membrane whereas Pbp3 inhibition induced elongated phenotypes with little internal division. Each HMW Pbp interacts with the Chlamydia cell division protein FtsK. Chlamydiae are coccoid yet contain MreB, a rod shape-determining protein linked to Pbp2 in bacilli. Using MreB-specific antibiotics, we show that MreB is essential for chlamydial growth and division. Importantly, co-treatment with MreB-specific and Pbp-specific antibiotics resulted in the MreB-inhibited phenotype, placing MreB upstream of Pbp function in chlamydial cell division. Finally, we showed that MreB also interacts with FtsK. We propose that, in Chlamydia, MreB acts as a central co-ordinator at the division site to substitute for the lack of FtsZ in this bacterium. © 2012 Blackwell Publishing Ltd.

  15. Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells

    International Nuclear Information System (INIS)

    Mamo, Tewodros; Mladek, Ann C.; Shogren, Kris L.; Gustafson, Carl; Gupta, Shiv K.; Riester, Scott M.; Maran, Avudaiappan; Galindo, Mario; Wijnen, Andre J. van; Sarkaria, Jann N.; Yaszemski, Michael J.

    2017-01-01

    Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PK CS ), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PK CS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PK CS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PK CS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PK CS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma. - Highlights: • DNA-PKcs is consistently expressed in human osteosarcoma tissue and cell lines. • The DNA-PKcs inhibitor, KU60648, effectively radiosensitizes osteosarcoma cells. • Combining KU60648 with radiation increases G2/M accumulation and DNA damage.

  16. Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1 exhibits slower rates of cell division and shoot induction

    Directory of Open Access Journals (Sweden)

    Schulz Burkhard

    2010-12-01

    Full Text Available Abstract Background Plant growth depends on both cell division and cell expansion. Plant hormones, including brassinosteroids (BRs, are central to the control of these two cellular processes. Despite clear evidence that BRs regulate cell elongation, their roles in cell division have remained elusive. Results Here, we report results emphasizing the importance of BRs in cell division. An Arabidopsis BR biosynthetic mutant, dwarf7-1, displayed various characteristics attributable to slower cell division rates. We found that the DWARF4 gene which encodes for an enzyme catalyzing a rate-determining step in the BR biosynthetic pathways, is highly expressed in the actively dividing callus, suggesting that BR biosynthesis is necessary for dividing cells. Furthermore, dwf7-1 showed noticeably slower rates of callus growth and shoot induction relative to wild-type control. Flow cytometric analyses of the nuclei derived from either calli or intact roots revealed that the cell division index, which was represented as the ratio of cells at the G2/M vs. G1 phases, was smaller in dwf7-1 plants. Finally, we found that the expression levels of the genes involved in cell division and shoot induction, such as PROLIFERATING CELL NUCLEAR ANTIGEN2 (PCNA2 and ENHANCER OF SHOOT REGENERATION2 (ESR2, were also lower in dwf7-1 as compared with wild type. Conclusions Taken together, results of callus induction, shoot regeneration, flow cytometry, and semi-quantitative RT-PCR analysis suggest that BRs play important roles in both cell division and cell differentiation in Arabidopsis.

  17. Induced pluripotent stem cells with a pathological mitochondrial DNA deletion

    Science.gov (United States)

    Cherry, Anne B. C.; Gagne, Katelyn E.; McLoughlin, Erin M.; Baccei, Anna; Gorman, Bryan; Hartung, Odelya; Miller, Justine D.; Zhang, Jin; Zon, Rebecca L.; Ince, Tan A.; Neufeld, Ellis J.; Lerou, Paul H.; Fleming, Mark D.; Daley, George Q.; Agarwal, Suneet

    2013-01-01

    In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. PMID:23400930

  18. Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs

    Science.gov (United States)

    Schatten, Heide; Schatten, Gerald; Balczon, Ron; Simerly, Calvin; Mazia, Daniel

    1986-01-01

    The behavior of centrosomes during the stages of fertilization and cell division in mouse oocytes and in sea urchin eggs was monitored in an immunofluorescence microscope, using autoimmune centrosomal antiserum derived from a patient with scleroderma to label the centrosomal material. These observations showed that centrosomes reproduce during the interphase and aggregate and separate during cell mitosis. Results supported the hypothesis of Mazia (1984), who proposed that centrosomes are 'flexible bodies'. It was also found that, while the sea urchin centrosomes are paternally inherited as was initially proposed by Bovery (1904), the mouse centrosomes are of maternal origin.

  19. DNA nanotechnology from the test tube to the cell.

    Science.gov (United States)

    Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A; Seelig, Georg

    2015-09-01

    The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology - applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems - lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

  20. DNA nanotechnology from the test tube to the cell

    Science.gov (United States)

    Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A.; Seelig, Georg

    2015-09-01

    The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology -- applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems -- lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

  1. ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

    Science.gov (United States)

    Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

    2010-10-01

    The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

  2. Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    Imbalanced dNTP pools are highly mutagenic due to a deleterious effect on DNA polymerase fidelity. Mitochondrial DNA defects, including mutations and deletions, are commonly found in a wide variety of different cancer types. In order to further study the interconnection between dNTP pools...... and mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces an S phase delay in different human osteosarcoma cell lines. The UV pulse also has a destabilizing effect...... shows that normal mitochondrial function is prerequisite for retaining stable dNTP pools upon DNA damage. Therefore it is likely that mitochondrial deficiency defects may cause an increase in DNA mutations by disrupting dNTP pool balance....

  3. DNA MUTAGENESIS IN PANAX GINSENG CELL CULTURES

    Directory of Open Access Journals (Sweden)

    Kiselev K.V.

    2012-08-01

    Full Text Available At the present time, it is well documented that plant tissue culture induces a number of mutations and chromosome rearrangements termed “somaclonal variations”. However, little is known about the nature and the molecular mechanisms of the tissue culture-induced mutagenesis and the effects of long-term subculturing on the rate and specific features of the mutagenesis. The aim of the present study was to investigate and compare DNA mutagenesis in different genes of Panax ginseng callus cultures of different age. It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during long-term cultivation of transgenic cell cultures of P. ginseng. In the present work, we analyzed nucleotide sequences of selected plant gene families in a 2-year-old and 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We analysed sequence variability between the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL and dammarenediol synthase (DDS genes, which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK genes, which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-year-old callus culture was markedly higher than that in cultivated plants but lower than that in the 20-year-old callus culture of P. ginseng. Most of the mutations in the 2- and 20-year-old P. ginseng calli were A↔G and T↔C transitions. The number of nonsynonymous mutations was higher in the 2- and 20-year-old callus cultures than the number of nonsynonymous mutations in the cultivated plants of P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using methylation-sensitive DNA fragmentation

  4. Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

    Science.gov (United States)

    Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Tamaru, Yutaka; Ogata, Shin; Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

    2015-12-01

    Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

  5. Circulating nucleic acids damage DNA of healthy cells by integrating ...

    Indian Academy of Sciences (India)

    2015-02-04

    Feb 4, 2015 ... detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic .... 2.7 Development of single-cell clones from DNAfs- ... DNA was isolated to generate whole genome libraries for.

  6. Application of DNA fingerprints for cell-line individualization.

    OpenAIRE

    Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

    1990-01-01

    DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they d...

  7. [DNA-dependent DNA polymerase induced by herpes virus papio (HVP) in producing cells].

    Science.gov (United States)

    D'iachenko, A G; Beriia, L Ia; Matsenko, L D; Kakubava, V V; Kokosh, L V

    1980-11-01

    A new DNA polymerase was found in the cells of suspension lymphoblastoid cultures, which produce lymphotropic baboon herpes virus (HVP). The enzyme was isolated in a partially purified form. In some properties the enzyme differs from other cellular DNA polymerases. The HVP-induced DNA polymerase has the molecular weight of 1,6 x 10(5) and sedimentation coefficient of about 8S. The enzyme is resistant to high salt concentrations and N-ethylmaleimide, but shows a pronounced sensitivity to phosphonoacetate. The enzyme effectively copies "activated" DNA and synthetic deoxyribohomopolymers. The attempts to detect the DNA polymerase activity in HVP virions were unsuccessful.

  8. The effect of vinblastine on DNA metabolism in tumour cells

    International Nuclear Information System (INIS)

    Cabela, E.; Klein, W.

    1976-01-01

    Studies on the influence of Vinblastine on normal and tumor cells show that semiconservative DNA-synthesis correlates with the enzymatic activity of thymidinkinase. DNA-repair-investigations performed with Yoshida-Ascites cells indicate an inhibition effect on the ligase system after Vinblastine-treatment and gamma irradiation. (author)

  9. Effect of microgravity environment on cell wall regeneration, cell divisions, growth, and differentiation of plants from protoplasts (7-IML-1)

    Science.gov (United States)

    Rasmussen, Ole

    1992-01-01

    The primary goal of this project is to investigate if microgravity has any influence on growth and differentiation of protoplasts. Formation of new cell walls on rapeseed protoplasts takes place within the first 24 hours after isolation. Cell division can be observed after 2-4 days and formation of cell aggregates after 5-7 days. Therefore, it is possible during the 7 day IML-1 Mission to investigate if cell wall formation, cell division, and cell differentiation are influenced by microgravity. Protoplasts of rapeseeds and carrot will be prepared shortly before launch and injected into 0.6 ml polyethylene bags. Eight bags are placed in an aluminum block inside the ESA Type 1 container. The containers are placed at 4 C in PTCU's and transferred to orbiter mid-deck. At 4 C all cell processes are slowed down, including cell wall formation. Latest access to the shuttle will be 12 hours before launch. In orbit the containers will be transferred from the PTC box to the 22 C Biorack incubator. The installation of a 1 g centrifuge in Biorack will make it possible to distinguish between effects of near weightlessness and effects caused by cosmic radiation and other space flight factors including vibrations. Parallel control experiments will be carried out on the ground. Other aspects of the experiment are discussed.

  10. Effect of human cell malignancy on activity of DNA polymerase iota.

    Science.gov (United States)

    Kazakov, A A; Grishina, E E; Tarantul, V Z; Gening, L V

    2010-07-01

    An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase iota (Pol iota) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg2+, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn2+, that activates homogeneous Pol iota preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg2+, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn2+ the activity of Pol iota was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol iota activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol iota in both cases, though to a different extent. In the presence of Mn2+ the Pol iota activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn2+-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol iota. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by

  11. EzrA: a spectrin-like scaffold in the bacterial cell division machinery

    Directory of Open Access Journals (Sweden)

    Robert M Cleverley

    2015-01-01

    Full Text Available Much progress has been made in identifying the components of the divisome, the assembly of proteins that undertakes the vital process of cell division in bacteria. However, how the highly interdependent processes on either side of the membrane are coordinated during division is a major unresolved question. How is the degradation and synthesis of the cell wall on the outside of the cell coordinated with cytokinesis and membrane fission, which are driven from the inside of the cell by the tubulin homologue FtsZ? A possible key mediator of such coordination is the membrane protein EzrA, as it interacts both with FtsZ and the penicillin binding proteins (PBPs that synthesize peptidoglycan. Cleverley et al. [Nature Communications (2014 5, 5421] have recently solved the crystal structure of the cytoplasmic domain of B. subtilis EzrA, which points to an important scaffolding role for EzrA in the divisome. The structure resembles the eukaryotic, cytoskeletal spectrin proteins, which link actin filaments in the cytoskeleton and also connect the actin cytoskeleton to membrane-bound integrin proteins.

  12. A specific role of iron in promoting meristematic cell division during adventitious root formation.

    Science.gov (United States)

    Hilo, Alexander; Shahinnia, Fahimeh; Druege, Uwe; Franken, Philipp; Melzer, Michael; Rutten, Twan; von Wirén, Nicolaus; Hajirezaei, Mohammad-Reza

    2017-07-10

    Adventitious root (AR) formation is characterized by a sequence of physiological and morphological processes and determined by external factors, including mineral nutrition, the impacts of which remain largely elusive. Morphological and anatomical evaluation of the effects of mineral elements on AR formation in leafy cuttings of Petunia hybrida revealed a striking stimulation by iron (Fe) and a promotive action of ammonium (NH4+). The optimal application period for these nutrients corresponded to early division of meristematic cells in the rooting zone and coincided with increased transcript levels of mitotic cyclins. Fe-localization studies revealed an enhanced allocation of Fe to the nuclei of meristematic cells in AR initials. NH4+ supply promoted AR formation to a lesser extent, most likely by favoring the availability of Fe. We conclude that Fe acts locally by promoting cell division in the meristematic cells of AR primordia. These results highlight a specific biological function of Fe in AR development and point to an unexploited importance of Fe for the vegetative propagation of plants from cuttings. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  13. The amount of DNA damage needed to activate the radiation-induced G2 checkpoint varies between single cells

    International Nuclear Information System (INIS)

    Tkacz-Stachowska, Kinga; Lund-Andersen, Christin; Velissarou, Angeliki; Myklebust, June H.; Stokke, Trond; Syljuåsen, Randi G.

    2011-01-01

    Background and purpose: The radiation-induced G2 checkpoint helps facilitate DNA repair before cell division. However, recent work has revealed that human cells often escape the G2 checkpoint with unrepaired DNA breaks. The purpose was to explore whether G2 checkpoint activation occurs according to a threshold level of DNA damage. Materials and methods: G2 checkpoint activation was assayed at 75–90 min and 24–48 h after X-ray irradiation of BJ diploid fibroblasts and U2OS osteosarcoma cells. Multiparameter flow cytometry with pacific blue barcoding, and flow cytometry-based sorting of phospho-H3 positive cells to microscope slides, were used to examine the DNA damage marker γ-H2AX in individual mitotic cells that had escaped the G2 checkpoint. Results: For all radiation doses and times tested, the number of γ-H2AX foci varied between individual mitotic cells. At 75 min the median levels of γ-H2AX in mitotic cells increased with higher radiation doses. At 24–48 h, following a prolonged G2 checkpoint, cells were more resistant to checkpoint re-activation by a second dose of radiation. Conclusion: Our results suggest that different amounts of DNA damage are needed to activate the G2 checkpoint in individual cells. Such single cell variation in checkpoint activation may potentially contribute to radiation-induced genomic instability.

  14. Influence of the circadian rhythm in cell division on radiation-induced mitotic delay in vivo

    International Nuclear Information System (INIS)

    Rubin, N.A.

    1980-01-01

    All mitotically active normal tissues in mammals investigated to date demonstrate a circadian rhythm in cell division. The murine corneal epithelium is a practical and advantageous tissue model for studying this phenomenon. In animals synchronized to a light-dark (LD) schedule, one sees predictably reproducible occurrences of peaks and troughs in the mitotic index (MI) within each 24-hour (h) period. One of the harmful effects of ionizing radiation on dividing cells is mitotic delay, reported to be a G 2 block in cells approaching mitosis. Affected cells are not killed but are inhibited from entering mitosis and are delayed for a span of time reported to be dose and cell cycle dependent. In the classical description of mitotic delay, MI of irradiated cells begins to drop in relation to the control, which is plotted as a straight line, uniform throughout the experiment. After the damage is repaired, delayed cells can enter mitosis along with other cells in the pool unaffected by the radiation, resulting in a MI higher than control levels. The span of delay and the occurrence of recovery are assumed to be constant for a given dose and tissue under similar experimental conditions. First described in asynchronously-dividing tissue culture cells, this concept is also extrapolated to the in vivo situation

  15. A general framework for modeling growth and division of mammalian cells.

    Science.gov (United States)

    Gauthier, John H; Pohl, Phillip I

    2011-01-06

    Modeling the cell-division cycle has been practiced for many years. As time has progressed, this work has gone from understanding the basic principles to addressing distinct biological problems, e.g., the nature of the restriction point, how checkpoints operate, the nonlinear dynamics of the cell cycle, the effect of localization, etc. Most models consist of coupled ordinary differential equations developed by the researchers, restricted to deal with the interactions of a limited number of molecules. In the future, cell-cycle modeling--and indeed all modeling of complex biologic processes--will increase in scope and detail. A framework for modeling complex cell-biologic processes is proposed here. The framework is based on two constructs: one describing the entire lifecycle of a molecule and the second describing the basic cellular machinery. Use of these constructs allows complex models to be built in a straightforward manner that fosters rigor and completeness. To demonstrate the framework, an example model of the mammalian cell cycle is presented that consists of several hundred differential equations of simple mass action kinetics. The model calculates energy usage, amino acid and nucleotide usage, membrane transport, RNA synthesis and destruction, and protein synthesis and destruction for 33 proteins to give an in-depth look at the cell cycle. The framework presented here addresses how to develop increasingly descriptive models of complex cell-biologic processes. The example model of cellular growth and division constructed with the framework demonstrates that large structured models can be created with the framework, and these models can generate non-trivial descriptions of cellular processes. Predictions from the example model include those at both the molecular level--e.g., Wee1 spontaneously reactivates--and at the system level--e.g., pathways for timing-critical processes must shut down redundant pathways. A future effort is to automatically estimate

  16. The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 → S transition

    International Nuclear Information System (INIS)

    Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay; Sarvepalli, Kavitha; Sadhale, Parag P.; Nath, Utpal

    2011-01-01

    Highlights: → TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. → TCP4 expression in yeast retards cell division by blocking G1 → S transition. → Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 → S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 → S arrest is discussed.

  17. DNA repair and radiation sensitivity in mammalian cells

    International Nuclear Information System (INIS)

    Chen, D.J.C.; Stackhouse, M.; Chen, D.S.

    1993-01-01

    Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population

  18. How bacterial cell division might cheat turgor pressure - a unified mechanism of septal division in Gram-positive and Gram-negative bacteria.

    Science.gov (United States)

    Erickson, Harold P

    2017-08-01

    An important question for bacterial cell division is how the invaginating septum can overcome the turgor force generated by the high osmolarity of the cytoplasm. I suggest that it may not need to. Several studies in Gram-negative bacteria have shown that the periplasm is isoosmolar with the cytoplasm. Indirect evidence suggests that this is also true for Gram-positive bacteria. In this case the invagination of the septum takes place within the uniformly high osmotic pressure environment, and does not have to fight turgor pressure. A related question is how the V-shaped constriction of Gram-negative bacteria relates to the plate-like septum of Gram-positive bacteria. I collected evidence that Gram-negative bacteria have a latent capability of forming plate-like septa, and present a model in which septal division is the basic mechanism in both Gram-positive and Gram-negative bacteria. © 2017 WILEY Periodicals, Inc.

  19. Life span prediction from the rate of age-related DNA demethylation in normal and cancer cell lines.

    Science.gov (United States)

    Mazin, A L

    1995-01-01

    A method has been proposed for the Hayflick Limit prediction by the analysis of the 5-methylcytosine content in DNA at earlier and later cell passages. The following facts were used as the basis of the method: (i) the rate of m5C loss from DNA remains approximately constant during cell divisions and it does not depend on the cell donor age; (ii) this rate is inversely proportional to the Hayflick Limit as well as to the life span of cell donor species; (iii) the period corresponded to loss of all m5C residues from the genome coincides with or somewhat exceeds the Hayflick Limit of normal cells. The prognosis of the Hayflick Limit has usually been found in good agreement with the experimental evidences for various human, hamster, and mouse cell lines. The method proposed may be used for early detection of precrisis and cancer cells. The age-related m5C loss may result from accumulation of the m5C-->T+C transitions occurring with DNA methylation in every cell division.

  20. Nek1 silencing slows down DNA repair and blocks DNA damage-induced cell cycle arrest.

    Science.gov (United States)

    Pelegrini, Alessandra Luíza; Moura, Dinara Jaqueline; Brenner, Bethânia Luise; Ledur, Pitia Flores; Maques, Gabriela Porto; Henriques, João Antônio Pegas; Saffi, Jenifer; Lenz, Guido

    2010-09-01

    Never in mitosis A (NIMA)-related kinases (Nek) are evolutionarily conserved proteins structurally related to the Aspergillus nidulans mitotic regulator NIMA. Nek1 is one of the 11 isoforms of the Neks identified in mammals. Different lines of evidence suggest the participation of Nek1 in response to DNA damage, which is also supported by the interaction of this kinase with proteins involved in DNA repair pathways and cell cycle regulation. In this report, we show that cells with Nek1 knockdown (KD) through stable RNA interference present a delay in DNA repair when treated with methyl-methanesulfonate (MMS), hydrogen peroxide (H(2)O(2)) and cisplatin (CPT). In particular, interstrand cross links induced by CPT take much longer to be resolved in Nek1 KD cells when compared to wild-type (WT) cells. In KD cells, phosphorylation of Chk1 in response to CPT was strongly reduced. While WT cells accumulate in G(2)/M after DNA damage with MMS and H(2)O(2), Nek1 KD cells do not arrest, suggesting that G(2)/M arrest induced by the DNA damage requires Nek1. Surprisingly, CPT-treated Nek1 KD cells arrest with a 4N DNA content similar to WT cells. This deregulation in cell cycle control in Nek1 KD cells leads to an increased sensitivity to genotoxic agents when compared to WT cells. These results suggest that Nek1 is involved in the beginning of the cellular response to genotoxic stress and plays an important role in preventing cell death induced by DNA damage.

  1. Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases astrogliogenesis

    Directory of Open Access Journals (Sweden)

    Geissy LL Araújo

    2014-03-01

    Full Text Available The morphogen Sonic Hedgehog (SHH plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

  2. Stem cell identity and template DNA strand segregation.

    Science.gov (United States)

    Tajbakhsh, Shahragim

    2008-12-01

    The quest for stem cell properties to distinguish their identity from that of committed daughters has led to a re-investigation of the notion that DNA strands are not equivalent, and 'immortal' DNA strands are retained in stem cells whereas newly replicated DNA strands segregate to the differentiating daughter cell during mitosis. Whether this process occurs only in stem cells, and also in all tissues, remains unclear. That individual chromosomes can be also partitioned non-randomly raises the question if this phenomenon is related to the immortal DNA hypothesis, and it underscores the need for high-resolution techniques to observe these events empirically. Although initially postulated as a mechanism to avoid DNA replication errors, alternative views including epigenetic regulation and sister chromatid silencing may provide insights into this process.

  3. The Relationship between Cell Number, Division Behavior and Developmental Potential of Cleavage Stage Human Embryos: A Time-Lapse Study.

    Directory of Open Access Journals (Sweden)

    Xiangyi Kong

    Full Text Available Day 3 cleavage embryo transfer is routine in many assisted reproductive technology centers today. Embryos are usually selected according to cell number, cell symmetry and fragmentation for transfer. Many studies have showed the relationship between cell number and embryo developmental potential. However, there is limited understanding of embryo division behavior and their association with embryo cell number and developmental potential. A retrospective and observational study was conducted to investigate how different division behaviors affect cell number and developmental potential of day 3 embryos by time-lapse imaging. Based on cell number at day 3, the embryos (from 104 IVF/intracytoplasmic sperm injection (ICSI treatment cycles, n = 799 were classified as follows: less than 5 cells (10C; n = 42. Division behavior, morphokinetic parameters and blastocyst formation rate were analyzed in 5 groups of day 3 embryos with different cell numbers. In 10C embryos increased compared to 7-8C embryos (45.8%, 33.3% vs. 11.1%, respectively. In ≥5C embryos, FR and DC significantly reduced developmental potential, whereas 10C. In NB embryos, the cell cycle elongation or shortening was the main cause for abnormally low or high cell number, respectively. After excluding embryos with abnormal division behaviors, the developmental potential, implantation rate and live birth rate of day 3 embryos increased with cell number.

  4. Live imaging of individual cell divisions in mouse neuroepithelium shows asymmetry in cilium formation and Sonic hedgehog response

    Directory of Open Access Journals (Sweden)

    Piotrowska-Nitsche Karolina

    2012-05-01

    Full Text Available Abstract Background Primary cilia are microtubule-based sensory organelles that play important roles in developmental signaling pathways. Recent work demonstrated that, in cell culture, the daughter cell that inherits the older mother centriole generates a primary cilium and responds to external stimuli prior to its sister cell. This asynchrony in timing of cilia formation could be especially critical during development as cell divisions are required for both differentiation and maintenance of progenitor cell niches. Methods Here we integrate several fluorescent markers and use ex vivo live imaging of a single cell division within the mouse E8.5 neuroepithelium to reveal both the formation of a primary cilium and the transcriptional response to Sonic hedgehog in the daughter cells. Results We show that, upon cell division, cilia formation and the Sonic hedgehog response are asynchronous between the daughter cells. Conclusions Our results demonstrate that we can directly observe single cell divisions within the developing neuroepithelium and concomitantly monitor cilium formation or Sonic hedgehog response. We expect this method to be especially powerful in examining whether cellular behavior can lead to both differentiation and maintenance of cells in a progenitor niche.

  5. Plasma Cell-Free DNA in Paediatric Lymphomas

    Science.gov (United States)

    Mussolin, Lara; Burnelli, Roberta; Pillon, Marta; Carraro, Elisa; Farruggia, Piero; Todesco, Alessandra; Mascarin, Maurizio; Rosolen, Angelo

    2013-01-01

    Background: Extracellular circulating DNA (cfDNA) can be found in small amounts in plasma of healthy individuals. Increased levels of cfDNA have been reported in patients with cancer of breast, cervix, colon, liver and it was shown that cfDNA can originate from both tumour and non-tumour cells. Objectives: Levels of cfDNA of a large series of children with lymphoma were evaluated and analyzed in relation with clinical characteristics. Methods: plasma cfDNA levels obtained at diagnosis in 201 paediatric lymphoma patients [43 Hodgkin lymphomas (HL), 45 anaplastic large cell lymphomas (ALCL), 88 Burkitt lymphomas (BL), 17 lymphoblastic (LBL), 8 diffuse large B cell lymphoma (DLBCL)] and 15 healthy individuals were determined using a quantitative PCR assay for POLR2 gene and, in addition, for NPM-ALK fusion gene in ALCL patients. Wilcoxon rank sum test was used to compare plasma levels among different patient subgroups and controls and to analyze relationship between levels of cfDNA and clinical characteristics. Results: Levels of cfDNA in lymphoma patients were significantly higher compared with controls (p<0.0001). CfDNA was associated with median age (p=0.01) in HL, and with stage in ALCL (p=0.01). In HL patients high cfDNA levels were correlated with poor prognosis (p=0.03). In ALCL we found that most of the cfDNA (77%) was non-tumor DNA. Conclusion: level of plasma cfDNA might constitute an important non-invasive tool at diagnosis in lymphoma patients' management; in particular in patients with HL, cfDNA seems to be a promising prognostic biomarker. PMID:23678368

  6. Effects of radiation on the cell division cycle. Using yeasts as models

    International Nuclear Information System (INIS)

    Mann, C.; Marsolier, M.C.

    2000-01-01

    The living organisms, since the appearance on earth of the simplest of them, are submitted to numerous attacks having different origin. They use response systems to the DNA damages coming from these attacks and especially radiations. The cell knows how to take stock of the situation, at different moment of its life, to slow down, eventually to stop its cycle before continuing, after repairing of its DNA and divided itself. These mechanisms have kept a remarkable similarity during the evolution. The study of these systems among yeasts is a precious help to understand the corresponding systems for man and to evaluate the limits but also the possibilities, particularly, in oncology. (N.C.)

  7. DNA replication and repair of Tilapia cells: Pt. 2

    International Nuclear Information System (INIS)

    Chen, J.D.; Yew, F.H.

    1988-01-01

    TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15-34 0 C). We report the effects of temperature on DNA replication and u.v. repair in TO-2 cells. When the cells were moved from 31 0 C to the sublethal high temperature of 37 0 C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8h at 37 0 C the rate of DNA synthesis overshot the 31 0 C control level by 180%. When moved to low temperature (18 0 C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. U.V. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3h at 37, 31 and 25 0 C, but not at 18 0 C. Initiation of nascent DNA synthesis was blocked at 4Jm -2 in TO-2 cells compared with ≤ 1Jm -2 in mammalian cells. After 9Jm -2 u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate. TO-2 cells showed low levels of u.v.-induced excision repair. (author)

  8. CDKL5 localizes at the centrosome and midbody and is required for faithful cell division.

    Science.gov (United States)

    Barbiero, Isabella; Valente, Davide; Chandola, Chetan; Magi, Fiorenza; Bergo, Anna; Monteonofrio, Laura; Tramarin, Marco; Fazzari, Maria; Soddu, Silvia; Landsberger, Nicoletta; Rinaldo, Cinzia; Kilstrup-Nielsen, Charlotte

    2017-07-24

    The cyclin-dependent kinase-like 5 (CDKL5) gene has been associated with rare neurodevelopmental disorders characterized by the early onset of seizures and intellectual disability. The CDKL5 protein is widely expressed in most tissues and cells with both nuclear and cytoplasmic localization. In post-mitotic neurons CDKL5 is mainly involved in dendritic arborization, axon outgrowth, and spine formation while in proliferating cells its function is still largely unknown. Here, we report that CDKL5 localizes at the centrosome and at the midbody in proliferating cells. Acute inactivation of CDKL5 by RNA interference (RNAi) leads to multipolar spindle formation, cytokinesis failure and centrosome accumulation. At the molecular level, we observed that, among the several midbody components we analyzed, midbodies of CDKL5-depleted cells were devoid of HIPK2 and its cytokinesis target, the extrachromosomal histone H2B phosphorylated at S14. Of relevance, expression of the phosphomimetic mutant H2B-S14D, which is capable of overcoming cytokinesis failure in HIPK2-defective cells, was sufficient to rescue spindle multipolarity in CDKL5-depleted cells. Taken together, these results highlight a hitherto unknown role of CDKL5 in regulating faithful cell division by guaranteeing proper HIPK2/H2B functions at the midbody.

  9. Aging of hematopoietic stem cells: DNA damage and mutations?

    Science.gov (United States)

    Moehrle, Bettina M; Geiger, Hartmut

    2016-10-01

    Aging in the hematopoietic system and the stem cell niche contributes to aging-associated phenotypes of hematopoietic stem cells (HSCs), including leukemia and aging-associated immune remodeling. Among others, the DNA damage theory of aging of HSCs is well established, based on the detection of a significantly larger amount of γH2AX foci and a higher tail moment in the comet assay, both initially thought to be associated with DNA damage in aged HSCs compared with young cells, and bone marrow failure in animals devoid of DNA repair factors. Novel data on the increase in and nature of DNA mutations in the hematopoietic system with age, the quality of the DNA damage response in aged HSCs, and the nature of γH2AX foci question a direct link between DNA damage and the DNA damage response and aging of HSCs, and rather favor changes in epigenetics, splicing-factors or three-dimensional architecture of the cell as major cell intrinsic factors of HSCs aging. Aging of HSCs is also driven by a strong contribution of aging of the niche. This review discusses the DNA damage theory of HSC aging in the light of these novel mechanisms of aging of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  10. Targeting the Wolbachia cell division protein FtsZ as a new approach for antifilarial therapy.

    Directory of Open Access Journals (Sweden)

    Zhiru Li

    2011-11-01

    Full Text Available The use of antibiotics targeting the obligate bacterial endosymbiont Wolbachia of filarial parasites has been validated as an approach for controlling filarial infection in animals and humans. Availability of genomic sequences for the Wolbachia (wBm present in the human filarial parasite Brugia malayi has enabled genome-wide searching for new potential drug targets. In the present study, we investigated the cell division machinery of wBm and determined that it possesses the essential cell division gene ftsZ which was expressed in all developmental stages of B. malayi examined. FtsZ is a GTPase thereby making the protein an attractive Wolbachia drug target. We described the molecular characterization and catalytic properties of Wolbachia FtsZ. We also demonstrated that the GTPase activity was inhibited by the natural product, berberine, and small molecule inhibitors identified from a high-throughput screen. Furthermore, berberine was also effective in reducing motility and reproduction in B. malayi parasites in vitro. Our results should facilitate the discovery of selective inhibitors of FtsZ as a novel anti-symbiotic approach for controlling filarial infection. NOTE: The nucleotide sequences reported in this paper are available in GenBank™ Data Bank under the accession number wAlB-FtsZ (JN616286.

  11. [Knockdown of DNA-PKcs inhibits cell cycle and its mechanism of drug-resistant Bel7402/5-Fu hepatocellular carcinoma cells].

    Science.gov (United States)

    Li, Dayu; Liu, Yun; Yu, Chunbo; Liu, Xiping; Fan, Fang

    2017-12-01

    Objective To study the effect of the knock-down of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) on the cell cycle of the multidrug-resistant (MDR) Bel7402/5-Fu hepatocellular carcinoma cells and its MDR mechanism. Methods After cationic liposome-mediated siDNA-PKcs oligonucleotide transfection, the drug sensitivity of Bel7402/5-Fu cells to 5-fluorouracil (5-Fu) and adriamycin (ADM) was determined by MTT assay; the cell cycle were detected by flow cytometry; meanwhile, the protein expressions of cell cycle-related proteins P21, cell cycle protein B1 (cyclin B1), cell cycle division protein 2 (CDC2) were tested by Western blotting; the expressions of ataxia telangiectasia mutated (ATM) and p53 at both mRNA and protein levels were detected by real-time PCR and Western blot analysis. Results The MTT results showed siDNA-PKcs increased the chemotherapeutic sensitivity of Bel7402/5-Fu cells to 5-Fu and ADM. The flow cytometric analysis showed siDNA-PKcs decreased the percentage of S-phase cells but increased the percentage of G2/M phase cells. Western blotting showed siDNA-PKcs increased the protein expression of P21 but decreased cyclinB1 and CDC2 proteins. In addition, siDNA-PKcs also increased the expressions of ATM and p53. Conclusion DNA-PKcs silencing increases P21 while decreases cyclin B1 and CDC2 expressions, and finally induces G2/M phase arrest in Bel7402/5-Fu cells, which may be related to ATM-p53 signaling pathway.

  12. The intersection between DNA damage response and cell death pathways.

    Science.gov (United States)

    Nowsheen, S; Yang, E S

    2012-10-01

    Apoptosis is a finely regulated process that serves to determine the fate of cells in response to various stresses. One such stress is DNA damage, which not only can signal repair processes but is also intimately involved in regulating cell fate. In this review we examine the relationship between the DNA damage/repair response in cell survival and apoptosis following insults to the DNA. Elucidating these pathways and the crosstalk between them is of great importance, as they eventually contribute to the etiology of human disease such as cancer and may play key roles in determining therapeutic response. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

  13. Application of DNA fingerprints for cell-line individualization.

    Science.gov (United States)

    Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

    1990-09-01

    DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.

  14. Simulation of E. coli gene regulation including overlapping cell cycles, growth, division, time delays and noise.

    Directory of Open Access Journals (Sweden)

    Ruoyu Luo

    Full Text Available Due to the complexity of biological systems, simulation of biological networks is necessary but sometimes complicated. The classic stochastic simulation algorithm (SSA by Gillespie and its modified versions are widely used to simulate the stochastic dynamics of biochemical reaction systems. However, it has remained a challenge to implement accurate and efficient simulation algorithms for general reaction schemes in growing cells. Here, we present a modeling and simulation tool, called 'GeneCircuits', which is specifically developed to simulate gene-regulation in exponentially growing bacterial cells (such as E. coli with overlapping cell cycles. Our tool integrates three specific features of these cells that are not generally included in SSA tools: 1 the time delay between the regulation and synthesis of proteins that is due to transcription and translation processes; 2 cell cycle-dependent periodic changes of gene dosage; and 3 variations in the propensities of chemical reactions that have time-dependent reaction rates as a consequence of volume expansion and cell division. We give three biologically relevant examples to illustrate the use of our simulation tool in quantitative studies of systems biology and synthetic biology.

  15. Analysis of epigenetic modifications of DNA in human cells

    DEFF Research Database (Denmark)

    Kristensen, Lasse Sommer; Treppendahl, Marianne Bach; Grønbæk, Kirsten

    2013-01-01

    Epigenetics, the study of somatically heritable changes in gene expression not related to changes in the DNA sequence, is a rapidly expanding research field that plays important roles in healthy as well as in diseased cells. DNA methylation and hydroxymethylation are epigenetic modifications found...

  16. Decreased stability of DNA in cells treated with alkylating agents

    Energy Technology Data Exchange (ETDEWEB)

    Frankfurt, O.S. (Cedars Medical Center, Miami, FL (United States))

    1990-12-01

    A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg{sup 2+}. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl{sub 2}. Thus, the presence of phosphates and MgCl{sub 2} during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S{sub 1} nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.

  17. DNA repair in human xeroderma pigmentosum and Chinese hamster cells

    NARCIS (Netherlands)

    B. Zelle (Bauke)

    1980-01-01

    textabstractAn important feature of living cells is their capacity to maintain the integrity of their hereditary material, the DNA. DNA can be damaged by a variety of physical and chemical agents, among which ultraviolet radiation (UV), ion1z1ng radiation and chemical carcinogens as

  18. DNA synthesis in permeabilized WI38 and MRC5 cells

    International Nuclear Information System (INIS)

    Griffiths, T.D.; Carpenter, J.G.

    1980-01-01

    DNA synthesis was examined in cultures of growing WI38 and MRC5 cells made permeable to deoxyribonucleotides. Cells from late passage cultures showed a reduced rate of deoxythymidine triphosphate (dTTP) uptake as compared to cells from early- to mid-passage cultures. This reduction became evident earlier in WI38 cultures (passage 33) than in MRC5 cultures (passage 41). Although this reduced rate of incorporation appeared to be primarily due to a reduced percentage of replicating (S phase) cells in later passage cultures, some effect on the rate of DNA synthesis in replicating cells was also evident

  19. Role of cell division and self-propulsion in self-organization of 2D cell co-cultures

    Science.gov (United States)

    Das, Moumita; Dey, Supravat; Wu, Mingming; Ma, Minglin

    Self-organization of cells is a key process in developmental and cancer biology. The differential adhesion hypothesis (DAH), which assumes cells as equilibrium liquid droplets and relates the self-assembly of cells to differences in inter-cellular adhesiveness, has been very successful in explaining cellular organization during morphogenesis where neighboring cells have the same non-equilibrium properties (motility, proliferation rate). However, recently it has been experimentally shown that for a co-culture of two different cell types proliferating at different rates, the resulting spatial morphologies cannot be explained using the DAH alone. Motivated by this, we develop and study a two-dimensional model of a cell co-culture that includes cell division and self-propulsion in addition to cell-cell adhesion, and systemically study how cells with significantly different adhesion, motility, and proliferation rate dynamically organize themselves in a spatiotemporal and context-dependent manner. Our results may help to understand how differential equilibrium and non-equilibrium properties cooperate and compete leading to different morphologies during tumor development, with important consequences for invasion and metastasis

  20. Dynamic single-cell NAD(P)H measurement reveals oscillatory metabolism throughout the E. coli cell division cycle.

    Science.gov (United States)

    Zhang, Zheng; Milias-Argeitis, Andreas; Heinemann, Matthias

    2018-02-01

    Recent work has shown that metabolism between individual bacterial cells in an otherwise isogenetic population can be different. To investigate such heterogeneity, experimental methods to zoom into the metabolism of individual cells are required. To this end, the autofluoresence of the redox cofactors NADH and NADPH offers great potential for single-cell dynamic NAD(P)H measurements. However, NAD(P)H excitation requires UV light, which can cause cell damage. In this work, we developed a method for time-lapse NAD(P)H imaging in single E. coli cells. Our method combines a setup with reduced background emission, UV-enhanced microscopy equipment and optimized exposure settings, overall generating acceptable NAD(P)H signals from single cells, with minimal negative effect on cell growth. Through different experiments, in which we perturb E. coli's redox metabolism, we demonstrated that the acquired fluorescence signal indeed corresponds to NAD(P)H. Using this new method, for the first time, we report that intracellular NAD(P)H levels oscillate along the bacterial cell division cycle. The developed method for dynamic measurement of NAD(P)H in single bacterial cells will be an important tool to zoom into metabolism of individual cells.

  1. Progesterone Receptor Membrane Component 1 (PGRMC1 in cell division: its role in bovine granulosa cells mitosis

    Directory of Open Access Journals (Sweden)

    Laura Terzaghi

    2015-07-01

    Full Text Available The present studies were aimed to assess Progesterone Receptor Membrane Component-1 (PGRMC1 role in regulating bovine granulosa cells (bGC mitosis. First, we performed immunofluorescence studies on in vitro cultured bGC collected from antral follicles, which showed that PGRMC1 localizes to the spindle apparatus in mitotic cells. Then, to evaluate PGRMC1 effect on cell proliferation we silenced its expression with RNA interference technique (RNAi. Quantitative RT-PCR and immunoblotting confirmed down-regulation of PGRMC1 expression, when compared to CTRL-RNAi treated bGC (p<0.05. After 72h of culture, PGRMC1 silencing determined a lower growth rate (p<0.05 and a higher percentage of cells arrested at G2/M phase as assessed by flowcytometry (p<0.05. Accordingly, live imaging studies revealed more aberrant mitosis and a delayed M-phase in PGRMC1-RNAi treated cells compared to CTRL-RNAi group (p<0.05. These data confirmed that PGRMC1 is directly involved in bGC mitosis and ongoing preliminary studies are aimed to elucidate its putative mechanisms of action. Since PGRMC1 is a membrane protein, we hypothesize its possible involvement in vesicular trafficking and endocytosis, which is in turn an important process to assure proper cell division. To assess this hypothesis, we have preliminarily conducted immunofluorescence and in situ proximity ligation assay experiments that showed PGRMC1 co-localization and direct interaction with clathrin. This is important since clathrin is an essential protein for both endosomes formation, and cell division acting directly on the spindle apparatus. Thus our studies set the stage for analysis aimed to further characterize PGRMC1’s mechanism of action in mitotic cell.

  2. DNA Charge Transport: From Chemical Principles to the Cell

    Science.gov (United States)

    Arnold, Anna R.; Grodick, Michael A.; Barton, Jacqueline K.

    2016-01-01

    The DNA double helix has captured the imagination of many, bringing it to the forefront of biological research. DNA has unique features that extend our interest into areas of chemistry, physics, material science and engineering. Our laboratory has focused on studies of DNA charge transport (CT), wherein charges can efficiently travel long molecular distances through the DNA helix while maintaining an exquisite sensitivity to base pair π-stacking. Because DNA CT chemistry reports on the integrity of the DNA duplex, this property may be exploited to develop electrochemical devices to detect DNA lesions and DNA-binding proteins. Furthermore, studies now indicate that DNA CT may also be used in the cell by, for example, DNA repair proteins, as a cellular diagnostic, in order to scan the genome to localize efficiently to damage sites. In this review, we describe this evolution of DNA CT chemistry from the discovery of fundamental chemical principles to applications in diagnostic strategies and possible roles in biology. PMID:26933744

  3. Achieving Precision Death with Cell-Cycle Inhibitors that Target DNA Replication and Repair.

    Science.gov (United States)

    Lin, Aimee Bence; McNeely, Samuel C; Beckmann, Richard P

    2017-07-01

    All cancers are characterized by defects in the systems that ensure strict control of the cell cycle in normal tissues. The consequent excess tissue growth can be countered by drugs that halt cell division, and, indeed, the majority of chemotherapeutics developed during the last century work by disrupting processes essential for the cell cycle, particularly DNA synthesis, DNA replication, and chromatid segregation. In certain contexts, the efficacy of these classes of drugs can be impressive, but because they indiscriminately block the cell cycle of all actively dividing cells, their side effects severely constrain the dose and duration with which they can be administered, allowing both normal and malignant cells to escape complete growth arrest. Recent progress in understanding how cancers lose control of the cell cycle, coupled with comprehensive genomic profiling of human tumor biopsies, has shown that many cancers have mutations affecting various regulators and checkpoints that impinge on the core cell-cycle machinery. These defects introduce unique vulnerabilities that can be exploited by a next generation of drugs that promise improved therapeutic windows in patients whose tumors bear particular genomic aberrations, permitting increased dose intensity and efficacy. These developments, coupled with the success of new drugs targeting cell-cycle regulators, have led to a resurgence of interest in cell-cycle inhibitors. This review in particular focuses on the newer strategies that may facilitate better therapeutic targeting of drugs that inhibit the various components that safeguard the fidelity of the fundamental processes of DNA replication and repair. Clin Cancer Res; 23(13); 3232-40. ©2017 AACR . ©2017 American Association for Cancer Research.

  4. Chromosomal Aberrations in DNA Repair Defective Cell Lines: Comparisons of Dose Rate and Radiation Quality

    Science.gov (United States)

    George, K. A.; Hada, M.; Patel, Z.; Huff, J.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase, DNA-PK activity. Chromosomes were analyzed using the fluorescence in-situ hybridization (FISH) chromosome painting method in cells at the first division post-irradiation and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma radiation induced higher yields of both simple and complex exchanges in the DSB repair defective cells than in the normal cells. The quadratic dose-response terms for both chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further

  5. DNA-mediated gene transfer into ataxia-telangiectasia cells

    International Nuclear Information System (INIS)

    Crescenzi, M.; Pulciani, S.; Carbonari, M.; Tedesco, L.; Russo, G.; Gaetano, C.; Fiorilli, M.

    1986-01-01

    The complete description of the genetic lesion(s) underlying the AT mutation might, therefore, highlight not only a DNA-repair pathwa, but also an important aspect of the physiology of lymphocytes. DNA-mediated gene transfer into eukaryotic cells has proved a powerful tool for the molecular cloning of certain mammalian genes. The possibility to clone a given gene using this technology depends, basically, on the availability of a selectable marker associated with the expression of the transfected gene in the recipient cell. Recently, a human DNA repair gene has been cloned in CHO mutant cells by taking advantage of the increased resistance to ultraviolet radiation of the transformants. As a preliminary step toward the molecular cloning of the AT gene(s), the authors have attempted to confer radioresistance to AT cells by transfection with normal human DNA

  6. DNA breaks early in replication in B cell cancers

    Science.gov (United States)

    Research by scientists at the NCI has identified a new class of DNA sites in cells that break early in the replication process. They found that these break sites correlate with damage often seen in B cell cancers, such as diffuse large B cell lymphoma.

  7. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    International Nuclear Information System (INIS)

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D.

    1991-01-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links

  8. Mitochondrial DNA mutations in human tumor cells

    OpenAIRE

    LI, HUI; HONG, ZE-HUI

    2012-01-01

    Mitochondria play significant roles in cellular energy metabolism, free radical generation and apoptosis. The dysfunction of mitochondria is correlated with the origin and progression of tumors; thus, mutations in the mitochondrial genome that affect mitochondrial function may be one of the causal factors of tumorigenesis. Although the role of mitochondrial DNA (mtDNA) mutations in carcinogenesis has been investigated extensively by various approaches, the conclusions remain controversial to ...

  9. Nonhomologous DNA End Joining in Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Sheetal Sharma

    2010-01-01

    Full Text Available Among various DNA damages, double-strand breaks (DSBs are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.

  10. Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice

    DEFF Research Database (Denmark)

    Sakamori, Ryotaro; Das, Soumyashree; Yu, Shiyan

    2012-01-01

    The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases...... reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells...... suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell-based approaches could be beneficial to infants with this often lethal condition....

  11. DNA damage-inducible transcripts in mammalian cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Alamo, I. Jr.; Hollander, M.C.

    1988-01-01

    Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury

  12. Cell-Free DNA and Active Rejection in Kidney Allografts.

    Science.gov (United States)

    Bloom, Roy D; Bromberg, Jonathan S; Poggio, Emilio D; Bunnapradist, Suphamai; Langone, Anthony J; Sood, Puneet; Matas, Arthur J; Mehta, Shikha; Mannon, Roslyn B; Sharfuddin, Asif; Fischbach, Bernard; Narayanan, Mohanram; Jordan, Stanley C; Cohen, David; Weir, Matthew R; Hiller, David; Prasad, Preethi; Woodward, Robert N; Grskovic, Marica; Sninsky, John J; Yee, James P; Brennan, Daniel C

    2017-07-01

    Histologic analysis of the allograft biopsy specimen is the standard method used to differentiate rejection from other injury in kidney transplants. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive test of allograft injury that may enable more frequent, quantitative, and safer assessment of allograft rejection and injury status. To investigate this possibility, we prospectively collected blood specimens at scheduled intervals and at the time of clinically indicated biopsies. In 102 kidney recipients, we measured plasma levels of dd-cfDNA and correlated the levels with allograft rejection status ascertained by histology in 107 biopsy specimens. The dd-cfDNA level discriminated between biopsy specimens showing any rejection (T cell-mediated rejection or antibody-mediated rejection [ABMR]) and controls (no rejection histologically), P rejection at a cutoff of 1.0% dd-cfDNA were 61% and 84%, respectively. The AUC for discriminating ABMR from samples without ABMR was 0.87 (95% CI, 0.75 to 0.97). Positive and negative predictive values for ABMR at a cutoff of 1.0% dd-cfDNA were 44% and 96%, respectively. Median dd-cfDNA was 2.9% (ABMR), 1.2% (T cell-mediated types ≥IB), 0.2% (T cell-mediated type IA), and 0.3% in controls ( P =0.05 for T cell-mediated rejection types ≥IB versus controls). Thus, dd-cfDNA may be used to assess allograft rejection and injury; dd-cfDNA levels rejection (T cell-mediated type ≥IB or ABMR) and levels >1% indicate a probability of active rejection. Copyright © 2017 by the American Society of Nephrology.

  13. UV stimulation of DNA-mediated transformation of human cells

    International Nuclear Information System (INIS)

    van Duin, M.; Westerveld, A.; Hoeijmakers, J.H.

    1985-01-01

    Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells, which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome

  14. Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation

    Directory of Open Access Journals (Sweden)

    Hanley Edward N

    2000-10-01

    Full Text Available Abstract Background The relationship between cell shape, proliferation, and extracellular matrix (ECM production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D culture. Results Three experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1 Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2 Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3 Cells on Matrigel™-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth. Conclusions The cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel™ showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior.

  15. Cell lines derived from a Medaka radiation-sensitive mutant have defects in DNA double-strand break responses

    International Nuclear Information System (INIS)

    Hidaka, Masayuki; Oda, Shoji; Mitani, Hiroshi; Kuwahara, Yoshikazu; Fukumoto, Manabu

    2010-01-01

    It was reported that the radiation-sensitive Medaka mutant 'ric1' has a defect in the repair of DNA double-strand breaks (DSBs) induced by γ-rays during early embryogenesis. To study the cellular response of a ric1 mutant to ionizing radiation (IR), we established the mutant embryonic cell lines RIC1-e9, RIC1-e42, RIC1-e43. Following exposure to γ-irradiation, the DSBs in wild-type cells were repaired within 1 h, while those in RIC1 cells were not rejoined even after 2 h. Cell death was induced in the wild-type cells with cell fragmentation, but only a small proportion of the RIC1 cells underwent cell death, and without cell fragmentation. Although both wild-type and RIC1 cells showed mitotic inhibition immediately after γ-irradiation, cell division was much slower to resume in the wild-type cells (20 h versus 12 h). In both wild-type and RIC1 cells, Ser139 phosphorylated H2AX (γH2AX) foci were formed after γ-irradiation, however, the γH2AX foci disappeared more quickly in the RIC1 cell lines. These results suggest that the instability of γH2AX foci in RIC1 cells cause an aberration of the DNA damage response. As RIC1 cultured cells showed similar defective DNA repair as ric1 embryos and RIC1 cells revealed defective cell death and cell cycle checkpoint, they are useful for investigating DNA damage responses in vitro. (author)

  16. Quantitation of DNA repair in brain cell cultures: implications for autoradiographic analysis of mixed cell populations

    International Nuclear Information System (INIS)

    Dambergs, R.; Kidson, C.

    1979-01-01

    Quantitation of DNA repair in the mixed cell population of mouse embryo brain cultures has been assessed by autoradiographic analysis of unscheduled DNA synthesis following UV-irradiation. The proportion of labelled neurons and the grain density over neuronal nuclei were both less than the corresponding values for glial cells. The nuclear geometries of these two classes of cell are very different. Partial correction for the different geometries by relating grain density to nuclear area brought estimates of neuronal and glial DNA repair synthesis more closely in line. These findings have general implications for autoradiographic measurement of DNA repair in mixed cell populations and in differentiated versus dividing cells. (author)

  17. Control of the proportion of inner cells by asymmetric divisions and the ensuing resilience of cloned rabbit embryos

    Science.gov (United States)

    Duranthon, Véronique

    2018-01-01

    ABSTRACT Mammalian embryo cloning by nuclear transfer has a low success rate. This is hypothesized to correlate with a high variability of early developmental steps that segregate outer cells, which are fated to extra-embryonic tissues, from inner cells, which give rise to the embryo proper. Exploring the cell lineage of wild-type embryos and clones, imaged in toto until hatching, highlights the respective contributions of cell proliferation, death and asymmetric divisions to phenotypic variability. Preferential cell death of inner cells in clones, probably pertaining to the epigenetic plasticity of the transferred nucleus, is identified as a major difference with effects on the proportion of inner cell. In wild type and clones, similar patterns of outer cell asymmetric divisions are shown to be essential to the robust proportion of inner cells observed in wild type. Asymmetric inner cell division, which is not described in mice, is identified as a regulator of the proportion of inner cells and likely gives rise to resilient clones. PMID:29567671

  18. From cell differentiation to cell collectives : Bacillus subtilis uses division of labor to migrate

    NARCIS (Netherlands)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In

  19. Uptake of DNA by cancer cells without a transfection reagent

    Directory of Open Access Journals (Sweden)

    Yanping Kong

    Full Text Available Abstract Background Cancer cells exhibit elevated levels of glucose uptake and may obtain pre-formed, diet-derived fatty acids from the bloodstream to boost their rapid growth; they may also use nucleic acid from their microenvironment. The study of processing nucleic acid by cancer cells will help improve the understanding of the metabolism of cancer. DNA is commonly packaged into a viral or lipid particle to be transferred into cells; this process is called transfection in laboratory. Cancer cells are known for having gene mutations and the evolving ability of endocytosis. Their uptake of DNAs might be different from normal cells; they may take in DNAs directly from the environment. In this report, we studied the uptake of DNAs in cancer cells without a transfection reagent. Methods A group of DNA fragments were prepared with PCR and labeled with isotope phosphorous-32 to test their uptake by Huh 7 (liver cancer and THLE3 (normal liver cells after incubation overnight by counting radioactivity of the cells’ genomic DNA. Multiple cell lines including breast cancer and lung cancer were tested with the same method. DNA molecules were also labeled with fluorescence to test the location in the cells using a kit of “label it fluorescence in situ hybridization (FISH” from Mirus (USA. Results The data demonstrated that hepatocellular carcinoma cells possess the ability to take in large DNA fragments directly without a transfection reagent whereas normal liver cells cannot. Huh7 and MDA-MB231 cells displayed a significantly higher Rhodamine density in the cytoplasmic phagosomes and this suggests that the mechanism of uptake of large DNA by cancer cells is likely endocytosis. The efficacy of uptake is related to the DNA’s size. Some cell lines of lung cancer and breast cancer also showed similar uptake of DNA. Conclusions In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA

  20. Utilization during mitotic cell division of loci controlling meiotic recombination and disjunction in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Baker, B.S.; Carpenter, A.T.C.; Ripoll, P.

    1978-01-01

    To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by uv and x rays. Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells

  1. ALIX and ESCRT-III coordinately control cytokinetic abscission during germline stem cell division in vivo.

    Directory of Open Access Journals (Sweden)

    Åsmund H Eikenes

    2015-01-01

    Full Text Available Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. Here we show that ALIX and the ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. We demonstrate that ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, we show that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. We conclude that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo.

  2. Cell division and endoreduplication play important roles in stem swelling of tuber mustard (Brassica juncea Coss. var. tumida Tsen et Lee).

    Science.gov (United States)

    Shi, H; Wang, L L; Sun, L T; Dong, L L; Liu, B; Chen, L P

    2012-11-01

    We investigated spatio-temporal variations in cell division and the occurrence of endoreduplication in cells of tuber mustard stems during development. Cells in the stem had 8C nuclei (C represents DNA content of a two haploid genome), since it is an allotetraploid species derived from diploid Brassica rapa (AA) and B. nigra (BB), thus indicating the occurrence of endoreduplication. Additionally, we observed a dynamic change of cell ploidy in different regions of the swollen stems, with a decrease in 4C proportion in P4-1 and a sharp increase in 8C cells that became the dominant cell type (86.33% at most) in the inner pith cells. Furthermore, cDNAs of 14 cell cycle genes and four cell expansion genes were cloned and their spatial transcripts analysed in order to understand their roles in stem development. The expression of most cell cycle genes peaked in regions of the outer pith (P2 or P3), some genes regulating S/G2 and G2/M (BjCDKB1;2, BjCYCB1;1 and BjCYCB1;2) significantly decrease in P5 and P6, while G1/S regulators (BjE2Fa, BjE2Fb and BjE2Fc) showed a relative high expression level in the inner pith (P5) where cells were undergoing endoreduplication. Coincidentally, BjXTH1and BjXTH2 were exclusively expressed in the endoreduplicated cells. Our results suggest that cells of outer pith regions (P2 and P3) mainly divide for cell proliferation, while cells of the inner pith expand through endoreduplication. Endoreduplication could trigger expression of BjXTH1 and BjXTH2 and thus function in cell expansion of the pith tissue. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

  3. Radiation effects on cultured mouse embryos in relation to cell division cycle

    International Nuclear Information System (INIS)

    Domon, M.

    1982-01-01

    The authors have worked with mouse embryos in vitro asking first, what are the suitable parameters to define the radiation sensitivity of embryos, and second what is a major factor determining it. The LD 50 was adopted as a parameter of the radiation sensitivity of a population in a mouse embryo system in culture. The fertilized ova were collected into Whitten's medium at various times during the pronuclear and 2-cell stages of development. They were irradiated in chambers with X-rays at doses of 0 to 800 rads. After the embryos were cultured, a set of the lethal fractions for various X-ray doses were obtained. Regarding the radiation sensitivity variation of the embryos, the LD 50 varied from 100 to 200 rads during the pronuclear stage and from 100 to 600 rads during the 2-cell stage. The embryos during the pronuclear stage were most radioresistant at early G 2 phase, followed by an increase in the sensitivity. The embryos during the 2-cell stage were also most radioresistant at early G 2 phase and were more sensitive when they got close to either the first or the second cleavage division. Furthermore, it seems that the factor 6 of the large variation was due to the extremely long G 2 period, 14 hrs for the 2-cell embryos. That is, the pooled 2-cell embryos were in a relative sense well synchronized with G 2 phase. In contrast, the synchrony was poor during the pronuclear stage, which led to less variation of the LD 50 for the pronuclear embryos. It is concluded that during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo. (Namekawa, K.)

  4. The cell wall and cell division gene cluster in the Mra operon of Pseudomonas aeruginosa: cloning, production, and purification of active enzymes.

    Science.gov (United States)

    Azzolina, B A; Yuan, X; Anderson, M S; El-Sherbeini, M

    2001-04-01

    We have cloned the Pseudomonas aeruginosa cell wall biosynthesis and cell division gene cluster that corresponds to the mra operon in the 2-min region of the Escherichia coli chromosome. The organization of the two chromosomal regions in P. aeruginosa and E. coli is remarkably similar with the following gene order: pbp3/pbpB, murE, murF, mraY, murD, ftsW, murG, murC, ddlB, ftsQ, ftsA, ftsZ, and envA/LpxC. All of the above P. aeruginosa genes are transcribed from the same strand of DNA with very small, if any, intragenic regions, indicating that these genes may constitute a single operon. All five amino acid ligases, MurC, MurD, MurE, MurF, and DdlB, in addition to MurG and MraY were cloned in expression vectors. The four recombinant P. aeruginosa Mur ligases, MurC, MurD, MurE, and MurF were overproduced in E. coli and purified as active enzymes. Copyright 2001 Academic Press.

  5. Distribution of DNA replication proteins in Drosophila cells

    Science.gov (United States)

    Easwaran, Hariharan P; Leonhardt, Heinrich; Cardoso, M Cristina

    2007-01-01

    Background DNA replication in higher eukaryotic cells is organized in discrete subnuclear sites called replication foci (RF). During the S phase, most replication proteins assemble at the RF by interacting with PCNA via a PCNA binding domain (PBD). This has been shown to occur for many mammalian replication proteins, but it is not known whether this mechanism is conserved in evolution. Results Fluorescent fusions of mammalian replication proteins, Dnmt1, HsDNA Lig I and HsPCNA were analyzed for their ability to target to RF in Drosophila cells. Except for HsPCNA, none of the other proteins and their deletions showed any accumulation at RF in Drosophila cells. We hypothesized that in Drosophila cells there might be some other peptide sequence responsible for targeting proteins to RF. To test this, we identified the DmDNA Lig I and compared the protein sequence with HsDNA Lig I. The two orthologs shared the PBD suggesting a functionally conserved role for this domain in the Drosophila counterpart. A series of deletions of DmDNA Lig I were analyzed for their ability to accumulate at RF in Drosophila and mammalian cells. Surprisingly, no accumulation at RF was observed in Drosophila cells, while in mammalian cells DmDNA Lig I accumulated at RF via its PBD. Further, GFP fusions with the PBD domains from Dnmt1, HsDNA Lig I and DmDNA Lig I, were able to target to RF only in mammalian cells but not in Drosophila cells. Conclusion We show that S phase in Drosophila cells is characterized by formation of RF marked by PCNA like in mammalian cells. However, other than PCNA none of the replication proteins and their deletions tested here showed accumulation at RF in Drosophila cells while the same proteins and deletions are capable of accumulating at RF in mammalian cells. We hypothesize that unlike mammalian cells, in Drosophila cells, replication proteins do not form long-lasting interactions with the replication machinery, and rather perform their functions via very

  6. Inhibition by 2-deoxy-D-ribose of DNA synthesis and growth in Raji cells

    International Nuclear Information System (INIS)

    Ulrich, F.

    1988-01-01

    When Raji cells were cultured for 3 days in serum-free medium, addition of 2-deoxy-D-ribose at the start of culture inhibited incorporation of [ 3 H]thymidine and cell division. At deoxyribose concentrations between 1 and 5 mM, viability was 80% or greater after 3 days of culture even though 5 mM deoxyribose inhibited thymidine incorporation 95-99%. Inhibition by deoxyribose could be completely reversed if the culture medium was replaced with fresh medium up to 8 hr after the start of culture. The inhibition was specific for deoxyribose since other monosaccharides had no effect. Inhibition of DNA synthesis did not appear to be due to depletion of essential nutrients in the medium since the percentage inhibition of thymidine incorporation by cells cultured either in suboptimal serum-free media or in media supplemented with 0.025-5% human AB serum was similar. When DNA repair synthesis was measured as hydroxyurea-resistant thymidine incorporation, addition of deoxyribose to Raji cultures caused increased thymidine incorporation. These results, together with data from others,suggest that deoxyribose damages DNA

  7. Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

    Science.gov (United States)

    Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R

    2001-01-01

    The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel

  8. DNA Origami: Folded DNA-Nanodevices That Can Direct and Interpret Cell Behavior

    Science.gov (United States)

    Kearney, Cathal J.; Lucas, Christopher R.; O'Brien, Fergal J.; Castro, Carlos E.

    2016-01-01

    DNA origami is a DNA-based nanotechnology that utilizes programmed combinations of short complementary oligonucleotides to fold a large single strand of DNA into precise 2-D and 3-D shapes. The exquisite nanoscale shape control of this inherently biocompatible material is combined with the potential to spatially address the origami structures with diverse cargos including drugs, antibodies, nucleic acid sequences, small molecules and inorganic particles. This programmable flexibility enables the fabrication of precise nanoscale devices that have already shown great potential for biomedical applications such as: drug delivery, biosensing and synthetic nanopore formation. In this Progress Report, we will review the advances in the DNA origami field since its inception several years ago and then focus on how these DNA-nanodevices can be designed to interact with cells to direct or probe their behavior. PMID:26840503

  9. Linear Association Between Cellular DNA and Epstein-Barr Virus DNA in a Human Lymphoblastoid Cell Line

    Science.gov (United States)

    Adams, Alice; Lindahl, Tomas; Klein, George

    1973-01-01

    High-molecular-weight DNA from cell line Raji (derived from Burkitt's lymphoma), which contains 50-60 copies of Epstein-Barr virus DNA per cell, was fractionated in neutral solution by several cycles of CsCl gradient centrifugation in fixed-angle rotors. Under the fractionation conditions used, intact Epstein-Barr virus DNA from virus particles can be separated from the less-dense cellular DNA. In contrast, a large proportion of the intrinsic Epstein-Barr virus DNA component of Raji cells remains associated with cellular DNA, as determined by nucleic acid hybridization. This interaction, which is resistant to Pronase and phenol treatment, is not the result of aggregation. When the molecular weight of Raji DNA is reduced by hydrodynamic shear, the amount of virus DNA associated with cell DNA decreases. However, some virus DNA still remains bound to fragments of cellular DNA after shearing. The association is completely destroyed in alkaline solution. Molecular weight analysis of Raji DNA after denaturation showed that the alkali-induced release of Epstein-Barr virus DNA was specific and not the result of random single-strand breaks. These data indicate that Epstein-Barr virus DNA is linearly integrated into Raji cell DNA by alkali-labile bonds. PMID:4355371

  10. Characterization of a null allelic mutant of the rice NAL1 gene reveals its role in regulating cell division.

    Directory of Open Access Journals (Sweden)

    Dan Jiang

    Full Text Available Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1 show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1, nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogeneous expression of NAL1 in fission yeast (Schizosaccharomyces pombe further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division.

  11. DNA ligase III is involved in a DNA-PK independent pathway of NHEJ in human cells

    International Nuclear Information System (INIS)

    Wang, H.; Perrault, A.R.; Qin, W.; Wang, H.; Iliakis, G.

    2003-01-01

    Full text: Double strand breaks (DSB) induced by ionizing radiation (IR) and other cytotoxic agents in the genome of higher eukaryotes are thought to be repaired either by homologous recombination repair (HRR), or non-homologous endjoining (NHEJ). We previously reported the operation of two components of NHEJ in vivo: a DNA-PK dependent component that operates with fast kinetics (D-NHEJ), and a DNA-PK independent component that acts as a backup (basic or B-NHEJ) and operates with kinetics an order of magnitude slower. To gain further insight into the mechanisms of B-NHEJ, we investigated DNA endjoining in extracts 180BR, a human cell line deficient in DNA ligase IV, using an in vitro plasmid-based DNA endjoining assay. An anti DNA ligase III antibody inhibited almost completely DNA endjoining activity in these extracts. On the other hand, an anti DNA ligase I antibody had no measurable effect in DNA endjoining activity. Immunodepletion of DNA ligase III from 180BR cell extracts abolished the DNA endjoining activity, which could be restored by addition of purified human DNA ligase IIIb. Full-length DNA ligase III bound to double stranded DNA and stimulated DNA endjoining in both intermolecular and intramolecular ligation. Furthermore, fractionation of HeLa cell extracts demonstrated the presence of an activity stimulating the function of DNA ligase III. Based on these observations we propose that DNA ligase III is the ligase operating in B-NHEJ

  12. Effect of gamma-irradiation and colchicine on cell division and differentiation of xylem elements in citrus limon juice vesicle cultures

    International Nuclear Information System (INIS)

    Khan, Aysha; Chauhan, Y.S.

    1999-01-01

    The effects of varying doses of gamma irradiation on cell division and cytodifferentiation of tracheary elements in cultured juice vesicles of Citrus limon (L) Burmann var. Assam lemon were investigated. Low radiation doses stimulated cell division and differentiation of xylem fibres, sclereids and tracheids in explants given up to 10 Gy of gamma rays. Although cell division and cytodifferentiation of fibers and sclereids occurred in explants exposed to 150 dose of Gy radiation, the intensity of differentiation was much less than that induced by 10 Gy radiation dose. Amongst the differential elements, tracheids were more sensitive to radiation than fibres and sclereids. The requirement of cell division for differentiation of xylem cells was also studied by using different concentrations of colchicine in Citrus limon juice vesicle cultures. It was found that the low concentrations of colchicine permitted normal cell division and also resulted in normal differentiation of xylem cells; higher colchicine concentration, however, inhibited cell division as well as differentiation and resulted in an abnormal differentiation of tracheary element. A positive correlation between intensity of nucleic acid staining and cell division in both the above-mentioned experiments was qualitatively confirmed by Azur B staining test of nucleic acid. Thus, it was concluded that juice vesicle parenchyma cells go through nucleic acid synthesis, followed by cell division before differentiation. (author)

  13. Intestinal Epithelial Cells Modulate Antigen-Presenting Cell Responses to Bacterial DNA

    Science.gov (United States)

    Campeau, J. L.; Salim, S. Y.; Albert, E. J.; Hotte, N.

    2012-01-01

    Intestinal epithelial cells and antigen-presenting cells orchestrate mucosal innate immunity. This study investigated the role of bacterial DNA in modulating epithelial and bone marrow-derived antigen-presenting cells (BM-APCs) and subsequent T-lymphocyte responses. Murine MODE-K epithelial cells and BM-APCs were treated with DNA from either Bifidobacterium breve or Salmonella enterica serovar Dublin directly and under coculture conditions with CD4+ T cells. Apical stimulation of MODE-K cells with S. Dublin DNA enhanced secretion of cytokines from underlying BM-APCs and induced interleukin-17 (IL-17) and gamma interferon (IFN-γ) secretion from CD4+ T cells. Bacterial DNA isolated from either strain induced maturation and increased cytokine secretion from BM-APCs. Conditioned medium from S. Dublin-treated MODE-K cells elicited an increase in cytokine secretion similar to that seen for S. Dublin DNA. Treatment of conditioned medium from MODE-K cells with RNase and protease prevented the S. Dublin-induced increased cytokine secretion. Oral feeding of mice with B. breve DNA resulted in enhanced levels of colonic IL-10 and transforming growth factor β (TGFβ) compared with what was seen for mice treated with S. Dublin DNA. In contrast, feeding mice with S. Dublin DNA increased levels of colonic IL-17 and IL-12p70. T cells from S. Dublin DNA-treated mice secreted high levels of IL-12 and IFN-γ compared to controls and B. breve DNA-treated mice. These results demonstrate that intestinal epithelial cells are able to modulate subsequent antigen-presenting and T-cell responses to bacterial DNA with pathogenic but not commensal bacterial DNA inducing effector CD4+ T lymphocytes. PMID:22615241

  14. Photoenzyme probes of photodamage to cells and cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, B. M.

    1979-01-01

    Development of photoenzyme probes for detection of ultraviolet damage to cells and DNA is reviewed with special emphasis on a process using polyethylene glycol to induce cell fusion. Polyethylene glycol is easy to obtain and handle, is gentle to the cells and does not induce latent or productive virus infection; therefore, it may be a general method for insertion of exogenous enzymes into mammalian cells. (PCS)

  15. Changes in radiosensitivity of V-79 cells accompanying growth and cell division

    International Nuclear Information System (INIS)

    Lehnert, S.

    1975-01-01

    The X-ray survival curve for asynchronous Chinese hamster V-79 cells at 17 to 20 hr after plating when cells are irradiated as microclones of two to four cells differs from the survival curve seen at short times after plating, when single cells are irradiated, in having higher D 0 (300 rad vs 160 rad) and negligible extrapolation number. As a consequence of the difference in D 0 the difference in survival between single cells and clones increases with increasing dose. Transient cyclic changes in survival occur at early times after plating and are probably related to partial synchronization induced by trypsinization. In addition there is a progressive increase in survival which develops with increasing time after plating, as the number of cells in the clones increases. Decrease in radiosensitivity with increasing number of cells irradiated is also observed for synchronous cells when cells at corresponding points in the cell cycle are irradiated. Accumulation and repair of sublethal damage is demonstrable in cells irradiated at short times after plating, but cannot be shown at 20 hr after plating when cells are irradiated as microclones. (U.S.)

  16. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Science.gov (United States)

    Makarova, Alena V; Grabow, Corinn; Gening, Leonid V; Tarantul, Vyacheslav Z; Tahirov, Tahir H; Bessho, Tadayoshi; Pavlov, Youri I

    2011-01-31

    Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  17. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Directory of Open Access Journals (Sweden)

    Alena V Makarova

    2011-01-01

    Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  18. Cell lethality after selective irradiation of the DNA replication fork

    International Nuclear Information System (INIS)

    Hofer, K.G.; Warters, R.L.

    1985-01-01

    It has been suggested that nascent DNA located at the DNA replication fork may exhibit enhanced sensitivity to radiation damage. To evaluate this hypothesis, Chinese hamster ovary cells (CHO) were labeled with 125 I-iododeoxyuridine ( 125 IUdR) either in the presence or absence of aphidicolin. Aphidicolin (5 μg/ml) reduced cellular 125 IUdR incorporation to 3-5% of the control value. The residual 125 I incorporation appeared to be restricted to low molecular weight (sub-replicon sized) fragments of DNA which were more sensitive to micrococcal nuclease attack and less sensitive to high salt DNase I digestion than randomly labeled DNA. These findings suggest that DNA replicated in the presence of aphidicolin remains localized at the replication fork adjacent to the nuclear matrix. Based on these observations an attempt was made to compare the lethal consequences of 125 I decays at the replication fork to that of 125 I decays randomly distributed over the entire genome. Regardless of the distribution of decay events, all treatment groups exhibited identical dose-response curves (D 0 : 101 125 I decays/cell). Since differential irradiation of the replication complex did not result in enhanced cell lethality, it can be concluded that neither the nascent DNA nor the protein components (replicative enzymes, nuclear protein matrix) associated with the DNA replication site constitute key radiosensitive targets within the cellular genome. (orig.)

  19. PDK1 Is a Regulator of Epidermal Differentiation that Activates and Organizes Asymmetric Cell Division

    Directory of Open Access Journals (Sweden)

    Teruki Dainichi

    2016-05-01

    Full Text Available Asymmetric cell division (ACD in a perpendicular orientation promotes cell differentiation and organizes the stratified epithelium. However, the upstream cues regulating ACD have not been identified. Here, we report that phosphoinositide-dependent kinase 1 (PDK1 plays a critical role in establishing ACD in the epithelium. Production of phosphatidyl inositol triphosphate (PIP3 is localized to the apical side of basal cells. Asymmetric recruitment of atypical protein kinase C (aPKC and partitioning defective (PAR 3 is impaired in PDK1 conditional knockout (CKO epidermis. PDK1CKO keratinocytes do not undergo calcium-induced activation of aPKC or IGF1-induced activation of AKT and fail to differentiate. PDK1CKO epidermis shows decreased expression of Notch, a downstream effector of ACD, and restoration of Notch rescues defective expression of differentiation-induced Notch targets in vitro. We therefore propose that PDK1 signaling regulates the basal-to-suprabasal switch in developing epidermis by acting as both an activator and organizer of ACD and the Notch-dependent differentiation program.

  20. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    Science.gov (United States)

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

  1. Melanin photosensitizes ultraviolet light (UVC) DNA damage in pigmented cells

    International Nuclear Information System (INIS)

    Huselton, C.A.; Hill, H.Z.

    1990-01-01

    Melanins, pigments of photoprotection and camouflage, are very photoreactive and can both absorb and emit active oxygen species. Nevertheless, black skinned individuals rarely develop skin cancer and melanin is assumed to act as a solar screen. Since DNA is the target for solar carcinogenesis, the effect of melanin on Ultraviolet (UV)-induced thymine lesions was examined in mouse melanoma and carcinoma cells that varied in melanin content. Cells prelabeled with 14C-dThd were irradiated with UVC; DNA was isolated, purified, degraded to bases by acid hydrolysis and analyzed by HPLC. Thymine dimers were detected in all of the extracts of irradiated cells. Melanotic and hypomelanotic but not mammary carcinoma cell DNA from irradiated cells contained hydrophilic thymine derivatives. The quantity of these damaged bases was a function of both the UVC dose and the cellular melanin content. One such derivative was identified by gas chromatography-mass spectroscopy as thymine glycol. The other appears to be derived from thymine glycol by further oxidation during acid hydrolysis of the DNA. The finding of oxidative DNA damage in melanin-containing cells suggests that melanin may be implicated in the etiology of caucasian skin cancer, particularly melanoma. Furthermore, the projected decrease in stratospheric ozone could impact in an unanticipated deleterious manner on dark-skinned individuals

  2. The deletion of bacterial dynamin and flotillin genes results in pleiotrophic effects on cell division, cell growth and in cell shape maintenance

    Directory of Open Access Journals (Sweden)

    Dempwolff Felix

    2012-12-01

    Full Text Available Abstract Background In eukaryotic cells, dynamin and flotillin are involved in processes such as endocytosis and lipid raft formation, respectively. Dynamin is a GTPase that exerts motor-like activity during the pinching off of vesicles, while flotillins are coiled coil rich membrane proteins with no known enzymatic activity. Bacteria also possess orthologs of both classes of proteins, but their function has been unclear. Results We show that deletion of the single dynA or floT genes lead to no phenotype or a mild defect in septum formation in the case of the dynA gene, while dynA floT double mutant cells were highly elongated and irregularly shaped, although the MreB cytoskeleton appeared to be normal. DynA colocalizes with FtsZ, and the dynA deletion strain shows aberrant FtsZ rings in a subpopulation of cells. The mild division defect of the dynA deletion is exacerbated by an additional deletion in ezrA, which affects FtsZ ring formation, and also by the deletion of a late division gene (divIB, indicating that DynA affects several steps in cell division. DynA and mreB deletions generated a synthetic defect in cell shape maintenance, showing that MreB and DynA play non-epistatic functions in cell shape maintenance. TIRF microscopy revealed that FloT forms many dynamic membrane assemblies that frequently colocalize with the division septum. The deletion of dynA did not change the pattern of localization of FloT, and vice versa, showing that the two proteins play non redundant roles in a variety of cellular processes. Expression of dynamin or flotillin T in eukaryotic S2 cells revealed that both proteins assemble at the cell membrane. While FloT formed patch structures, DynA built up tubulated structures extending away from the cells. Conclusions Bacillus subtilis dynamin ortholog DynA plays a role during cell division and in cell shape maintenance. It shows a genetic link with flotillin T, with both proteins playing non-redundant functions at

  3. DNA amplification is rare in normal human cells

    International Nuclear Information System (INIS)

    Wright, J.A.; Watt, F.M.; Hudson, D.L.; Stark, G.R.; Smith, H.S.; Hancock, M.C.

    1990-01-01

    Three types of normal human cells were selected in tissue culture with three drugs without observing a single amplification event from a total of 5 x 10 8 cells. No drug-resistant colonies were observed when normal foreskin keratinocytes were selected with N-(phosphonacetyl)-L-aspartate or with hydroxyurea or when normal mammary epithelial cells were selected with methotrexate. Some slightly resistant colonies with limited potential for growth were obtained when normal diploid fibroblast cells derived from fetal lung were selected with methotrexate or hydroxyurea but careful copy-number analysis of the dihydrofolate reductase and ribonucleotide reductase genes revealed no evidence of amplification. The rarity of DNA amplification in normal human cells contrasts strongly with the situation in tumors and in established cell lines, where amplification of onogenes and of genes mediating drug resistance is frequent. The results suggest that tumors and cell lines have acquired the abnormal ability to amplify DNA with high frequency

  4. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

    Moritomi-Yano, Keiko; Yano, Ken-ichi

    2010-01-01

    Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) that are the most severe form of DNA damages. Recently, live cell imaging techniques coupled with laser micro-irradiation were used to analyze the spatio-temporal behavior of the NHEJ core factors upon DSB induction in living cells. Based on the live cell imaging studies, we proposed a novel two-phase model for DSB sensing and protein assembly in the NHEJ pathway. This new model provides a novel view of the dynamic protein behavior on DSBs and broad implications for the molecular mechanism of NHEJ. (author)

  5. Model-Based Analysis of Arabidopsis Leaf Epidermal Cells Reveals Distinct Division and Expansion Patterns for Pavement and Guard Cells1[W][OA

    Science.gov (United States)

    Asl, Leila Kheibarshekan; Dhondt, Stijn; Boudolf, Véronique; Beemster, Gerrit T.S.; Beeckman, Tom; Inzé, Dirk; Govaerts, Willy; De Veylder, Lieven

    2011-01-01

    To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery. PMID:21693673

  6. Sunlight-induced DNA damage in human mononuclear cells

    DEFF Research Database (Denmark)

    Møller, Peter; Wallin, Hakan; Holst, Erik

    2002-01-01

    of sunlight was comparable to the interindividual variation, indicating that sunlight exposure and the individual's background were the two most important determinants for the basal level of DNA damage. Influence of other lifestyle factors such as exercise, intake of foods, infections, and age could......In this study of 301 blood samples from 21 subjects, we found markedly higher levels of DNA damage (nonpyrimidine dimer types) in the summer than in the winter detected by single-cell gel electrophoresis. The level of DNA damage was influenced by the average daily influx of sunlight ... to blood sampling. The 3 and 6 day periods before sampling influenced DNA damage the most. The importance of sunlight was further emphasized by a positive association of the DNA damage level to the amount of time the subjects had spent in the sun over a 3 day period prior to the sampling. The effect...

  7. DNA double strand break repair in a radioresistant cell line

    International Nuclear Information System (INIS)

    Koval, T.M.; Kazmar, E.R.

    1987-01-01

    TN-368 lepidopteran insect cells are on the order of 100 times more resistant to the lethal effects of ionizing radiation than cultured mammalian cells. DNA double strand breaks (DSB) are believed by many to be the critical molecular lesion leading to cell death. The authors therefore measured the rejoining of DSB in TN-368 and V79 Chinese hamster cells. Cells were irradiated on ice with /sup 137/Cs γ rays at a dose rate of 2.5 Gy/min, incubated for various periods of time, and assayed for DNA DSB using the method of neutral elution. The kinetics of DSB rejoining following a dose of 90.2 Gy are similar for both cell lines. Approximately 80% of the DSB are rejoined in both lines by 1 hr postirradiation. However, no further rejoining occurs in the TN-368 cells through at least 6 hr postirradiation, whereas 90% of the DSB are rejoined in the V79 cells by 2 hr postirradiation. Other studies (from 22.6 to 226 Gy) demonstrate that the amount of rejoining of DSB varies inversely with dose for the V79 cells but remains constant for the TN-368 cells. These findings do not support the hypothesis that unrejoined DNA DSB represent the major lesion resulting in cell death

  8. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    International Nuclear Information System (INIS)

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K.

    1990-01-01

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling

  9. Mode division multiplexing over 19-cell hollow-core photonic bandgap fibre by employing integrated mode multiplexer

    NARCIS (Netherlands)

    Chen, H.; Uden, van R.G.H.; Okonkwo, C.M.; Jung, Y.; Wheeler, N.V.; Fokoua, E.N.; Baddela, N.; Petrovich, M.N.; Poletti, F.; Richardson, D.J.; Raz, O.; Waardt, de H.; Koonen, A.M.J.

    2014-01-01

    A photonic integrated mode coupler based on silicon-on-insulator is employed for mode division multiplexing (MDM) over a 193 m 19-cell hollow-core photonic bandgap fibre (HC-PBGF) with a -3 dB bandwidth >120 nm. Robust MDM transmissions using LP01 and LP11 modes, and two degenerate LP11 modes (LP11a

  10. Evolutionary transition towards permanent chloroplasts? - Division of kleptochloroplasts in starved cells of two species of Dinophysis (Dinophyceae.

    Directory of Open Access Journals (Sweden)

    Pernille Møller Rusterholz

    Full Text Available Species within the marine toxic dinoflagellate genus Dinophysis are phagotrophic organisms that exploit chloroplasts (kleptochloroplasts from other protists to perform photosynthesis. Dinophysis spp. acquire the kleptochloroplasts from the ciliate Mesodinium rubrum, which in turn acquires the chloroplasts from a unique clade of cryptophytes. Dinophysis spp. digest the prey nuclei and all other cell organelles upon ingestion (except the kleptochloroplasts and they are therefore believed to constantly acquire new chloroplasts as the populations grow. Previous studies have, however, indicated that Dinophysis can keep the kleptochloroplasts active during long term starvation and are able to produce photosynthetic pigments when exposed to prey starvation. This indicates a considerable control over the kleptochloroplasts and the ability of Dinophysis to replicate its kleptochloroplasts was therefore re-investigated in detail in this study. The kleptochloroplasts of Dinophysis acuta and Dinophysis acuminata were analyzed using confocal microscopy and 3D bioimaging software during long term starvation experiments. The cell concentrations were monitored to confirm cell divisions and samples were withdrawn each time a doubling had occurred. The results show direct evidence of kleptochloroplastidic division and that the decreases in total kleptochloroplast volume, number of kleptochloroplasts and number of kleptochloroplast centers were not caused by dilution due to cell divisions. This is the first report of division of kleptochloroplasts in any protist without the associated prey nuclei. This indicates that Dinophysis spp. may be in a transitional phase towards possessing permanent chloroplasts, which thereby potentially makes it a key organism to understand the evolution of phototrophic protists.

  11. LocZ is a new cell division protein involved in proper septum placement in Streptococcus pneumoniae

    Czech Academy of Sciences Publication Activity Database

    Holečková, Nela; Doubravová, Linda; Massidda, Orietta; Molle, Virginie; Buriánková, Karolína; Benada, Oldřich; Kofroňová, Olga; Ulrych, Aleš; Branny, Pavel

    2015-01-01

    Roč. 6, č. 1 (2015), s. 1-13 ISSN 2150-7511 R&D Projects: GA ČR GAP207/12/1568; GA ČR GAP302/12/0256 Institutional support: RVO:61388971 Keywords : cell division * septum placement * Streptococcus pneumoniae Subject RIV: EE - Microbiology, Virology Impact factor: 6.975, year: 2015

  12. DNA damage responses in human induced pluripotent stem cells and embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Olga Momcilovic

    2010-10-01

    Full Text Available Induced pluripotent stem (iPS cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability.Here, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB, and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2 phase of the cell cycle, displaying a lack of the G(1/S cell cycle arrest similar to human embryonic stem (ES cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts.High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G(1/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.

  13. Phytochemicals radiosensitize cancer cells by inhibiting DNA repair

    International Nuclear Information System (INIS)

    Singh, Rana P.

    2017-01-01

    Solid tumors are mostly treated with radiotherapy. Radiotherapy is toxic to normal tissues and also promote the invasiveness and radioresistance in cancer cells. The resistance against radiotherapy and adverse effects to normal cells reduce the overall therapeutic effects of the treatment. Radiosensitizing agents usually show limited success during clinical trials. Therefore, the search and development of new radiosensitizers showing selective response to only cancer cells is desirable. We analyzed the radiosensitizing effects including cell death effect of silibinin, a phytochemical on prostate cancer cells. Silibinin enhanced gamma radiation (2.5-10 Gy) induced inhibition in colony formation selectively in prostate cancer cells. In cell cycle progression, G2/M phase is the most sensitive phase for radiation-induced damage which was delayed by the compound treatment in radiation exposed cells. The lower concentrations of silibinin substantially enhanced radiation-induced apoptosis. A prolonged reactive oxygen species production was also observed in these treatments EGFR signaling pathway can contribute to radiation-induced pro-survival mechanisms and to the therapeutic resistance. Agent treatment reduced the IR-induced EGFR phosphorylation and consequently reversed the resistance mediating mechanisms within the cancer cell. Thus, inhibiting DNA repair in cancer cells would enhance therapeutic response of radiation in cancer cells. Silibinin affected the localization of EGFR and DNA-dependent protein kinase, the DNA-PK is known to be an important mediator of DSB repair in human cells, and showed increased number of pH2AX (ser139) foci, and thus indicating lower DNA repair in these cancer cells. This was also confirmed in the tumor xenograft study. Our findings suggest that a combination of silibinin with radiation could be an effective treatment of radioresistant human prostate cancer and warrants further investigation. (author)

  14. Controls to validate plasma samples for cell free DNA quantification

    DEFF Research Database (Denmark)

    Pallisgaard, Niels; Spindler, Karen-Lise Garm; Andersen, Rikke Fredslund

    2015-01-01

    , are diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use. The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample...... preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values...

  15. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  16. DNA repair in lens cells during chick embryo development

    International Nuclear Information System (INIS)

    Counis, M.F.; Chaudun, E.; Simonneau, L.; Courtois, Y.

    1979-01-01

    When chick lens epithelium is cultured in vitro, differentiation into lens fiber cells is accompanied by DNA degradation. This phenomenon of terminal differentiation was studied in the epithelium from embryos at the 6th and 11th days of development. DNA size and the ability of the cells to repair DNA damage induced by X-rays were analysed in alkaline sucrose gradients. In the 6-day epithelium a rapid degradation and complete lack of DNA repair were recorded. Similar observations have been made in previous studies on the 11-day sample, but here degradation is progressive and occurs after a lag of several days. In the younger epithelium, internal irradiation by [ 3 H)thymidine also had a drastic effect resembling that caused by X-rays. In order to assess the process of differentiation in the experimental system the synthesis of delta- and αcrystallins was monitored. Stage-related modifications in the rates of synthesis were recorded. The results confirm that the DNA repair system is impaired during terminal differentiation. The differences observed between the two stages may reflect either a developmental modification in DNA repair mechanisms or a change in the relative proportions of differentiating cells. An hypothesis is proposed in support of the latter case. (Auth.)

  17. Auxin as an inducer of asymmetrical division generating the subsidiary cells in stomatal complexes of Zea mays.

    Science.gov (United States)

    Livanos, Pantelis; Giannoutsou, Eleni; Apostolakos, Panagiotis; Galatis, Basil

    2015-01-01

    The data presented in this work revealed that in Zea mays the exogenously added auxins indole-3-acetic acid (IAA) and 1-napthaleneacetic acid (NAA), promoted the establishment of subsidiary cell mother cell (SMC) polarity and the subsequent subsidiary cell formation, while treatment with auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and 1-napthoxyacetic acid (NOA) specifically blocked SMC polarization and asymmetrical division. Furthermore, in young guard cell mother cells (GMCs) the PIN1 auxin efflux carriers were mainly localized in the transverse GMC faces, while in the advanced GMCs they appeared both in the transverse and the lateral ones adjacent to SMCs. Considering that phosphatidyl-inositol-3-kinase (PI3K) is an active component of auxin signal transduction and that phospholipid signaling contributes in the establishment of polarity, treatments with the specific inhibitor of the PI3K LY294002 were carried out. The presence of LY294002 suppressed polarization of SMCs and prevented their asymmetrical division, whereas combined treatment with exogenously added NAA and LY294002 restricted the promotional auxin influence on subsidiary cell formation. These findings support the view that auxin is involved in Z. mays subsidiary cell formation, probably functioning as inducer of the asymmetrical SMC division. Collectively, the results obtained from treatments with auxin transport inhibitors and the appearance of PIN1 proteins in the lateral GMC faces indicate a local transfer of auxin from GMCs to SMCs. Moreover, auxin signal transduction seems to be mediated by the catalytic function of PI3K.

  18. Patterns of oriented cell division during the steady-state morphogenesis of the body column in hydra.

    Science.gov (United States)

    Shimizu, H; Bode, P M; Bode, H R

    1995-12-01

    In an adult hydra, the tissue of the body column is in a dynamic state. The epithelial cells of both layers are constantly in the mitotic cycle. As the tissue expands, it is continuously displaced along the body axis in either an apical or basal direction, but not in a circumferential direction. Using a modified whole mount method we examined the orientation of mitotic spindles to determine what role the direction of cell division plays in axial displacement. Surprisingly, the direction of cell division was found to differ in the two epithelial layers. In the ectoderm it was somewhat biased in an axial direction. In the endoderm it was strongly biased in a circumferential direction. For both layers, the directional biases occurred throughout the length of the body column, with some regional variation in its extent. As buds developed into adults, the bias in each layer increased from an almost random distribution to the distinctly different orientations of the adult. Thus, to maintain the observed axial direction of tissue displacement, rearrangement of the epithelial cells of both layers must occur continuously in the adult as well as in developing animals. How the locomotory and contractile behavior of the muscle processes of the epithelial cells may effect changes in cell shape, and thereby influence the direction of cell division in each layer, is discussed.

  19. Differential sensitivity to aphidicolin of replicative DNA synthesis and ultraviolet-induced unscheduled DNA synthesis in vivo in mammalian cells

    International Nuclear Information System (INIS)

    Seki, Shuji; Hosogi, Nobuo; Oda, Takuzo

    1984-01-01

    In vivo in mammalian cells, ultraviolet-induced unscheduled DNA synthesis was less sensitive to aphidicolin than was replicative DNA synthesis. Replicative DNA synthesis in HeLa, HEp-2, WI-38 VA-13 and CV-1 cells was inhibited more than 97 % by aphidicolin at 10 μg/ml, whereas aphidicolin inhibition of DNA synthesis in ultraviolet-irradiated cells varied between 30 % and 90 % depending on cell types and assay conditions. Aphidicolin inhibition of unscheduled DNA synthesis (UDS) in HeLa cells increased gradually with increasing aphidicolin concentration and reached approximately 90 % at 100 μg/ml aphidicolin. A significant fraction of UDS in ultraviolet-irradiated HEp-2 cells was resistant to aphidicolin even at 300 μg/ml. Considered along with related information reported previously, the present results suggest that both aphidicolin-sensitive and insensitive DNA polymerases, DNA polymerase α and a non-α DNA polymerase (possibly DNA polymerase β), are involved in in situ UDS in these ultraviolet-irradiated cells. Comparison of staphylococcal nuclease sensitivity between DNAs repaired in the presence and in the absence of aphidicolin in HEp-2 cells suggested that the involvement of DNA polymerase α in UDS favored DNA synthesis in the intranucleosomal region. (author)

  20. Unscheduled DNA synthesis and elimination of DNA damage in liver cells of. gamma. -irradiated senescent mice

    Energy Technology Data Exchange (ETDEWEB)

    Gaziev, A.I.; Malakhova, L.V. (AN SSSR, Pushchino-na-Oke. Inst. Biologicheskoj Fiziki)

    1982-10-01

    The level of 'spontaneous' and ..gamma..-radiation-induced DNA synthesis which is not inhibited with hydroxyurea (unscheduled synthesis) is considerably lower in hepatocytes of 18-22-month-old mice than that of 1.5-2-month-old mice. The dose-dependent increase (10-300 Gy) of unscheduled DNA synthesis (UDS) in hepatocytes of senescent mice is higher than in young animals. The elimination of damage in DNA of ..gamma..-irradiated hepatocytes (100 Gy) was examined by using an enzyme system (M. luteus extract and DNA-polymerase I of E. coli). It was found that the rate of elimination of the DNA damage in hepatocytes of 20-month-old mice is lower than that of 2-month-old mice although the activities of DNA-polymerase ..beta.. and apurinic endonuclease remain equal in the liver of both senescent and young mice. However, the nucleoids from ..gamma..-irradiated liver nuclei of 2-month-old mice are relaxed to a greater extent (as judged by the criterion of ethidium-binding capacity) than those of 20-month-old mice. The results suggest that there are limitations in the functioning of repair enzymes and in their access to damaged DNA sites in the chromatin of senescent mouse liver cells.

  1. Un-repairable DNA damage in cell due to irradiation

    International Nuclear Information System (INIS)

    Yoshii, Giichi

    1992-01-01

    Radiation-induced cell reproductive deactivation is caused by damage to DNA. In a cell, cellular DNA radical reacts with diffusion controlled rate and generates DNA peroxide radical. The chemical repair of DNA radical with hydrogen donation by thiol competes with the reaction of oxygen with same radicals in the DNA molecules. From the point reaction rates, the prolongation of radical life time is not as great as expected from the reduction in the glutathione content of the cell. This indicates that further reducting compounds (protein bound thiol) are present in the cell. The residual radicals are altered to strand breaks, base damages and so on. The effective lesions for a number of endpoints is un-repaired double strand break, which has been discovered in a cluster. This event gives risk to high LET radiation or to a track end of X-rays. For X- or electron irradiations the strand breaks are frequently induced by the interactions between sublesions on two strands in DNA. A single strand break followed by radical action may be unstable excited state, because of remaining sugar radical action and of having negative charged phosphates, in which strands breaks will be rejoined in a short time to stable state. On the same time, a break in the double helix will be immediately produced if two breaks are on either or approximately opposite locations. The formation of a double strand break in the helix depends on the ion strength of the cell. The potassium ions are largely released from polyanionic strand during irradiation, which results in the induction of denatured region. Double strand break with the denatured region seems to be un-repairable DNA damage. (author)

  2. Cell-free DNA in a three-dimensional spheroid cell culture model

    DEFF Research Database (Denmark)

    Aucamp, Janine; Calitz, Carlemi; Bronkhorst, Abel J.

    2017-01-01

    Background Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo...... cultures can serve as effective, simplified in vivo-simulating “closed-circuit” models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. Biological...... significance 3D cell cultures can be used to translate “closed-circuit” in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth...

  3. The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

    Science.gov (United States)

    Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

    2017-07-15

    Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

  4. In tobacco BY-2 cells xyloglucan oligosaccharides alter the expression of genes involved in cell wall metabolism, signalling, stress responses, cell division and transcriptional control.

    Science.gov (United States)

    González-Pérez, Lien; Perrotta, Lara; Acosta, Alexis; Orellana, Esteban; Spadafora, Natasha; Bruno, Leonardo; Bitonti, Beatrice M; Albani, Diego; Cabrera, Juan Carlos; Francis, Dennis; Rogers, Hilary J

    2014-10-01

    Xyloglucan oligosaccharides (XGOs) are breakdown products of XGs, the most abundant hemicelluloses of the primary cell walls of non-Poalean species. Treatment of cell cultures or whole plants with XGOs results in accelerated cell elongation and cell division, changes in primary root growth, and a stimulation of defence responses. They may therefore act as signalling molecules regulating plant growth and development. Previous work suggests an interaction with auxins and effects on cell wall loosening, however their mode of action is not fully understood. The effect of an XGO extract from tamarind (Tamarindus indica) on global gene expression was therefore investigated in tobacco BY-2 cells using microarrays. Over 500 genes were differentially regulated with similar numbers and functional classes of genes up- and down-regulated, indicating a complex interaction with the cellular machinery. Up-regulation of a putative XG endotransglycosylase/hydrolase-related (XTH) gene supports the mechanism of XGO action through cell wall loosening. Differential expression of defence-related genes supports a role for XGOs as elicitors. Changes in the expression of genes related to mitotic control and differentiation also support previous work showing that XGOs are mitotic inducers. XGOs also affected expression of several receptor-like kinase genes and transcription factors. Hence, XGOs have significant effects on expression of genes related to cell wall metabolism, signalling, stress responses, cell division and transcriptional control.

  5. Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

    Directory of Open Access Journals (Sweden)

    Sharma Shikhar

    2012-01-01

    Full Text Available Abstract Background DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. Results Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation. Conclusions Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.

  6. Cellular aging of mitochondrial DNA-depleted cells

    International Nuclear Information System (INIS)

    Park, Sun Young; Choi, Bongkun; Cheon, Hwanju; Pak, Youngmi Kim; Kulawiec, Mariola; Singh, Keshav K.; Lee, Myung-Shik

    2004-01-01

    We have reported that mitochondrial DNA-depleted ρ 0 cells are resistant to cell death. Because aged cells have frequent mitochondrial DNA mutations, the resistance of ρ 0 cells against cell death might be related to the apoptosis resistance of aged cells and frequent development of cancers in aged individuals. We studied if ρ 0 cells have features simulating aged cells. SK-Hep1 hepatoma ρ 0 cells showed typical morphology associated with aging such as increased size and elongated appearance. They had increased senescence-associated β-Gal activity, lipofuscin pigment, and plasminogen activator inhibitor-1 expression. Consistent with their decreased proliferation, the expression of mitotic cyclins was decreased and that of cdk inhibitors was increased. Rb hypophosphorylation and decreased telomerase activity were also noted. Features simulating aged cells were also observed in MDA-MB-435 ρ 0 cells. These results support the mitochondrial theory of aging, and suggest that ρ 0 cells could serve as an in vitro model for aged cells

  7. DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

    International Nuclear Information System (INIS)

    Kim, Hak Jae; Kim, Jin Ho; Chie, Eui Kyu; Da Young, Park; Kim, In Ah; Kim, Il Han

    2012-01-01

    Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry. Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone. Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair

  8. Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells

    International Nuclear Information System (INIS)

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Musarrat, Javed

    2012-01-01

    Highlights: ► Butachlor exhibited strong binding affinity with DNA and produced 8-oxodG adducts. ► Butachlor induced DNA strand breaks and micronuclei formation in PBMN cells. ► Butachlor induced ROS and dissipation of mitochondrial membrane potential in cells. ► Butachlor resulted in cell cycle arrest and eventually caused cellular necrosis. -- Abstract: Butachlor is a systemic herbicide widely applied on rice, tea, wheat, beans and other crops; however, it concurrently exerts toxic effects on beneficial organisms like earthworms, aquatic invertebrates and other non-target animals including humans. Owing to the associated risk to humans, this chloroacetanilide class of herbicide was investigated with the aim to assess its potential for the (i) interaction with DNA, (ii) mitochondria membrane damage and DNA strand breaks and (iii) cell cycle arrest and necrosis in butachlor treated human peripheral blood mononuclear (PBMN) cells. Fluorescence quenching data revealed the binding constant (Ka = 1.2 × 10 4 M −1 ) and binding capacity (n = 1.02) of butachlor with ctDNA. The oxidative potential of butachlor was ascertained based on its capacity of inducing reactive oxygen species (ROS) and substantial amounts of promutagenic 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) adducts in DNA. Also, the discernible butachlor dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) and increased fluorescence intensity of 2′,7′-dichlorodihydro fluorescein diacetate (DCFH-DA) in treated cells signifies decreased mitochondrial membrane potential (ΔΨm) due to intracellular ROS generation. The comet data revealed significantly greater Olive tail moment (OTM) values in butachlor treated PBMN cells vs untreated and DMSO controls. Treatment of cultured PBMN cells for 24 h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). The flow

  9. Emp is a component of the nuclear matrix of mammalian cells and undergoes dynamic rearrangements during cell division

    International Nuclear Information System (INIS)

    Bala, Shashi; Kumar, Ajay; Soni, Shivani; Sinha, Sudha; Hanspal, Manjit

    2006-01-01

    Emp, originally detected in erythroblastic islands, is expressed in numerous cell types and tissues suggesting a functionality not limited to hematopoiesis. To study the function of Emp in non-hematopoietic cells, an epitope-tagged recombinant human Emp was expressed in HEK cells. Preliminary studies revealed that Emp partitioned into both the nuclear and Triton X-100-insoluble cytoskeletal fractions in approximately a 4:1 ratio. In this study, we report investigations of Emp in the nucleus. Sequential extractions of interphase nuclei showed that recombinant Emp was present predominantly in the nuclear matrix. Immunofluorescence microscopy showed that Emp was present in typical nuclear speckles enriched with the spliceosome assembly factor SC35 and partially co-localized with actin staining. Coimmunoprecipitation and GST-pull-down assays confirmed the apparent close association of Emp with nuclear actin. During mitosis, Emp was detected at the mitotic spindle/spindle poles, as well as in the contractile ring during cytokinesis. These results suggest that Emp undergoes dynamic rearrangements within the nuclear architecture that are correlated with cell division

  10. Molecular targets, DNA breakage, DNA repair: Their roles in mutation induction in mammalian germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.

    1989-01-01

    Variability in genetic sensitivity among different germ-cell stages in the mammal to various mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. Several chemicals have been found to bind very strongly to protamine in late-spermatid and early-spermatozoa stages in the mouse. The chemicals also produce their greatest genetic damage in these same germ-cell stages. While chemical binding to DNA has not been correlated with the level of induced genetic damage, DNA breakage in the sensitive stages has been shown to increase. This DNA breakage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus. 22 refs., 5 figs

  11. Nuclear and cell division in Bacillus subtilis. Antibiotic-induced morphological changes

    NARCIS (Netherlands)

    van Iterson, W.; Aten, J. A.

    1976-01-01

    Incubation of Bacillus subtilis after outgrowth from spores in the presence of four different antibiotics in two different concentrations, showed that septation can occur without termination of nuclear division. Septation is then only partially uncoupled from the normal division cycle. Observations

  12. Effect of various 3H-thymidine concentrations on the kinetics of chinese hamster cell division

    International Nuclear Information System (INIS)

    Yuzhakov, V.V.; Lychev, V.A.

    1985-01-01

    A study of the asynchronous culture of Chinese hamster fibroblasts by autoradiography has shown that the pulse (15 min) incorporation of 3 H-thymidine in nuclear DNA influences the kinetics of labelled cell proliferation. The results obtained suggest that one of the early biological effects of the pulse incorporation of 3 H-thymidine is a delay in the occurrence of the first mitosis. With the concentration of 3 H-thymidine 37 kBq/ml the slowing down of the movement of labelled cells in the cycle is detected by a shift and overlapping of waves of labelled and unlabelled mitotic cells. In an increase of the concentration up to 370-925 kBq/ml the pattern of the curves of labelled mitotic cells is distorted. These distortions are well interpreted by the nature of change of the index of labelled and unlabelled mitotic cells. After an increase in 3 H-thymidine concentration from 37 up to 370-925 kBq/ml the mitotic activity of cells labelled at the end of S-phase decreases from 1 to o0.6-0.1% respectively. With the concentration of 925 kBq/ml for these cells incorporating 3 H-thymidine at the end of S-phase, a delay of the entry into mitosis reaches 6-8 h. Autoradiography data with assessment of granule density suggest that mitotic activity and the period of delay in the occurrence of mitosis depend on the dose of irradiation with intranuclear tritium

  13. DNA damage by carbonyl stress in human skin cells

    International Nuclear Information System (INIS)

    Roberts, Michael J.; Wondrak, Georg T.; Laurean, Daniel Cervantes; Jacobson, Myron K.; Jacobson, Elaine L.

    2003-01-01

    Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the α-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N ε -(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress

  14. DNA damage by carbonyl stress in human skin cells

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, Michael J.; Wondrak, Georg T.; Laurean, Daniel Cervantes; Jacobson, Myron K.; Jacobson, Elaine L

    2003-01-28

    Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the {alpha}-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N{sup {epsilon}}-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.

  15. The simulation model of growth and cell divisions for the root apex with an apical cell in application to Azolla pinnata.

    Science.gov (United States)

    Piekarska-Stachowiak, Anna; Nakielski, Jerzy

    2013-12-01

    In contrast to seed plants, the roots of most ferns have a single apical cell which is the ultimate source of all cells in the root. The apical cell has a tetrahedral shape and divides asymmetrically. The root cap derives from the distal division face, while merophytes derived from three proximal division faces contribute to the root proper. The merophytes are produced sequentially forming three sectors along a helix around the root axis. During development, they divide and differentiate in a predictable pattern. Such growth causes cell pattern of the root apex to be remarkably regular and self-perpetuating. The nature of this regularity remains unknown. This paper shows the 2D simulation model for growth of the root apex with the apical cell in application to Azolla pinnata. The field of growth rates of the organ, prescribed by the model, is of a tensor type (symplastic growth) and cells divide taking principal growth directions into account. The simulations show how the cell pattern in a longitudinal section of the apex develops in time. The virtual root apex grows realistically and its cell pattern is similar to that observed in anatomical sections. The simulations indicate that the cell pattern regularity results from cell divisions which are oriented with respect to principal growth directions. Such divisions are essential for maintenance of peri-anticlinal arrangement of cell walls and coordinated growth of merophytes during the development. The highly specific division program that takes place in merophytes prior to differentiation seems to be regulated at the cellular level.

  16. Patterns of DNA Methylation in Development, Division of Labor and Hybridization in an Ant with Genetic Caste Determination

    OpenAIRE

    Smith, Chris R.; Mutti, Navdeep S.; Jasper, W. Cameron; Naidu, Agni; Smith, Christopher D.; Gadau, Jürgen

    2012-01-01

    BACKGROUND: DNA methylation is a common regulator of gene expression, including acting as a regulator of developmental events and behavioral changes in adults. Using the unique system of genetic caste determination in Pogonomyrmex barbatus, we were able to document changes in DNA methylation during development, and also across both ancient and contemporary hybridization events. METHODOLOGY/PRINCIPAL FINDINGS: Sodium bisulfite sequencing demonstrated in vivo methylation of symmetric CG dinucle...

  17. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    Directory of Open Access Journals (Sweden)

    José J. Gaforio

    2011-10-01

    Full Text Available Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A or breast cancer cells (MDA-MB-231 and MCF7. We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  18. The repair of damage to DNA in different cell types

    International Nuclear Information System (INIS)

    Karran, P.

    1974-01-01

    DNA single strand breaks induced by either X-ray irradiation or by methyl methanesulphonate (MMS) were studied in different lymphoid cell populations directly taken from the animal and maintained in tissue culture merely for the duration of the experiment. The results obtained from these cell populations were compared with those obtained with L5178Y cells maintained in tissue culture. All cell types studied were found to possess at least one class of enzymes required for repair of DNA damage, namely those enzymes involved in the rejoining of X-ray induced by MMS is different in each cell type. Repair replication was at much reduced levels and the endonucleolytic degradation was at much reduced levels and the endonucleolytic degradation was initiated at lower MMS concentration in the lymphoid cells as compared to L5178Y cells. It is suggested that the overall ''repair capacity'' of a population may be related to the number of cells in a cycle which, moreover, might be the only ones to have the ability to repair damage to DNA induced by MMS (G.G.)

  19. Conformations of MutS in DNA mismatch repair

    NARCIS (Netherlands)

    F.S. Groothuizen (Flora)

    2015-01-01

    markdownabstract__Abstract__ Prior to cell division, the DNA containing the genetic information of a cell has to be copied. During this process, errors are sometimes incorporated (so-called mismatches), which may cause genetic abnormalities in future cells. To prevent this, cells contain a DNA

  20. Yeast cells contain a heterogeneous population of peroxisomes that segregate asymmetrically during cell division

    NARCIS (Netherlands)

    Kumar, Sanjeev; de Boer, Rinse; van der Klei, Ida J

    2018-01-01

    Here we used fluorescence microscopy and a peroxisome-targeted tandem fluorescent protein timer to determine the relative age of peroxisomes in yeast. Our data indicate that yeast cells contain a heterogeneous population of relatively old and younger peroxisomes. During budding the peroxisome

  1. Cell division factors from crown gall tumors: a strategy for structural elucidation

    International Nuclear Information System (INIS)

    Manning, K.S.

    1985-01-01

    Mitogenic compounds present in extracts of Vinca rosea crown gall tumor tissue were investigated. An isolation procedure, consisting of solvent partitions and reverse phase chromatography, has yielded a group of isomeric compounds which show activity in the tobacco pith bioassay. Initial characterizations revealed an unsaturated base, a sugar residue, a β-linked glucose, an allylic alcohol, and two methyl groups. A two part strategy of mass spectrometry (MS) in combination with proton nuclear magnetic resonance ( 1 H NMR) was envisioned. The aglycone structure would be determined by MS and the regiochemical relationships among the structural units would be defined by 1 H NMR data. The utility of this approach was demonstrated by the structure assignment of a specific inhibitor of β-D-glucuronidase, 2(S)-carboxy-3(R),4(R),5(S)-trihydroxypiperidine. The relative stereochemistry of the hydroxyls was revealed by 1 H NMR and the absolute configuration was deduced by a comparison of Cotton effects with a model compound. The use of 1 H NMR to establish regiochemical relationships was investigated. Terpenes containing quaternary carbons and methyl groups were excellent models for the regiochemical problems presented by the mitogenic factors. This 1 H NMR spectroscopy has been applied to the cell division factor structure problem. These data, with information from two dimensional nOe experiments, have defined some of the regio-relationships among the structural units present in the isolated factors

  2. Asymmetric cell division and Notch signaling specify dopaminergic neurons in Drosophila.

    Directory of Open Access Journals (Sweden)

    Murni Tio

    Full Text Available In Drosophila, dopaminergic (DA neurons can be found from mid embryonic stages of development till adulthood. Despite their functional involvement in learning and memory, not much is known about the developmental as well as molecular mechanisms involved in the events of DA neuronal specification, differentiation and maturation. In this report we demonstrate that most larval DA neurons are generated during embryonic development. Furthermore, we show that loss of function (l-o-f mutations of genes of the apical complex proteins in the asymmetric cell division (ACD machinery, such as inscuteable and bazooka result in supernumerary DA neurons, whereas l-o-f mutations of genes of the basal complex proteins such as numb result in loss or reduction of DA neurons. In addition, when Notch signaling is reduced or abolished, additional DA neurons are formed and conversely, when Notch signaling is activated, less DA neurons are generated. Our data demonstrate that both ACD and Notch signaling are crucial mechanisms for DA neuronal specification. We propose a model in which ACD results in differential Notch activation in direct siblings and in this context Notch acts as a repressor for DA neuronal specification in the sibling that receives active Notch signaling. Our study provides the first link of ACD and Notch signaling in the specification of a neurotransmitter phenotype in Drosophila. Given the high degree of conservation between Drosophila and vertebrate systems, this study could be of significance to mechanisms of DA neuronal differentiation not limited to flies.

  3. Manganese(II) induces cell division and increases in superoxide dismutase and catalase activities in an aging deinococcal culture

    International Nuclear Information System (INIS)

    Chou, F.I.; Tan, S.T.

    1990-01-01

    Addition of Mn(II) at 2.5 microM or higher to stationary-phase cultures of Deinococcus radiodurans IR was found to trigger at least three rounds of cell division. This Mn(II)-induced cell division (Mn-CD) did not occur when the culture was in the exponential or death phase. The Mn-CD effect produced daughter cells proportionally reduced in size, pigmentation, and radioresistance but proportionally increased in activity and amount of the oxygen toxicity defense enzymes superoxide dismutase and catalase. In addition, the concentration of an Mn-CD-induced protein was found to remain high throughout the entire Mn-CD phase. It was also found that an untreated culture exhibited a growth curve characterized by a very rapid exponential-stationary transition and that cells which had just reached the early stationary phase were synchronous. Our results suggest the presence of an Mn(II)-sensitive mechanism for controlling cell division. The Mn-CD effect appears to be specific to the cation Mn(II) and the radioresistant bacteria, deinococci

  4. Nanoscale imaging of the growth and division of bacterial cells on planar substrates with the atomic force microscope

    Energy Technology Data Exchange (ETDEWEB)

    Van Der Hofstadt, M. [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Hüttener, M.; Juárez, A. [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Departament de Microbiologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona (Spain); Gomila, G., E-mail: ggomila@ibecbarcelona.eu [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Departament d' Electronica, Universitat de Barcelona, C/ Marti i Franqués 1, 08028 Barcelona (Spain)

    2015-07-15

    With the use of the atomic force microscope (AFM), the Nanomicrobiology field has advanced drastically. Due to the complexity of imaging living bacterial processes in their natural growing environments, improvements have come to a standstill. Here we show the in situ nanoscale imaging of the growth and division of single bacterial cells on planar substrates with the atomic force microscope. To achieve this, we minimized the lateral shear forces responsible for the detachment of weakly adsorbed bacteria on planar substrates with the use of the so called dynamic jumping mode with very soft cantilever probes. With this approach, gentle imaging conditions can be maintained for long periods of time, enabling the continuous imaging of the bacterial cell growth and division, even on planar substrates. Present results offer the possibility to observe living processes of untrapped bacteria weakly attached to planar substrates. - Highlights: • Gelatine coatings used to weakly attach bacterial cells onto planar substrates. • Use of the dynamic jumping mode as a non-perturbing bacterial imaging mode. • Nanoscale resolution imaging of unperturbed single living bacterial cells. • Growth and division of single bacteria cells on planar substrates observed.

  5. Exposure of Human CD8+ T Cells to Type-2 Cytokines Impairs Division and Differentiation and Induces Limited Polarization

    Directory of Open Access Journals (Sweden)

    Annette Fox

    2018-05-01

    Full Text Available Effector CD8+ T cells generally produce type-1 cytokines and mediators of the perforin/granzyme cytolytic pathway, yet type-2-polarized CD8+ cells (Tc2 are detected in type-2 (T2 cytokine-driven diseases such as asthma. It is unclear whether T2 cytokine exposure during activation is sufficient to polarize human CD8+ T cells. To address this question, a protocol was developed for high-efficiency activation of human CD8+ T cells in which purified single cells or populations were stimulated with plate-bound anti-CD3 and anti-CD11a mAb for up to 8 days in T2 polarizing or neutral conditions, before functional analysis. Activation of CD8+ naïve T cells (TN in T2 compared with neutral conditions decreased the size of single-cell clones, although early division kinetics were equivalent, indicating an effect on overall division number. Activation of TN in T2 conditions followed by brief anti-CD3 mAb restimulation favored expression of T2 cytokines, GATA3 and Eomes, and lowered expression of type-1 cytokines, Prf1, Gzmb, T-BET, and Prdm1. However, IL-4 was only weakly expressed, and PMA and ionomycin restimulation favored IFN-γ over IL-4 expression. Activation of TN in T2 compared with neutral conditions prevented downregulation of costimulatory (CD27, CD28 and lymph-node homing receptors (CCR7 and CD95 acquisition, which typically occur during differentiation into effector phenotypes. CD3 was rapidly and substantially induced after activation in neutral, but not T2 conditions, potentially contributing to greater division and differentiation in neutral conditions. CD8+ central memory T cells (TCM were less able to enter division upon reactivation in T2 compared with neutral conditions, and were more refractory to modulating IFN-γ and IL-4 production than CD8+ TN. In summary, while activation of TN in T2 conditions can generate T2 cytokine-biased cells, IL-4 expression is weak, T2 bias is lost upon strong restimulation, differentiation, and division

  6. Blood cell mitochondrial DNA content and premature ovarian aging.

    Directory of Open Access Journals (Sweden)

    Marco Bonomi

    Full Text Available Primary ovarian insufficiency (POI is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA content in a group of women undergoing ovarian hyperstimulation (OH, and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF and 42 poor responders (PR to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001 in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction.

  7. Deliberate ROS production and auxin synergistically trigger the asymmetrical division generating the subsidiary cells in Zea mays stomatal complexes.

    Science.gov (United States)

    Livanos, Pantelis; Galatis, Basil; Apostolakos, Panagiotis

    2016-07-01

    Subsidiary cell generation in Poaceae is an outstanding example of local intercellular stimulation. An inductive stimulus emanates from the guard cell mother cells (GMCs) towards their laterally adjacent subsidiary cell mother cells (SMCs) and triggers the asymmetrical division of the latter. Indole-3-acetic acid (IAA) immunolocalization in Zea mays protoderm confirmed that the GMCs function as local sources of auxin and revealed that auxin is polarly accumulated between GMCs and SMCs in a timely-dependent manner. Besides, staining techniques showed that reactive oxygen species (ROS) exhibit a closely similar, also time-dependent, pattern of appearance suggesting ROS implication in subsidiary cell formation. This phenomenon was further investigated by using the specific NADPH-oxidase inhibitor diphenylene iodonium, the ROS scavenger N-acetyl-cysteine, menadione which leads to ROS overproduction, and H2O2. Treatments with diphenylene iodonium, N-acetyl-cysteine, and menadione specifically blocked SMC polarization and asymmetrical division. In contrast, H2O2 promoted the establishment of SMC polarity and subsequently subsidiary cell formation in "younger" protodermal areas. Surprisingly, H2O2 favored the asymmetrical division of the intervening cells of the stomatal rows leading to the creation of extra apical subsidiary cells. Moreover, H2O2 altered IAA localization, whereas synthetic auxin analogue 1-napthaleneacetic acid enhanced ROS accumulation. Combined treatments with ROS modulators along with 1-napthaleneacetic acid or 2,3,5-triiodobenzoic acid, an auxin efflux inhibitor, confirmed the crosstalk between ROS and auxin functioning during subsidiary cell generation. Collectively, our results demonstrate that ROS are critical partners of auxin during development of Z. mays stomatal complexes. The interplay between auxin and ROS seems to be spatially and temporarily regulated.

  8. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

  9. Stable radioresistance in ataxia-telangiectasia cells containing DNA from normal human cells

    International Nuclear Information System (INIS)

    Kapp, L.N.; Painter, R.B.

    1989-01-01

    SV40-transformed ataxia-telangiectasia (AT) cells were transfected with a cosmid containing a normal human DNA library and selectable marker, the neo gene, which endows successfully transformed mammalian cells with resistance to the antibiotic G418. Cells from this line were irradiated with 50 Gy of X-rays and fused with non-transfected AT cells. Among the G418-resistant colonies recovered was one stably resistant to radiation. Resistance to ionizing radiation of both primary transfectant line and its fusion derivative was intermediate between that of AT cells and normal cells, as assayed by colony-forming ability and measurement of radiation-induced G 2 chromatic aberrations; both cell lines retained AT-like radioresistant DNA synthesis. Results suggest that, because radioresistance in transfected cells was not as great as in normal human cells, two hallmarks of AT, radiosensitivity and radioresistant DNA synthesis, may still be the result of a single defective AT gene. (author)

  10. DNA-repair, cell killing and normal tissue damage

    International Nuclear Information System (INIS)

    Dahm-Daphi, J.; Dikomey, E.; Brammer, I.

    1998-01-01

    Background: Side effects of radiotherapy in normal tissue is determined by a variety of factors of which cellular and genetic contributions are described here. Material and methods: Review. Results: Normal tissue damage after irradiation is largely due to loss of cellular proliferative capacity. This can be due to mitotic cell death, apoptosis, or terminal differentiation. Dead or differentiated cells release cytokines which additionally modulate the tissue response. DNA damage, in particular non-reparable or misrepaired double-strand breaks are considered the basic lesion leading to G1-arrest and ultimately to cell inactivation. Conclusion: Evidence for genetic bases of normal tissue response, cell killing and DNA-repair capacity is presented. However, a direct link of all 3 endpoints has not yet been proved directly. (orig.) [de

  11. DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

    International Nuclear Information System (INIS)

    Yaki, T.

    1982-01-01

    DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

  12. Mannosylated biodegradable polyethyleneimine for targeted DNA delivery to dendritic cells

    Directory of Open Access Journals (Sweden)

    Sun X

    2012-06-01

    Full Text Available Xun Sun, Simu Chen, Jianfeng Han, Zhirong ZhangKey Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People’s Republic of ChinaBackground: To establish a potential gene-delivery system with the ability to deliver plasmid DNA to dendritic cells (DCs more efficiently and specifically, we designed and synthesized a low-molecular-weight polyethyleneimine and triethyleneglycol polymer (PEI–TEG and a series of its mannosylated derivatives.Methods: PEI–TEG was synthesized from PEI2000 and PEI600 with TEG as the cross-linker. PEI–TEG was then linked to mannose via a phenylisothiocyanate bridge to obtain man-PEI–TEG conjugates. The DNA conveyance abilities of PEI–TEG, man-PEI–TEG, as well as control PEI25k were evaluated by measuring their zeta potential, particle size, and DNA-binding abilities. The in vitro cytotoxicity, cell uptake, and transfection efficiency of these PEI/DNA complexes were examined on the DC2.4 cell line. Finally, a maturation experiment evaluated the effect of costimulatory molecules CD40, CD80, and CD86 on murine bone marrow-derived DCs (BMDCs using flow cytometry.Results: PEI–TEG and man-PEI–TEG were successfully synthesized and were shown to retain the excellent properties of PEI25k for condensing DNA. Compared with PEI–TEG as well as PEI25k, the man-PEI–TEG had less cytotoxicity and performed better in both cellular uptake and transfection assays in vitro. The results of the maturation experiment showed that all the PEI/DNA complexes induced an adequate upregulation of surface markers for DC maturation.Conclusion: These results demonstrated that man-PEI–TEG can be employed as a DC-targeting gene-delivery system.Keywords: dendritic cells, DCs, mannose, polyethyleneimine, PEI, gene delivery

  13. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    tetrazolium bromide (MTT) test. Production of ROS was determined by the oxidation of 2',7'-dichlorodihydrofluorescein to a fluorescent 2',7'-dichlorofluorescein and measured by fluorescence reading and visualized by fluorescence microscopy. DNA stability was determined by single cell gel electrophoresis...

  14. DNA fingerprinting of the NCI-60 cell line panel.

    Science.gov (United States)

    Lorenzi, Philip L; Reinhold, William C; Varma, Sudhir; Hutchinson, Amy A; Pommier, Yves; Chanock, Stephen J; Weinstein, John N

    2009-04-01

    The National Cancer Institute's NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Because many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also show that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes.

  15. Damage-induced DNA repair processes in Escherichia coli cells

    International Nuclear Information System (INIS)

    Slezarikova, V.

    1986-01-01

    The existing knowledge is summed up of the response of Escherichia coli cells to DNA damage due to various factors including ultraviolet radiation. So far, three inducible mechanisms caused by DNA damage are known, viz., SOS induction, adaptation and thermal shock induction. Greatest attention is devoted to SOS induction. Its mechanism is described and the importance of the lexA recA proteins is shown. In addition, direct or indirect role is played by other proteins, such as the ssb protein binding the single-strand DNA sections. The results are reported of a study of induced repair processes in Escherichia coli cells repeatedly irradiated with UV radiation. A model of induction by repeated cell irradiation discovered a new role of induced proteins, i.e., the elimination of alkali-labile points in the daughter DNA synthetized on a damaged model. The nature of the alkali-labile points has so far been unclear. In the adaptation process, regulation proteins are synthetized whose production is induced by the presence of alkylation agents. In the thermal shock induction, new proteins synthetize in cells, whose function has not yet been clarified. (E.S.)

  16. Flow cytometric DNA ploidy analysis of ovarian granulosa cell tumors

    NARCIS (Netherlands)

    D. Chadha; C.J. Cornelisse; A. Schabert (A.)

    1990-01-01

    textabstractAbstract The nuclear DNA content of 50 ovarian tumors initially diagnosed as granulosa cell tumors was measured by flow cytometry using paraffin-embedded archival material. The follow-up period of the patients ranged from 4 months to 19 years. Thirty-eight tumors were diploid or

  17. Ionizing radiation sensitivity of DNA polymerase lambda-deficient cells.

    NARCIS (Netherlands)

    Vermeulen, C.; Bertocci, B.; Begg, A.C.; Vens, C.

    2007-01-01

    Ionizing radiation induces a diverse spectrum of DNA lesions, including strand breaks and oxidized bases. In mammalian cells, ionizing radiation-induced lesions are targets of non-homologous end joining, homologous recombination, and base excision repair. In vitro assays show a potential involvement

  18. DNA repair in a Fanconi's anemia fibroblast cell strain

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Little, J.B.; Weichselbaum, R.R.

    1979-01-01

    DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution. (Auth.)

  19. Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

    Directory of Open Access Journals (Sweden)

    María Berdasco

    Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

  20. DNA repair in a Fanconi's anemia fibroblast cell strain

    Energy Technology Data Exchange (ETDEWEB)

    Fornace, Jr, A J; Little, J B [Harvard School of Public Health, Boston, MA (USA); Weichselbaum, R R [Harvard Medical School, Boston, MA (USA)

    1979-01-26

    DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution.

  1. GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Kostyuk, Svetlana; Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

    2015-01-01

    Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose-derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

  2. Genome-Derived Cytosolic DNA Mediates Type I Interferon-Dependent Rejection of B Cell Lymphoma Cells

    Directory of Open Access Journals (Sweden)

    Yu J. Shen

    2015-04-01

    Full Text Available The DNA damage response (DDR induces the expression of type I interferons (IFNs, but the underlying mechanisms are poorly understood. Here, we show the presence of cytosolic DNA in different mouse and human tumor cells. Treatment of cells with genotoxic agents increased the levels of cytosolic DNA in a DDR-dependent manner. Cloning of cytosolic DNA molecules from mouse lymphoma cells suggests that cytosolic DNA is derived from unique genomic loci and has the potential to form non-B DNA structures, including R-loops. Overexpression of Rnaseh1, which resolves R-loops, reduced the levels of cytosolic DNA, type I Ifn transcripts, and type I IFN-dependent rejection of lymphoma cells. Live-cell imaging showed a dynamic contact of cytosolic DNA with mitochondria, an important organelle for innate immune recognition of cytosolic nucleotides. In summary, we found that cytosolic DNA is present in many tumor cells and contributes to the immunogenicity of tumor cells.

  3. The value of cell-free DNA for molecular pathology.

    Science.gov (United States)

    Stewart, Caitlin M; Kothari, Prachi D; Mouliere, Florent; Mair, Richard; Somnay, Saira; Benayed, Ryma; Zehir, Ahmet; Weigelt, Britta; Dawson, Sarah-Jane; Arcila, Maria E; Berger, Michael F; Tsui, Dana Wy

    2018-04-01

    Over the past decade, advances in molecular biology and genomics techniques have revolutionized the diagnosis and treatment of cancer. The technological advances in tissue profiling have also been applied to the study of cell-free nucleic acids, an area of increasing interest for molecular pathology. Cell-free nucleic acids are released from tumour cells into the surrounding body fluids and can be assayed non-invasively. The repertoire of genomic alterations in circulating tumour DNA (ctDNA) is reflective of both primary tumours and distant metastatic sites, and ctDNA can be sampled multiple times, thereby overcoming the limitations of the analysis of single biopsies. Furthermore, ctDNA can be sampled regularly to monitor response to treatment, to define the evolution of the tumour genome, and to assess the acquisition of resistance and minimal residual disease. Recently, clinical ctDNA assays have been approved for guidance of therapy, which is an exciting first step in translating cell-free nucleic acid research tests into clinical use for oncology. In this review, we discuss the advantages of cell-free nucleic acids as analytes in different body fluids, including blood plasma, urine, and cerebrospinal fluid, and their clinical applications in solid tumours and haematological malignancies. We will also discuss practical considerations for clinical deployment, such as preanalytical factors and regulatory requirements. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  4. Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

    Energy Technology Data Exchange (ETDEWEB)

    Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

    2008-01-18

    Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

  5. Increased genome instability is not accompanied by sensitivity to DNA damaging agents in aged yeast cells

    NARCIS (Netherlands)

    Novarina, Daniele; Mavrova, Sara N.; Janssens, Georges E.; Rempel, Irina L.; Veenhoff, Liesbeth M.; Chang, Michael

    The budding yeast Saccharomyces cerevisiae divides asymmetrically, producing a new daughter cell from the original mother cell. While daughter cells are born with a full lifespan, a mother cell ages with each cell division and can only generate on average 25 daughter cells before dying. Aged yeast

  6. DNA fragmentation and cytotoxicity by recombinant human tumor necrosis factor in L929 fibroblast cells

    International Nuclear Information System (INIS)

    Kosaka, T.; Kuwabara, M.; Koide, F.

    1992-01-01

    Induction of cell DNA fragmentation by treatment of recombinant human Tumor Necrosis Factor alpha (rhTNF alpha) was examined by using mouse L929 cells derived from mouse fibroblast cells. The amount of DNA fragments derived from rhTNF alpha-treated cells, detected by alkaline elution technique, was smaller than that derived from X-irradiated cells. The rhTNF alpha caused the DNA fragmentation depending on its incubation time and concentration. The DNA damage caused by rhTNF alpha treatment correlated with its cytotoxicity. This result suggested that the DNA fragmentation is one of causes of cell death. The treatment with proteinase K of DNA obtained from rhTNF alpha-treated cells did not increase the amount of DNA fragmentation, which indicates that rhTNF alpha causes DNA-fragmentation but not DNA-protein cross-linking

  7. Droplet Microfluidics for Compartmentalized Cell Lysis and Extension of DNA from Single-Cells

    Science.gov (United States)

    Zimny, Philip; Juncker, David; Reisner, Walter

    Current single cell DNA analysis methods suffer from (i) bias introduced by the need for molecular amplification and (ii) limited ability to sequence repetitive elements, resulting in (iii) an inability to obtain information regarding long range genomic features. Recent efforts to circumvent these limitations rely on techniques for sensing single molecules of DNA extracted from single-cells. Here we demonstrate a droplet microfluidic approach for encapsulation and biochemical processing of single-cells inside alginate microparticles. In our approach, single-cells are first packaged inside the alginate microparticles followed by cell lysis, DNA purification, and labeling steps performed off-chip inside this microparticle system. The alginate microparticles are then introduced inside a micro/nanofluidic system where the alginate is broken down via a chelating buffer, releasing long DNA molecules which are then extended inside nanofluidic channels for analysis via standard mapping protocols.

  8. The Antibacterial Cell Division Inhibitor PC190723 Is an FtsZ Polymer-stabilizing Agent That Induces Filament Assembly and Condensation*

    OpenAIRE

    Andreu, José M.; Schaffner-Barbero, Claudia; Huecas, Sonia; Alonso, Dulce; Lopez-Rodriguez, María L.; Ruiz-Avila, Laura B.; Núñez-Ramírez, Rafael; Llorca, Oscar; Martín-Galiano, Antonio J.

    2010-01-01

    Cell division protein FtsZ can form single-stranded filaments with a cooperative behavior by self-switching assembly. Subsequent condensation and bending of FtsZ filaments are important for the formation and constriction of the cytokinetic ring. PC190723 is an effective bactericidal cell division inhibitor that targets FtsZ in the pathogen Staphylococcus aureus and Bacillus subtilis and does not affect Escherichia coli cells, which apparently binds to a zone equivalent to the binding site of ...

  9. Quantitative live imaging of endogenous DNA replication in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Andrew Burgess

    Full Text Available Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.

  10. Aqueous humor from traumatized eyes triggers cell division in the epithelia of cultured lenses

    International Nuclear Information System (INIS)

    Reddan, J.R.; Weinsieder, A.; Wilson, D.

    1979-01-01

    Experiments were designed to gain a better understanding of the relationship between ocular inflammation and cell proliferation in the lens epithelium. Aqueous humor (AH) was collected from rabbit eyes that had been subjected to a variety of traumata, including paracentesis, needle injury, X-irradiation and the intravitreal administration of an antigen. In all cases the protein content of the AH increased, reflecting a breakdown in the blood aqueous barrier. Rabbit lenses from non-traumatized eyes were isolated and cultured in medium KEI-4 containing samples of the various aqueous humors noted above. Control lenses were cultured in medium KEI-4 alone or in KEI-4 containing rabbit serum albumen at a protein concentration equivalent to that used in the AH studies. In contrast to controls, the epithelial cells of lenses exposed to AH from injured or inflamed eyes exhibited mitosis throughout the normally amitotic regions of epithelium. Moreover, the specific activity of AH collected 15 min after initial paracentesis, relative to both DNA synthesis and mitosis, exceeded that of rabbit serum. An identification of the mitogenic factor(s) in the AH may help in understanding the environmental conditions that regulate the mitotic response which normally precedes wound healing in the lens in situ, and may help in elucidating the mechanism which controls mitosis and differentiation in the lens in vivo. (author)

  11. Polyphosphate is a key factor for cell survival after DNA damage in eukaryotic cells.

    Science.gov (United States)

    Bru, Samuel; Samper-Martín, Bàrbara; Quandt, Eva; Hernández-Ortega, Sara; Martínez-Laínez, Joan M; Garí, Eloi; Rafel, Marta; Torres-Torronteras, Javier; Martí, Ramón; Ribeiro, Mariana P C; Jiménez, Javier; Clotet, Josep

    2017-09-01

    Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Mitochondrial DNA deletion mutations in adult mouse cardiac side population cells

    International Nuclear Information System (INIS)

    Lushaj, Entela B.; Lozonschi, Lucian; Barnes, Maria; Anstadt, Emily; Kohmoto, Takushi

    2012-01-01

    We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.

  13. Cell cycle phase dependent role of DNA polymerase beta in DNA repair and survival after ionizing radiation.

    NARCIS (Netherlands)

    Vermeulen, C.; Verwijs-Janssen, M.; Begg, A.C.; Vens, C.

    2008-01-01

    PURPOSE: The purpose of the present study was to determine the role of DNA polymerase beta in repair and response after ionizing radiation in different phases of the cell cycle. METHODS AND MATERIALS: Synchronized cells deficient and proficient in DNA polymerase beta were irradiated in different

  14. DNA damage in synchronized hela cells irradiated with ultraviolet

    International Nuclear Information System (INIS)

    Downes, C.S.; Collins, A.R.S.; Johnson, R.T.

    1979-01-01

    The lethal effect of uv radiation on HeLa cells is least in mitosis and greatest in late G 1 -early S. Photochemical damage to HeLa DNA, as measured by thymine-containing dimer formation and by alkaline sucrose sedimentation, also increases from mitosis towards early S phase. Computer simulations of uv absorption by an idealized HeLa cell at different stages of the cell cycle indicate that changes in damage could be due solely to changes in chromatin geometry. But survival is not exclusively a function of damage

  15. Selective induction of DNA repair pathways in human B cells activated by CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Xiaosheng Wu

    Full Text Available Greater than 75% of all hematologic malignancies derive from germinal center (GC or post-GC B cells, suggesting that the GC reaction predisposes B cells to tumorigenesis. Because GC B cells acquire expression of the highly mutagenic enzyme activation-induced cytidine deaminase (AID, GC B cells may require additional DNA repair capacity. The goal of this study was to investigate whether normal human B cells acquire enhanced expression of DNA repair factors upon AID induction. We first demonstrated that several DNA mismatch repair, homologous recombination, base excision repair, and ATR signaling genes were overexpressed in GC B cells relative to naïve and memory B cells, reflecting activation of a process we have termed somatic hyperrepair (SHR. Using an in vitro system, we next characterized activation signals required to induce AID expression and SHR. Although AID expression was induced by a variety of polyclonal activators, SHR induction strictly required signals provided by contact with activated CD4+ T cells, and B cells activated in this manner displayed reduced levels of DNA damage-induced apoptosis. We further show the induction of SHR is independent of AID expression, as GC B cells from AID-/-mice retained heightened expression of SHR proteins. In consideration of the critical role that CD4+ T cells play in inducing the SHR process, our data suggest a novel role for CD4+ T cells in the tumor suppression of GC/post-GC B cells.

  16. Global DNA methylation synergistically regulates the nuclear and mitochondrial genomes in glioblastoma cells.

    Science.gov (United States)

    Sun, Xin; Johnson, Jacqueline; St John, Justin C

    2018-05-02

    Replication of mitochondrial DNA is strictly regulated during differentiation and development allowing each cell type to acquire its required mtDNA copy number to meet its specific needs for energy. Undifferentiated cells establish the mtDNA set point, which provides low numbers of mtDNA copy but sufficient template for replication once cells commit to specific lineages. However, cancer cells, such as those from the human glioblastoma multiforme cell line, HSR-GBM1, cannot complete differentiation as they fail to enforce the mtDNA set point and are trapped in a 'pseudo-differentiated' state. Global DNA methylation is likely to be a major contributing factor, as DNA demethylation treatments promote differentiation of HSR-GBM1 cells. To determine the relationship between DNA methylation and mtDNA copy number in cancer cells, we applied whole genome MeDIP-Seq and RNA-Seq to HSR-GBM1 cells and following their treatment with the DNA demethylation agents 5-azacytidine and vitamin C. We identified key methylated regions modulated by the DNA demethylation agents that also induced synchronous changes to mtDNA copy number and nuclear gene expression. Our findings highlight the control exerted by DNA methylation on the expression of key genes, the regulation of mtDNA copy number and establishment of the mtDNA set point, which collectively contribute to tumorigenesis.

  17. The Bright and the Dark Sides of DNA Repair in Stem Cells

    OpenAIRE

    Frosina, Guido

    2010-01-01

    DNA repair is a double-edged sword in stem cells. It protects normal stem cells in both embryonic and adult tissues from genetic damage, thus allowing perpetuation of intact genomes into new tissues. Fast and efficient DNA repair mechanisms have evolved in normal stem and progenitor cells. Upon differentiation, a certain degree of somatic mutations becomes more acceptable and, consequently, DNA repair dims. DNA repair turns into a problem when stem cells transform and become cancerous. Tran...

  18. Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division

    KAUST Repository

    Robertson, Anthony J.

    2013-03-25

    In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

  19. Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division

    KAUST Repository

    Robertson, Anthony J.; Coluccio, Alison; Jensen, Sarah; Rydlizky, Katarina; Coffman, James A.

    2013-01-01

    In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

  20. Composition and Dynamics of the Nucleolinus, a Link between the Nucleolus and Cell Division Apparatus in Surf Clam (Spisula) Oocytes*

    Science.gov (United States)

    Alliegro, Mark C.; Hartson, Steven; Alliegro, Mary Anne

    2012-01-01

    The nucleolinus is a little-known cellular structure, discovered over 150 years ago (Agassiz, L. (1857) Contributions to the Natural History of the United States of America, First Monograph, Part IIL, Little, Brown and Co., Boston) and thought by some investigators in the late 19th to mid-20th century to function in the formation of the centrosomes or spindle. A role for the nucleolinus in formation of the cell division apparatus has recently been confirmed in oocytes of the surf clam, Spisula solidissima (Alliegro, M. A., Henry, J. J., and Alliegro, M. C. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 13718–13723). However, we know so little about the composition and dynamics of this compartment, it is difficult to construct mechanistic hypotheses or even to be sure that prior reports were describing analogous structures in the cells of mammals, amphibians, plants, and other organisms where it was observed. Surf clam oocytes are an attractive model to approach this problem because the nucleolinus is easily visible by light microscopy, making it accessible by laser microsurgery as well as isolation by common cell fractionation techniques. In this report, we analyze the macromolecular composition of isolated Spisula nucleolini and examine the relationship of this structure to the nucleolus and cell division apparatus. Analysis of nucleolinar RNA and protein revealed a set of molecules that overlaps with but is nevertheless distinct from the nucleolus. The proteins identified were primarily ones involved in nucleic acid metabolism and cell cycle regulation. Monoclonal antibodies generated against isolated nucleolini revealed centrosomal forerunners in the oocyte cytoplasm. Finally, induction of damage to the nucleolinus by laser microsurgery altered the trafficking of α- and γ-tubulin after fertilization. These observations strongly support a role for the nucleolinus in cell division and represent our first clues regarding mechanism. PMID:22219192

  1. Composition and dynamics of the nucleolinus, a link between the nucleolus and cell division apparatus in surf clam (Spisula) oocytes.

    Science.gov (United States)

    Alliegro, Mark C; Hartson, Steven; Alliegro, Mary Anne

    2012-02-24

    The nucleolinus is a little-known cellular structure, discovered over 150 years ago (Agassiz, L. (1857) Contributions to the Natural History of the United States of America, First Monograph, Part IIL, Little, Brown and Co., Boston) and thought by some investigators in the late 19th to mid-20th century to function in the formation of the centrosomes or spindle. A role for the nucleolinus in formation of the cell division apparatus has recently been confirmed in oocytes of the surf clam, Spisula solidissima (Alliegro, M. A., Henry, J. J., and Alliegro, M. C. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 13718-13723). However, we know so little about the composition and dynamics of this compartment, it is difficult to construct mechanistic hypotheses or even to be sure that prior reports were describing analogous structures in the cells of mammals, amphibians, plants, and other organisms where it was observed. Surf clam oocytes are an attractive model to approach this problem because the nucleolinus is easily visible by light microscopy, making it accessible by laser microsurgery as well as isolation by common cell fractionation techniques. In this report, we analyze the macromolecular composition of isolated Spisula nucleolini and examine the relationship of this structure to the nucleolus and cell division apparatus. Analysis of nucleolinar RNA and protein revealed a set of molecules that overlaps with but is nevertheless distinct from the nucleolus. The proteins identified were primarily ones involved in nucleic acid metabolism and cell cycle regulation. Monoclonal antibodies generated against isolated nucleolini revealed centrosomal forerunners in the oocyte cytoplasm. Finally, induction of damage to the nucleolinus by laser microsurgery altered the trafficking of α- and γ-tubulin after fertilization. These observations strongly support a role for the nucleolinus in cell division and represent our first clues regarding mechanism.

  2. DNA-radiosensitivity and repair in mammolian cells

    International Nuclear Information System (INIS)

    Proskuryakov, S.Ya.; Ivannik, B.P.; Ryabchenko, N.I.

    1979-01-01

    Determination was made of the formation and repair of single-stranded DNA breaks (SB) in cells of rat thymus and liver and Ehrlich's ascites tumor (EAT) with the use of the method of low-gradient viscosimetry of alkaline cell lysates. The radiochemical yield of single-stranded breaks (Gsub(SB)) induced by irradiation of animals is 41.2 eV/break for hepatocytes, 96.8 eV/break, for thymocytes, and 129.7 eV/break, for EAT cells. The half-recovery time of single-stranded DNA breaks for cells of thymus and EAT exposed in vivo is 16.0 and 5.1 s -1 , correspondingly. In hepatocytes exposed in vivo and in vitro no repairs occurs for 3 h. Under conditions of inhibition of SB repair, when suspensions of thymocytes and hepatocytes were exposed in vitro at 4 deg C, Gsub(SB) is 35.5 and 38.7 eV/break, respectively. The analysis of the data obtained prompts the conclusion that under in vivo conditions, there is a correlation between DNA radiosensitivity and the rate of repair processes

  3. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    Science.gov (United States)

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

  4. Cell sensitivity to irradiation and DNA repair processes. II

    International Nuclear Information System (INIS)

    Kozubek, S.; Krasavin, E.A.

    1984-01-01

    A new model of DNA single-strand break (SSB) and double-strand break (DSB) induction by radiations of different linear energy transfer (LET) has been developed. Utilizing quadratic dependence of the dose that delta-electrons depart in the track of heavy particles the fraction of heavy particle energy deposited in the target of DNA dimensions has been calculated. SSBs arise from energy depositions in one strand of DNA, direct DSBs arise from two SSBs on opposite strands of DNA in the track of one particle. It is concluded that DSB's induced by γ-radiation are mostly of enzymatic origin, meanwhile DSB's induced by high-LET radiation are direct DSB's. The dependence of radiosensitivity D 0 -1 on LET (L) for isogenic mutants of E. coli with different sensitivity to γ-radiation has been determined on the bases of the model and considering microscopic energy fluctuations. The shape of D 0 -1 (L) function is formed both by physical characteristics of radiation and by the ability of cells to repair some types of DNA damage. The model provides a basis for further investigation. (author)

  5. Deficient repair of chemical adducts in alpha DNA of monkey cells

    International Nuclear Information System (INIS)

    Zolan, M.E.; Cortopassi, G.A.; Smith, C.A.; Hanawalt, P.C.

    1982-01-01

    Researchers have examined excision repair of DNA damage in the highly repeated alpha DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in alpha DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for alpha DNA and bulk DNA. In cells treated with furocoumarins and long-wave-length ultraviolet light, however, repair synthesis in alpha DNA was only 30% of that in bulk DNA, although it followed the same time course. Researchers found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as researchers found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from alpha DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in alpha DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences

  6. Disorganization of cell division of methicillin-resistant Staphylococcus aureus by methanolic extract from Phyllanthus columnaris stem bark

    Energy Technology Data Exchange (ETDEWEB)

    Adnalizawati, A. Siti Noor; Nazlina, I. [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Yaacob, W. A. [School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)

    2013-11-27

    The in vitro activity of methanolic extract from Phyllanthus columnaris stem bark was studied against Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 and MRSA BM1 (clinical strain) using time-kill curves in conjunction with scanning and transmission electron microscopy. The extract showed more markedly bactericidal activity in MRSA BM1 clinical strain within less than 4 h by 6.25-12.5 mg/mL and within 6 h by 1.56 mg/mL. Scanning electron microscopy of MRSA BM1 revealed distortion of cell whilst transmission electron microscopy revealed disruption in cell wall division.

  7. Disorganization of cell division of methicillin-resistant Staphylococcus aureus by methanolic extract from Phyllanthus columnaris stem bark

    International Nuclear Information System (INIS)

    Adnalizawati, A. Siti Noor; Nazlina, I.; Yaacob, W. A.

    2013-01-01

    The in vitro activity of methanolic extract from Phyllanthus columnaris stem bark was studied against Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 and MRSA BM1 (clinical strain) using time-kill curves in conjunction with scanning and transmission electron microscopy. The extract showed more markedly bactericidal activity in MRSA BM1 clinical strain within less than 4 h by 6.25-12.5 mg/mL and within 6 h by 1.56 mg/mL. Scanning electron microscopy of MRSA BM1 revealed distortion of cell whilst transmission electron microscopy revealed disruption in cell wall division

  8. DNA damage in oral cancer and normal cells induced by nitrogen atmospheric pressure plasma jets

    Science.gov (United States)

    Han, Xu; Kapaldo, James; Liu, Yueying; Stack, M. Sharon; Ptasinska, Sylwia

    2015-09-01

    Nitrogen atmospheric pressure plasma jets (APPJs) have been shown to effectively induce DNA double strand breaks in SCC25 oral cancer cells. The APPJ source constructed in our laboratory operates based on dielectric barrier discharge. It consists of two copper electrodes alternatively wrapping around a fused silica tube with nitrogen as a feed gas. It is generally more challenging to ignite plasma in N2 atmosphere than in noble gases. However, N2 provides additional advantages such as lower costs compared to noble gases, thus this design can be beneficial for the future long-term clinical use. To compare the effects of plasma on cancer cells (SCC25) and normal cells (OKF), the cells from both types were treated at the same experimental condition for various treatment times. The effective area with different damage levels after the treatment was visualized as 3D maps. The delayed damage effects were also explored by varying the incubation times after the treatment. All of these studies are critical for a better understanding of the damage responses of cellular systems exposed to the plasma radiation, thus are useful for the development of the advanced plasma cancer therapy. The research described herein was supported by the Division of Chemical Sciences, Geosciences and Biosciences, Basic Energy Sciences, Office of Science, United States Department of Energy through Grant No. DE-FC02-04ER15533.

  9. File list: Oth.ALL.10.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.10.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.10.DNA-RNA_hybrids.AllCell.bed ...

  10. File list: Oth.ALL.50.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.50.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.50.DNA-RNA_hybrids.AllCell.bed ...

  11. File list: Oth.ALL.05.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.05.DNA-RNA_hybrids.AllCell.bed ...

  12. File list: Oth.ALL.20.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids All cell ...types http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.20.DNA-RNA_hybrids.AllCell.bed ...

  13. Inhibition of Cell Survival by Curcumin Is Associated with Downregulation of Cell Division Cycle 20 (Cdc20) in Pancreatic Cancer Cells.

    Science.gov (United States)

    Zhang, Yu; Xue, Ying-Bo; Li, Hang; Qiu, Dong; Wang, Zhi-Wei; Tan, Shi-Sheng

    2017-02-04

    Pancreatic cancer is one of the most aggressive human tumors in the United States. Curcumin, a polyphenol derived from the Curcuma longa plant, has been reported to exert its antitumor activity in pancreatic cancer. However, the molecular mechanisms of curcumin-mediated tumor suppressive function have not been fully elucidated. In the current study, we explore whether curcumin exhibits its anti-cancer function through inhibition of oncoprotein cell division cycle 20 (Cdc20) in pancreatic cancer cells. We found that curcumin inhibited cell growth, enhanced apoptosis, induced cell cycle arrest and retarded cell invasion in pancreatic cancer cells. Moreover, we observed that curcumin significantly inhibited the expression of Cdc20 in pancreatic cancer cells. Furthermore, our results demonstrated that overexpression of Cdc20 enhanced cell proliferation and invasion, and abrogated the cytotoxic effects induced by curcumin in pancreatic cancer cells. Consistently, downregulation of Cdc20 promoted curcumin-mediated anti-tumor activity. Therefore, our findings indicated that inhibition of Cdc20 by curcumin could be useful for the treatment of pancreatic cancer patients.

  14. DNA repair and its coupling to DNA replication in eukaryotic cells. [UV, x ray

    Energy Technology Data Exchange (ETDEWEB)

    Cleaver, J.E.

    1978-01-01

    This review article with 184 references presents the view that mammalian cells have one major repair system, excision repair, with many branches (nucleotide excision repair, base excision repair, crosslink repair, etc.) and a multiplicity of enzymes. Any particular carcinogen makes a spectrum of damaged sites and each kind of damage may be repaired by one or more branches of excision repair. Excision repair is rarely complete, except at very low doses, and eukaryotic cells survive and replicate DNA despite the presence of unrepaired damage. An alteration in a specific biochemical pathway seen in damaged or mutant cells will not always be the primary consequence of damage or of the biochemical defect of the cells. Detailed kinetic data are required to understand comprehensively the various facets of excision repair and replication. Correlation between molecular events of repair and cytological and cellular changes such as chromosomal damage, mutagenesis, transformation, and carcinogenesis are also rudimentary.

  15. Nitric oxide mediated DNA double strand breaks induced in proliferating bystander cells after {alpha}-particle irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Han Wei [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Chen Shaopeng [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China); Yu, K.N., E-mail: peter.yu@cityu.edu.hk [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Wu Lijun [Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China)

    2010-02-03

    Low-dose {alpha}-particle exposures comprise 55% of the environmental dose to the human population and have been shown to induce bystander responses. Previous studies showed that bystander effect could induce stimulated cell growth or genotoxicity, such as excessive DNA double strand breaks (DSBs), micronuclei (MN), mutation and decreased cell viability, in the bystander cell population. In the present study, the stimulated cell growth, detected with flow cytometry (FCM), and the increased MN and DSB, detected with p53 binding protein 1 (53BP1) immunofluorescence, were observed simultaneously in the bystander cell population, which were co-cultured with cells irradiated by low-dose {alpha}-particles (1-10 cGy) in a mixed system. Further studies indicated that nitric oxide (NO) and transforming growth factor {beta}1 (TGF-{beta}1) played very important roles in mediating cell proliferation and inducing MN and DSB in the bystander population through treatments with NO scavenger and TGF-{beta}1 antibody. Low-concentrations of NO, generated by spermidine, were proved to induce cell proliferation, DSB and MN simultaneously. The proliferation or shortened cell cycle in bystander cells gave them insufficient time to repair DSBs. The increased cell division might increase the probability of carcinogenesis in bystander cells since cell proliferation increased the probability of mutation from the mis-repaired or un-repaired DSBs.

  16. Nitric oxide mediated DNA double strand breaks induced in proliferating bystander cells after α-particle irradiation

    International Nuclear Information System (INIS)

    Han Wei; Chen Shaopeng; Yu, K.N.; Wu Lijun

    2010-01-01

    Low-dose α-particle exposures comprise 55% of the environmental dose to the human population and have been shown to induce bystander responses. Previous studies showed that bystander effect could induce stimulated cell growth or genotoxicity, such as excessive DNA double strand breaks (DSBs), micronuclei (MN), mutation and decreased cell viability, in the bystander cell population. In the present study, the stimulated cell growth, detected with flow cytometry (FCM), and the increased MN and DSB, detected with p53 binding protein 1 (53BP1) immunofluorescence, were observed simultaneously in the bystander cell population, which were co-cultured with cells irradiated by low-dose α-particles (1-10 cGy) in a mixed system. Further studies indicated that nitric oxide (NO) and transforming growth factor β1 (TGF-β1) played very important roles in mediating cell proliferation and inducing MN and DSB in the bystander population through treatments with NO scavenger and TGF-β1 antibody. Low-concentrations of NO, generated by spermidine, were proved to induce cell proliferation, DSB and MN simultaneously. The proliferation or shortened cell cycle in bystander cells gave them insufficient time to repair DSBs. The increased cell division might increase the probability of carcinogenesis in bystander cells since cell proliferation increased the probability of mutation from the mis-repaired or un-repaired DSBs.

  17. DNA mismatch repair and its many roles in eukaryotic cells

    DEFF Research Database (Denmark)

    Liu, Dekang; Keijzers, Guido; Rasmussen, Lene Juel

    2017-01-01

    in the clinic, and as a biomarker of cancer susceptibility in animal model systems. Prokaryotic MMR is well-characterized at the molecular and mechanistic level; however, MMR is considerably more complex in eukaryotic cells than in prokaryotic cells, and in recent years, it has become evident that MMR plays...... novel roles in eukaryotic cells, several of which are not yet well-defined or understood. Many MMR-deficient human cancer cells lack mutations in known human MMR genes, which strongly suggests that essential eukaryotic MMR components/cofactors remain unidentified and uncharacterized. Furthermore......, the mechanism by which the eukaryotic MMR machinery discriminates between the parental (template) and the daughter (nascent) DNA strand is incompletely understood and how cells choose between the EXO1-dependent and the EXO1–independent subpathways of MMR is not known. This review summarizes recent literature...

  18. CbtA toxin of Escherichia coli inhibits cell division and cell elongation via direct and independent interactions with FtsZ and MreB.

    Science.gov (United States)

    Heller, Danielle M; Tavag, Mrinalini; Hochschild, Ann

    2017-09-01

    The toxin components of toxin-antitoxin modules, found in bacterial plasmids, phages, and chromosomes, typically target a single macromolecule to interfere with an essential cellular process. An apparent exception is the chromosomally encoded toxin component of the E. coli CbtA/CbeA toxin-antitoxin module, which can inhibit both cell division and cell elongation. A small protein of only 124 amino acids, CbtA, was previously proposed to interact with both FtsZ, a tubulin homolog that is essential for cell division, and MreB, an actin homolog that is essential for cell elongation. However, whether or not the toxic effects of CbtA are due to direct interactions with these predicted targets is not known. Here, we genetically separate the effects of CbtA on cell elongation and cell division, showing that CbtA interacts directly and independently with FtsZ and MreB. Using complementary genetic approaches, we identify the functionally relevant target surfaces on FtsZ and MreB, revealing that in both cases, CbtA binds to surfaces involved in essential cytoskeletal filament architecture. We show further that each interaction contributes independently to CbtA-mediated toxicity and that disruption of both interactions is required to alleviate the observed toxicity. Although several other protein modulators are known to target FtsZ, the CbtA-interacting surface we identify represents a novel inhibitory target. Our findings establish CbtA as a dual function toxin that inhibits both cell division and cell elongation via direct and independent interactions with FtsZ and MreB.

  19. Role of DNA deletion length in mutation and cell survival

    International Nuclear Information System (INIS)

    Braby, L.A.; Morgan, T.L.

    1992-01-01

    A model is presented which is based on the assumption that malignant transformation, mutation, chromosome aberration, and reproductive death of cells are all manifestations of radiation induced deletions in the DNA of the cell, and that the size of the deletion in relation to the spacing of essential genes determines the consequences of that deletion. It is assumed that two independent types of potentially lethal lesions can result in DNA deletions, and that the relative numbers of these types of damage is dependent on radiation quality. The repair of the damage reduces the length of a deletion, but does not always eliminate it. The predictions of this model are in good agreement with a wide variety of experimental evidence. (author)

  20. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng

    2010-01-01

    DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per...... strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found...... research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies....

  1. DNA Damage by Radiation in Tradescantia Leaf Cells

    International Nuclear Information System (INIS)

    Han, Min; Hyun, Kyung Man; Ryu, Tae Ho; Kim, Jin Kyu; Nili, Mohammad

    2010-01-01

    The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Tradescantia tests are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay. The development of comet assay has enabled investigators to detect DNA damage at the levels of cells. To adapt this assay to plant cells, nuclei were directly obtained from Tradescantia leaf samples. A significant dose-dependent increase in the average tail moment values over the negative control was observed. Recently the adaptation of this technique to plant cells opens new possibilities for studies in variety area. The future applications of the comet assay could impact some other important areas, certainly, one of the limiting factors to its utility is the imagination of the investigator.

  2. DNA Damage by Radiation in Tradescantia Leaf Cells

    Energy Technology Data Exchange (ETDEWEB)

    Han, Min; Hyun, Kyung Man; Ryu, Tae Ho; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

    2010-04-15

    The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Tradescantia tests are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay. The development of comet assay has enabled investigators to detect DNA damage at the levels of cells. To adapt this assay to plant cells, nuclei were directly obtained from Tradescantia leaf samples. A significant dose-dependent increase in the average tail moment values over the negative control was observed. Recently the adaptation of this technique to plant cells opens new possibilities for studies in variety area. The future applications of the comet assay could impact some other important areas, certainly, one of the limiting factors to its utility is the imagination of the investigator.

  3. The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Tetsuya, E-mail: suzukite@hiroshima-u.ac.jp [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Grúz, Petr; Honma, Masamitsu [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Adachi, Noritaka [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Nohmi, Takehiko [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan)

    2016-09-15

    Highlights: • Human cells knockout (KO) and expressing catalytically dead (CD) variant of DNA polymerase ζ (Pol ζ) have been established by gene targeting techniques with Nalm-6 cells. • Both Pol ζ KO and CD cells displayed prolonged cell cycle and higher incidence of micronucleus formation than the wild-type cells in the absence of exogenous genotoxic treatments. • Pol ζ protects human cells from genotoxic stresses that induce bulky DNA lesions and cross-links. • Pol ζ plays quite limited roles in protection against strand-breaks in DNA. - Abstract: Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD > KO > WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N′-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were

  4. The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells

    International Nuclear Information System (INIS)

    Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

    2016-01-01

    Highlights: • Human cells knockout (KO) and expressing catalytically dead (CD) variant of DNA polymerase ζ (Pol ζ) have been established by gene targeting techniques with Nalm-6 cells. • Both Pol ζ KO and CD cells displayed prolonged cell cycle and higher incidence of micronucleus formation than the wild-type cells in the absence of exogenous genotoxic treatments. • Pol ζ protects human cells from genotoxic stresses that induce bulky DNA lesions and cross-links. • Pol ζ plays quite limited roles in protection against strand-breaks in DNA. - Abstract: Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD > KO > WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N′-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were

  5. Molecular dosimetry of chemical mutagens: measurement of molecular dose and DNA repair germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.

    1975-01-01

    Molecular dosimetry in the germ cells of male mice is reviewed with regard to in vivo alkylation of sperm heads, in vivo alkylation of sperm DNA, and possible alkylation of sperm protamine. DNA repair in male germ cells is reviewed with regard to basic design of experiments, DNA repair in various stages of spermatogenesis, effect of protamine on DNA repair following treatment with EMS or x radiation, and induction of DNA repair by methyl methanesulfonate, propyl methanesulfonate, and isopropyl methanesulfonate

  6. Heavy ion induced DNA transfer in biological cells

    International Nuclear Information System (INIS)

    Vilaithong, T.; Yu, L.D.; Apavatjrut, P.; Phanchaisri, B.; Sangyuenyongpipat, S.; Anuntalabhochai, S.; Brown, I.G.

    2004-01-01

    Low-energy ion beam bombardment of biological materials for genetic modification purposes has experienced rapid growth in the last decade, particularly for the direct DNA transfer into living organisms including both plants and bacteria. Attempts have been made to understand the mechanisms involved in ion-bombardment-induced direct gene transfer into biological cells. Here we summarize the present status of the application of low-energy ions for genetic modification of living sample materials

  7. Systemic control of cell division and endoreduplication by NAA and BAP by modulating CDKs in root tip cells of Allium cepa.

    Science.gov (United States)

    Tank, Jigna G; Thaker, Vrinda S

    2014-01-01

    Molecular mechanism regulated by auxin and cytokinin during endoreduplication, cell division, and elongation process is studied by using Allium cepa roots as a model system. The activity of CDK genes modulated by auxin and cytokinin during cell division, elongation, and endoreduplication process is explained in this research work. To study the significance of auxin and cytokinin in the management of cell division and endoreduplication process in plant meristematic cells at molecular level endoreduplication was developed in root tips of Allium cepa by giving colchicine treatment. There were inhibition of vegetative growth, formation of c-tumor at root tip, and development of endoreduplicated cells after colchicine treatment. This c-tumor was further treated with NAA and BAP to reinitiate vegetative growth in roots. BAP gave positive response in reinitiation of vegetative growth of roots from center of c-tumor. However, NAA gave negative response in reinitiation of vegetative growth of roots from c-tumor. Further, CDKs gene expression analysis from normal, endoreduplicated, and phytohormone (NAA or BAP) treated root tip was done and remarkable changes in transcription level of CDK genes in normal, endoreduplicated, and phytohormones treated cells were observed.

  8. Postirradiation DNA synthesis is inversely related to cell survival

    International Nuclear Information System (INIS)

    Kapiszewska, M.; Lange, C.S.

    1987-01-01

    Postirradiation (PI) events which might lead to cellular reproductive death or survival were studied in L5178Y-S (LY-S) cells. PI incubation at 25 0 C protects LY-S cells against the PLD fixation which takes place at 37 0 C. An optimal condition for the repair of PLD is 1h at 37 0 C followed by 4h holding at 25 0 C prior to the second half of a split dose, or 5L holding at 25 0 C without a 37 0 C incubation. Longer incubations at 37 0 C resulted in progressively decreased survivals. Postirradiation inhibition of DNA synthesis at 37 0 C was observed only during the first 30 min; thereafter, /sup 3/H-dThd incorporation was higher than in unirradiated controls. This excess synthesis effect was removed by shifting irradiated cells to 25 0 C holding. The inhibition observed at 25 0 C was reversed by shifting to 37 0 C. Thus the degree of postirradiation DNA synthesis is inversely related to PLD/SLD repair. DNA filter elution shows complete SSB repair by 3h at both temperatures (with faster kinetics at 37 0 C), and DSB repair plateaus at 80% (37 0 C) and 60% (25 0 C) after 90 min

  9. Metformin (dimethyl-biguanide induced DNA damage in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rubem R. Amador

    2012-01-01

    Full Text Available Metformin (dimethyl-biguanide is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays and in mice (micronucleus assays. Concentrations of 114.4 µg/mL and 572 µg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 µg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.

  10. Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

    International Nuclear Information System (INIS)

    Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

    2005-01-01

    Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [ 14 C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [ 14 C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 (micro)g of DNA. This sensitivity may allow the method to be used for clinical applications

  11. Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

    2005-12-29

    Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [{sup 14}C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [{sup 14}C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 {micro}g of DNA. This sensitivity may allow the method to be used for clinical applications.

  12. DNA hybrids suggesting a recombination process repairing radiation-induced DNA double-strand breaks in Ehrlich Ascites tumor cells

    International Nuclear Information System (INIS)

    Barthel, H.R.

    1984-01-01

    The results presented suggest the possibility of repair of DNA double-strand breaks by recombination, at least in the S and G 2 -phases of the cell cycle, in mammalian cells. Further experiments with synchronized cell cultures will have to show whether this process may also occur in the G 1 -phase of the cell cycle. (orig./AJ) [de

  13. Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

    Science.gov (United States)

    Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

    2015-01-01

    Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

  14. Observations of the first postirradiation division of HeLa cells following continuous or fractionated exposure to γ rays

    International Nuclear Information System (INIS)

    Mitchell, J.B.; Bedford, J.S.; Bailey, S.M.

    1979-01-01

    The first postirradiation division of synchronized S3 HeLa cells was studied using both continuous and fractionated irradiation treatments. Synchronized HeLa cells continuously irradiated at a dose rate of 37 rad/hr eventually accumulate in mitosis. If the continuous irradiation is stopped before the cells enter G2 or even after they have progressed for a limited time into the G2 arrest that develops, very little subsequent accumulation of cells in mitosis occurs. If they progress for a longer time into the G2 arrest, then some mitotic accumulation does occur after the irradiation is stopped. When synchronized cells were allowed to progress through G1 and S before the irradiation was started, very little cell division occurred during subsequent continuous irradiation and extensive mitotic accumulation was observed. Thus, for continuous irradiation of HeLa cells, the dose received by a cell during G2 or a G2 delay apparently determines whether it will be able to divide if it reaches mitosis. Arguing against the notion that continuous irradiation during G2 is required to produce a mitotic accumulation was the result of an expriment which showed that a similar effect was obtained using two acute doses: the first to produce a G2 delay and the second to give the necessary dose during the delay. The first dose alone resulted in little mitotic accumulation. The time of delivery of the second dose during the G2 delay affected the extent of mitotic accumulation observed. There was less mitotic accumulation when second acute doses were given early or at intermediate times during the delay than when they were given late during the G2 delay. An accumulation of cells in mitosis was also observed by using a combination of low-dose-rate irradiation to induce a G2 delay, followed immediately by an acute dose of either 500 or 1000 rad. The low-dose-rate treatment alone resulted in no mitotic accumulation

  15. Raman Spectroscopy of DNA Packaging in Individual Human Sperm Cells distinguishes Normal from Abnormal Cells

    Energy Technology Data Exchange (ETDEWEB)

    Huser, T; Orme, C; Hollars, C; Corzett, M; Balhorn, R

    2009-03-09

    Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

  16. Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

    International Nuclear Information System (INIS)

    Dai Heqiao; Liu Jianying; Malkas, Linda H.; Catalano, Jennifer; Alagharu, Srilakshmi; Hickey, Robert J.

    2009-01-01

    Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC 50 of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases α, δ and ε is 15, 45 and 125 μM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC 50 = 88 μM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication

  17. A specific role for the ZipA protein in cell division: stabilization of the FtsZ protein.

    Science.gov (United States)

    Pazos, Manuel; Natale, Paolo; Vicente, Miguel

    2013-02-01

    In Escherichia coli, the cell division protein FtsZ is anchored to the cytoplasmic membrane by the action of the bitopic membrane protein ZipA and the cytoplasmic protein FtsA. Although the presence of both ZipA and FtsA is strictly indispensable for cell division, an FtsA gain-of-function mutant FtsA* (R286W) can bypass the ZipA requirement for cell division. This observation casts doubts on the role of ZipA and its need for cell division. Maxicells are nucleoid-free bacterial cells used as a whole cell in vitro system to probe protein-protein interactions without the need of protein purification. We show that ZipA protects FtsZ from the ClpXP-directed degradation observed in E. coli maxicells and that ZipA-stabilized FtsZ forms membrane-attached spiral-like structures in the bacterial cytoplasm. The overproduction of the FtsZ-binding ZipA domain is sufficient to protect FtsZ from degradation, whereas other C-terminal ZipA partial deletions lacking it are not. Individual overproduction of the proto-ring component FtsA or its gain-of-function mutant FtsA* does not result in FtsZ protection. Overproduction of FtsA or FtsA* together with ZipA does not interfere with the FtsZ protection. Moreover, neither FtsA nor FtsA* protects FtsZ when overproduced together with ZipA mutants lacking the FZB domain. We propose that ZipA protects FtsZ from degradation by ClpP by making the FtsZ site of interaction unavailable to the ClpX moiety of the ClpXP protease. This role cannot be replaced by either FtsA or FtsA*, suggesting a unique function for ZipA in proto-ring stability.

  18. An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

    Science.gov (United States)

    Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

    2012-07-01

    cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

  19. SecA is required for membrane targeting of the cell division protein DivIVA in vivo

    Directory of Open Access Journals (Sweden)

    Sven eHalbedel

    2014-02-01

    Full Text Available The conserved protein DivIVA is involved in different morphogenetic processes in Gram-positive bacteria. In Bacillus subtilis, the protein localises to the cell division site and cell poles, and functions as a scaffold for proteins that regulate division site selection, and for proteins that are required for sporulation. To identify other proteins that bind to DivIVA, we performed an in vivo cross-linking experiment. A possible candidate that emerged was the secretion motor ATPase SecA. SecA mutants have been described that inhibit sporulation, and since DivIVA is necessary for sporulation, we examined the localisation of DivIVA in these mutants. Surprisingly, DivIVA was delocalised, suggesting that SecA is required for DivIVA targeting. To further corroborate this, we performed SecA depletion and inhibition experiments, which provided further indications that DivIVA localisation depends on SecA. Cell fractionation experiments showed that SecA is important for binding of DivIVA to the cell membrane. This was unexpected since DivIVA does not contain a signal sequence, and is able to bind to artificial lipid membranes in vitro without support of other proteins. SecA is required for protein secretion and membrane insertion, and therefore its role in DivIVA localisation is likely indirect. Possible alternative roles of SecA in DivIVA folding and/or targeting are discussed.

  20. Transfer of unstable chromosomal aberrations in human peripheral lymphocytes at cell division and their significance for the aberration frequency

    International Nuclear Information System (INIS)

    Stephan, G.; Chang Tsangpi.

    1986-04-01

    In 48 h cultures, the fraction of human lymphocytes in 2nd mitosis was found to be between 0 and 42.5% (mean value 8.7%). The X-ray exposure from irradiating with 2 Gy resulted in a cell cycle delay which varied from donor to donor. A loss of nearly 50% of dicentric chromosomes and acentric fragments from unstable chromosomes occurred at cell division, while centric rings were not impeded. When dicentric chromosomes, or acentric fragments are found in 2nd mitosis, they show a characteristic differential staining, which means that chromatides at cell division fall free and are replicated in daughter cells. When plotting dose effect curves of dicentric chromosomes, up to 20% of 2nd mitosis fractions have little influence on the aberration rate. This may be additionally verified as part of the 'biological dosimetry' in a person with 24% of 2nd mitosis. When the rates of dicentric chromosomes exclusively evaluated from 1st mitosis after irradiation with 2.0 Gy were related to the donors age, no age-dependent sensitivity to radiation could be observed. Aberration rates which deviate from person to person are comparable to the results achieved by conventional staining methods. (orig./MG) [de

  1. Salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA.

    Science.gov (United States)

    Gao, Qiuqiang; Liou, Liang-Chun; Ren, Qun; Bao, Xiaoming; Zhang, Zhaojie

    2014-03-03

    The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ 0 ). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ 0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ 0 cells to SDS after salt treatment, while overexpression of SCW11 results in higher sensitivity. In addition, salt stress in ρ 0 cells induces high levels of reactive oxygen species (ROS), which further damages the cell wall, causing cells to become more sensitive towards the cell wall-perturbing agent.

  2. Investigation of epstein-barr virus and parvovirus b19 DNA in allogeneic stem cell transplant patients.

    Science.gov (United States)

    Atalay, Altay; Gökahmetoğlu, Selma; Durmaz, Süleyman; Kandemir, Idris; Sağlam, Derya; Kaynar, Leylagül; Eser, Bülent; Cetin, Mustafa; Kılıç, Hüseyin

    2014-06-01

    We aimed to investigate posttransplant Epstein-Barr virus (EBV) and parvovirus B19 DNA in allogeneic stem cell transplant patients between 2009 and 2010. Forty-five adult patients in whom allogeneic stem cell transplantation was performed between April 2009 and November 2010 in the Erciyes University Faculty of Medicine, Department of Internal Medicine, Division of Hematology and Oncology, were included in the study. EBV and parvovirus B19 DNA positivity was investigated by using real-time polymerase chain reaction technique in 135 plasma samples obtained after transplantation at between 1 and 6 months. Pretransplant serological markers of EBV and parvovirus B19 were provided from patient files. In 32 (71.1%) of the patients, EBV antibodies in the pretransplantation period were as follows: anti-EBNA-1 IgG (+), VCA IgM (-), and VCA IgG (+). In 2 patients (4.45%), these antibodies were as follows: anti-EBNA-1 IgG (+), VCA IgM (-), and VCA IgG (-). In 1 patient (2.2%), they were as follows: anti-EBNA-1 IgG (-), VCA IgM (-), and VCA IgG (+). EBV serological markers were negative in 2 (2.2%) out of 45 patients before transplantation. There was low DNA positivity (parvovirus B19 IgM was negative and IgG was positive, parvovirus B19 IgM was positive and IgG was negative in 1 (2.3%) patient. Parvovirus B19 DNA was not identified in any of the samples obtained from these 45 patients. In this study, EBV and parvovirus B19 DNA were investigated in allogeneic stem cell transplant patients. None of the patients developed PTLD and parvovirus B19 DNA positivity was not detected. However, this issue needs to be further evaluated in prospective, multicenter studies with larger series of patients.

  3. Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines.

    Science.gov (United States)

    Yamamoto, Kimiyo N; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P; Witt, Kristine L; Tice, Raymond R

    2011-08-01

    Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. Copyright © 2011 Wiley-Liss, Inc.

  4. Fibre autoradiography of repair and replication in DNA from single cells: the effect of DNA synthesis inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Ockey, C.H.

    1982-04-01

    DNA fibre autoradiography, after incorporation of high specific activity /sup 3/H-thymidine and /sup 3/H-deoxycytidine, has been used to investigate repair in DNA fibres from single cells following UV, or methyl-methane sulphonate (MMS) treatment. Asynchronously growing human fibroblasts, leucocytes, and HeLa cells at different phases of the cell cycle have been investigated. Isotope incorporation in repair could be differentiated from that involved in replication by the distribution and density of silver grains along the DNA fibres. Grain distribution due to repair was continuous over long stretches of the fibres and was at a low density, occasionally interspersed with short slightly denser segments. Replication labelling on the other hand, was dense and usually in short tandem segments. Repair labelling was of a similar overall density in fibres from a single cell, but differed in intensity from cell to cell. In mutagen treated Go (leucocytes) of G/sub 1/ (HeLa cells), repair labelling was not increased by the presence of the DNA inhibitors, hydroxyurea (HU) or 5-fluorodeoxyuridine (FUdR). Repair was not detectable in S cells however without the use of these inhibitors to reduce endogenous nucleoside production. FUdR enhanced the repair labelling in S cells only slightly, while HU increased it beyond that observed in UV irradiated, HU treated, G/sub 1/ cells. The intensity of repair labelling in fibres from mutagen treated S cells appears to be proportional to the degree of reduction of DNA chain elongation in replicons.

  5. Adenovirus DNA replication in vitro is stimulated by RNA from uninfected HeLa cells

    NARCIS (Netherlands)

    Vliet, P.C. van der; Dam, D. van; Kwant, M.M.

    1984-01-01

    Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA-binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat-stable,

  6. Different Amounts of DNA in Newborn Cells of Escherichia coli Preclude a Role for the Chromosome in Size Control According to the "Adder" Model.

    Science.gov (United States)

    Huls, Peter G; Vischer, Norbert O E; Woldringh, Conrad L

    2018-01-01

    According to the recently-revived adder model for cell size control, newborn cells of Escherichia coli will grow and divide after having added a constant size or length, ΔL , irrespective of their size at birth. Assuming exponential elongation, this implies that large newborns will divide earlier than small ones. The molecular basis for the constant size increment is still unknown. As DNA replication and cell growth are coordinated, the constant ΔL could be based on duplication of an equal amount of DNA, ΔG , present in newborn cells. To test this idea, we measured amounts of DNA and lengths of nucleoids in DAPI-stained cells growing in batch culture at slow and fast rates. Deeply-constricted cells were divided in two subpopulations of longer and shorter lengths than average; these were considered to represent large and small prospective daughter cells, respectively. While at slow growth, large and small prospective daughter cells contained similar amounts of DNA, fast growing cells with multiforked replicating chromosomes, showed a significantly higher amount of DNA (20%) in the larger cells. This observation precludes the hypothesis that Δ L is based on the synthesis of a constant ΔG . Growth curves were constructed for siblings generated by asymmetric division and growing according to the adder model. Under the assumption that all cells at the same growth rate exhibit the same time between initiation of DNA replication and cell division (i.e., constant C+D -period), the constructions predict that initiation occurs at different sizes ( Li ) and that, at fast growth, large newborn cells transiently contain more DNA than small newborns, in accordance with the observations. Because the state of segregation, measured as the distance between separated nucleoids, was found to be more advanced in larger deeply-constricted cells, we propose that in larger newborns nucleoid separation occurs faster and at a shorter length, allowing them to divide earlier. We propose

  7. DNA Replication and Cell Cycle Progression Regulatedby Long Range Interaction between Protein Complexes bound to DNA.

    Science.gov (United States)

    Matsson, L

    2001-12-01

    A nonstationary interaction that controlsDNA replication and the cell cycle isderived from many-body physics in achemically open T cell. The model predictsa long range force F'(ξ) =- (κ/2) ξ(1 - ξ)(2 - ξ)between thepre-replication complexes (pre-RCs) boundby the origins in DNA, ξ = ϕ/N being the relativedisplacement of pre-RCs, ϕ the number of pre-RCs, N the number of replicons to be replicated,and κ the compressibilitymodulus in the lattice of pre-RCs whichbehaves dynamically like an elasticallybraced string. Initiation of DNAreplication is induced at the thresholdϕ = N by a switch ofsign of F''(ξ), fromattraction (-) and assembly in the G(1) phase (0force at ϕ = 2N, from repulsion inS phase back to attraction in G(2), when all primed replicons havebeen duplicated once. F'(0) = 0corresponds to a resting cell in theabsence of driving force at ϕ= 0. The model thus ensures that the DNAcontent in G(2) cells is exactlytwice that of G(1) cells. The switch of interaction at the R-point, at which N pre-RCs have been assembled, starts the release of Rb protein thus also explaining the shift in the Rb phosphorylation from mitogen-dependent cyclinD to mitogen-independent cyclin E.Shape,slope and scale of the response curvesderived agree well with experimental datafrom dividing T cells and polymerising MTs,the variable length of which is due to anonlinear dependence of the growthamplitude on the initial concentrations oftubulin dimers and guanosine-tri-phosphate(GTP). The model also explains the dynamic instabilityin growing MTs.

  8. Cells immobilized on patterns printed in DNA by an inkjet printer.

    Science.gov (United States)

    Sakurai, Kengo; Teramura, Yuji; Iwata, Hiroo

    2011-05-01

    The ability to two-dimensionally align various kinds of cells freely onto substrate would be a useful tool for analysis of cell-cell interactions. In this study, we aimed to establish a method for attaching cells to the substrate, in which the pattern is drawn by an inkjet printer. Poly-deoxyribonucleic acid (DNA) was immobilized onto the cell surface by use of DNA-conjugated poly(ethylene) glycol-phospholipid (DNA-PEG-lipid), which is the amphiphilic conjugate of PEG-lipid and single-stranded DNA. The surface of the substrate was then modified with the complementary DNA using an inkjet printer. Finally, DNA-immobilized cells were attached onto the substrate through DNA hybridization. The use of the inkjet printer enabled us to draw the DNA pattern accurately on the substrate with a resolution of a few hundred micrometers. DNA-immobilized cells could be attached precisely along the DNA pattern on the substrate. In addition, various kinds of cells could be attached simultaneously by using various sequences of DNA. Our technique is promising for analysis of cell-cell interactions and differentiation induction in stem cell research. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Sedimentation properties of DNA-membrane complexes and yield of DNA breaks at irradiation of mammalian cells

    International Nuclear Information System (INIS)

    Erzgraber, G.; Kozubek, S.; Lapidus, I.L.

    1985-01-01

    The dependence of the relative sedimentation velocity of DNA-membrane complexes on the dose of irradiation and time of incubation of Chinese Hamster cells is analysed. It is concluded that the initial part of the curve provides the information on the occurrence of single strand breaks in DNA; the position of the local maximum allows us to calculate the yield of DNA double strand breaks. The reparation decay constant can be estimated as well

  10. DNA demethylation by 5-aza-2-deoxycytidine treatment abrogates 17 beta-estradiol-induced cell growth and restores expression of DNA repair genes in human breast cancer cells.

    Science.gov (United States)

    Singh, Kamaleshwar P; Treas, Justin; Tyagi, Tulika; Gao, Weimin

    2012-03-01

    Prolonged exposure to elevated levels of estrogen is a risk factor for breast cancer. Though increased cell growth and loss of DNA repair capacity is one of the proposed mechanisms for estrogen-induced cancers, the mechanism through which estrogen induces cell growth and decreases DNA repair capacity is not clear. DNA hypermethylation is known to inactivate DNA repair genes and apoptotic response in cancer cells. Therefore, the objective of this study was to determine the role of DNA hypermethylation in estrogen-induced cell growth and regulation of DNA repair genes expression in breast cancer cells. To achieve this objective, the estrogen-responsive MCF-7 cells either pretreated with 5-aza-2-deoxycytidine (5-aza-dC) or untreated (as control) were exposed to 17 beta-estradiol (E2), and its effect on cell growth and expression of DNA repair genes were measured. The result revealed that 5-aza-dC abrogates the E2-induced growth in MCF-7 cells. An increased expression of OGG1, MSH4, and MLH1 by 5-aza-dC treatment alone, suggest the DNA hypermethylation as a potential cause for decreased expression of these genes in MCF-7 cells. The decreased expression of ERCC1, XPC, OGG1, and MLH1 by E2 alone and its restoration by co-treatment with 5-aza-dC further suggest that E2 reduces the expression of these DNA repair genes potentially through promoter hypermethylation. Reactivation of mismatch repair (MMR) gene MLH1 and abrogation of E2-induced cell growth by 5-aza-dC treatment suggest that estrogen causes increased growth in breast cancer cells potentially through the inhibition of MMR-mediated apoptotic response. In summary, this study suggests that estrogen increases cell growth and decreases the DNA repair capacity in breast cancer cells, at least in part, through epigenetic mechanism. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  11. Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells.

    Science.gov (United States)

    Kannappan, Ramaswamy; Mattapally, Saidulu; Wagle, Pooja A; Zhang, Jianyi

    2018-05-18

    The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human iPS cells has never been studied. p53-TAD was knocked out in iPS cells using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD KO cells were characterized by: accelerated proliferation, decreased population doubling time, and unaltered Bcl2, BBC3, IGF1R, Bax and altered Mdm2, p21, and PIDD transcripts expression. In p53-TAD KO cells p53 regulated DNA repair proteins XPA, DNA polH and DDB2 expression were found to be reduced compared to p53-WT cells. Exposure to low dose of doxorubicin (Doxo) induced similar DNA damage and DNA damage response (DDR) measured by RAD50 and MRE11 expression, Checkpoint kinase 2 activation and γH2A.X recruitment at DNA strand breaks in both the cell groups indicating silencing p53-TAD do not affect DDR mechanism upstream of p53. Following removal of Doxo p53-WT hiPS cells underwent DNA repair, corrected their damaged DNA and restored DNA integrity. Conversely, p53-TAD KO hiPS cells did not undergo complete DNA repair and failed to restore DNA integrity. More importantly continuous culture of p53-TAD KO hiPS cells underwent G2/M cell cycle arrest and expressed cellular senescent marker p16 INK4a . Our data clearly shows that silencing transactivation domain of p53 did not affect DDR but affected the DNA repair process implying the crucial role of p53 transactivation domain in maintaining DNA integrity. Therefore, activating p53-TAD domain using small molecules may promote DNA repair and integrity of cells and prevent senescence.

  12. Tetracycline hypersensitivity of an ezrA mutant links GalE and TseB (YpmB to cell division

    Directory of Open Access Journals (Sweden)

    Pamela eGamba

    2015-04-01

    Full Text Available Cell division in bacteria is initiated by the polymerization of FtsZ into a ring-like structure at midcell that functions as a scaffold for the other cell division proteins. In Bacillus subtilis, the conserved cell division protein EzrA is involved in modulation of Z-ring formation and coordination of septal peptidoglycan synthesis. Here, we show that an ezrA mutant is hypersensitive to tetracycline, even when the tetracycline efflux pump TetA is present. This effect is not related to the protein translation inhibiting activity of tetracycline. Overexpression of FtsL suppresses this phenotype, which appears to be related to the intrinsic low FtsL levels in an ezrA mutant background. A transposon screen indicated that the tetracycline effect can also be suppressed by overproduction of the cell division protein ZapA. In addition, tetracycline sensitivity could be suppressed by transposon insertions in galE and the unknown gene ypmB, which was renamed tseB (tetracycline sensitivity suppressor of ezrA. GalE is an epimerase using UDP-glucose and UDP-N-acetylglucosamine as substrate. Deletion of this protein bypasses the synthetic lethality of zapA ezrA and sepF ezrA double mutations, indicating that GalE influences cell division. The transmembrane protein TseB contains an extracytoplasmic peptidase domain, and a GFP fusion shows that the protein is enriched at cell division sites. A tseB deletion causes a shorter cell phenotype, indicating that TseB plays a role in cell division. Why a deletion of ezrA renders B. subtilis cells hypersensitive for tetracycline remains unclear. We speculate that this phenomenon is related to the tendency of tetracycline analogues to accumulate into the lipid bilayer, which may destabilize certain membrane proteins.

  13. Predicting Variation of DNA Shape Preferences in Protein-DNA Interaction in Cancer Cells with a New Biophysical Model.

    Science.gov (United States)

    Batmanov, Kirill; Wang, Junbai

    2017-09-18

    DNA shape readout is an important mechanism of transcription factor target site recognition, in addition to the sequence readout. Several machine learning-based models of transcription factor-DNA interactions, considering DNA shape features, have been developed in recent years. Here, we present a new biophysical model of protein-DNA interactions by integrating the DNA shape properties. It is based on the neighbor dinucleotide dependency model BayesPI2, where new parameters are restricted to a subspace spanned by the dinucleotide form of DNA shape features. This allows a biophysical interpretation of the new parameters as a position-dependent preference towards specific DNA shape features. Using the new model, we explore the variation of DNA shape preferences in several transcription factors across various cancer cell lines and cellular conditions. The results reveal that there are DNA shape variations at FOXA1 (Forkhead Box Protein A1) binding sites in steroid-treated MCF7 cells. The new biophysical model is useful for elucidating the finer details of transcription factor-DNA interaction, as well as for predicting cancer mutation effects in the future.

  14. Dose dependency of the frequency of micronucleated binucleated clone cells and of division related median clone sizes difference. Pt. 2

    International Nuclear Information System (INIS)

    Hagemann, G,; Kreczik, A.; Treichel, M.

    1996-01-01

    Following irradiation of the progenitor cells the clone growth of CHO cells decreases as a result of cell losses. Lethally acting expressions of micronuclei are produced by heritable lethal mutations. The dependency of the frequency of micronucleated binucleated clone cells and of the median clone sizes difference on the radiation dose was measured and compared to non-irradiated controls. Using the cytokinesis-block-micronucleus-method binucleated cells with micronuclei were counted as ratio of all binucleated cells within a clone size distribution. This ratio (shortened: micronucleus yield) was determined for all clone size distributions, which had been exposed to different irradiation doses and incubation times. The micronucleus yields were compared to the corresponding median clone sizes differences. The micronucleus yield is linearly dependent on the dose and is independent of the incubation time. The same holds true for the division related median clone sizes difference, which as a result is also linearly dependent on the micronucleus yield. Due to the inevitably errors of the cell count of micronucleated binucleated cells, an automatic measurement of the median clone sizes differences is the preferred method for evaluation of cellular radiation sensitivity for heritable lethal mutations. This value should always be determined in addition, if clone survival fractions are used as predictive test because it allows for an estimation of the remission probability of surviving cells. (orig.) [de

  15. DNA-crosslinker cisplatin eradicates bacterial persister cells.

    Science.gov (United States)

    Chowdhury, Nityananda; Wood, Thammajun L; Martínez-Vázquez, Mariano; García-Contreras, Rodolfo; Wood, Thomas K

    2016-09-01

    For all bacteria, nearly every antimicrobial fails since a subpopulation of the bacteria enter a dormant state known as persistence, in which the antimicrobials are rendered ineffective due to the lack of metabolism. This tolerance to antibiotics makes microbial infections the leading cause of death worldwide and makes treating chronic infections, including those of wounds problematic. Here, we show that the FDA-approved anti-cancer drug cisplatin [cis-diamminodichloroplatinum(II)], which mainly forms intra-strand DNA crosslinks, eradicates Escherichia coli K-12 persister cells through a growth-independent mechanism. Additionally, cisplatin is more effective at killing Pseudomonas aeruginosa persister cells than mitomycin C, which forms inter-strand DNA crosslinks, and cisplatin eradicates the persister cells of several pathogens including enterohemorrhagic E. coli, Staphylococcus aureus, and P. aeruginosa. Cisplatin was also highly effective against clinical isolates of S. aureus and P. aeruginosa. Therefore, cisplatin has broad spectrum activity against persister cells. Biotechnol. Bioeng. 2016;113: 1984-1992. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. Aberrant methylation of cell-free circulating DNA in plasma predicts poor outcome in diffuse large B cell lymphoma

    DEFF Research Database (Denmark)

    Sommer Kristensen, Lasse; Hansen, Jakob Werner; Kristensen, Søren Sommer

    2016-01-01

    BACKGROUND: The prognostic value of aberrant DNA methylation of cell-free circulating DNA in plasma has not previously been evaluated in diffuse large B cell lymphoma (DLBCL). The aim of this study was to investigate if aberrant promoter DNA methylation can be detected in plasma from DLBCL patients...

  17. Adoptive cancer immunotherapy using DNA-demethylated T helper cells as antigen-presenting cells

    DEFF Research Database (Denmark)

    Kirkin, Alexei F.; Dzhandzhugazyan, Karine N.; Guldberg, Per

    2018-01-01

    In cancer cells, cancer/testis (CT) antigens become epigenetically derepressed through DNA demethylation and constitute attractive targets for cancer immunotherapy. Here we report that activated CD4+ T helper cells treated with a DNA-demethylating agent express a broad repertoire of endogenous CT...... antigens and can be used as antigen-presenting cells to generate autologous cytotoxic T lymphocytes (CTLs) and natural killer cells. In vitro, activated CTLs induce HLA-restricted lysis of tumor cells of different histological types, as well as cells expressing single CT antigens. In a phase 1 trial of 25...... patients with recurrent glioblastoma multiforme, cytotoxic lymphocytes homed to the tumor, with tumor regression ongoing in three patients for 14, 22, and 27 months, respectively. No treatment-related adverse effects were observed. This proof-of-principle study shows that tumor-reactive effector cells can...

  18. Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli.

    Science.gov (United States)

    Fenton, Andrew K; Gerdes, Kenn

    2013-07-03

    How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin-MreB while cell division is governed by tubulin-FtsZ. A ring-like structure containing FtsZ (the Z ring) at mid-cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid-cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB-FtsZ interaction is required for transfer of cell-wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB.

  19. Quantification of residual host cell DNA in adenoviral vectors produced on PER.C6 cells

    NARCIS (Netherlands)

    Gijsbers, Linda; Koel, Björn; Weggeman, Miranda; Goudsmit, Jaap; Havenga, Menzo; Marzio, Giuseppe

    2005-01-01

    Recombinant adenoviral vectors for gene therapy and vaccination are routinely prepared on cultures of immortalized cells, allowing the production of vector batches of high titer and consistent quality. Quantification of residual DNA from the producing cell line is part of the purity tests for

  20. GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Svetlana Kostyuk

    2015-01-01

    Full Text Available Background. Cell free DNA (cfDNA circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Principal Findings. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci. As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR, PCNA (FACS and antiapoptotic genes (BCL2 (RT-PCR and FACS, BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR. Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs. Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR, in the level of fatty acid binding protein FABP4 (FACS analysis and in the level of fat (Oil Red O. Conclusions. GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose—derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

  1. In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication

    NARCIS (Netherlands)

    Baltus, Andrew E.; Menke, Douglas B.; Hu, Yueh-Chiang; Goodheart, Mary L.; Carpenter, Anne E.; de Rooij, Dirk G.; Page, David C.

    2006-01-01

    The transition from mitosis to meiosis is a defining juncture in the life cycle of sexually reproducing organisms. In yeast, the decision to enter meiosis is made before the single round of DNA replication that precedes the two meiotic divisions. We present genetic evidence of an analogous decision

  2. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Ann-Sofie, E-mail: ann-sofie.gustafsson@bms.uu.se; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-15

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  3. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

    International Nuclear Information System (INIS)

    Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

    2014-01-01

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  4. Pathogenic Chlamydia Lack a Classical Sacculus but Synthesize a Narrow, Mid-cell Peptidoglycan Ring, Regulated by MreB, for Cell Division.

    Science.gov (United States)

    Liechti, George; Kuru, Erkin; Packiam, Mathanraj; Hsu, Yen-Pang; Tekkam, Srinivas; Hall, Edward; Rittichier, Jonathan T; VanNieuwenhze, Michael; Brun, Yves V; Maurelli, Anthony T

    2016-05-01

    The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe's developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.

  5. Pathogenic Chlamydia Lack a Classical Sacculus but Synthesize a Narrow, Mid-cell Peptidoglycan Ring, Regulated by MreB, for Cell Division.

    Directory of Open Access Journals (Sweden)

    George Liechti

    2016-05-01

    Full Text Available The peptidoglycan (PG cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe's developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.

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    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.20.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Yeast str...ain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.20.DNA-RNA_hybrids.AllCell.bed ...

  13. File list: Oth.YSt.10.DNA-RNA_hybrids.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.10.DNA-RNA_hybrids.AllCell sacCer3 TFs and others DNA-RNA hybrids Yeast str...ain http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.10.DNA-RNA_hybrids.AllCell.bed ...

  14. Effect of 5-fluorodeoxyuridine on DNA replication in ultraviolet-irradiated HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Brozmanova, J.; Masek, F.; Synzynys, B.I.; Saenko, A.S.

    1985-11-05

    In HeLa cells precultivated for 6 hours with 5-fluorodeoxyuridine (FUdR) and for 18 hours in FUdR-free medium, DNA synthesis was much more resistant to UV irradiation than that of untreated cells. DNA synthesized in FUdR-pretreated and UV irradiated cells represents a semiconservative DNA replication and shows more rapid shift of the pulse-labelled chased DNA to high molecular weight. This DNA synthesis is not induced by synchronization of the cell cycle. It is assumed that either the changes of chromatine structure, or an enhanced level of some enzymes might be involved in the replication of the damaged template. (author).

  15. Effect of chronic fractionated low-dose gamma irradiation on division potential of human embryonic cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Masami; Suzuki, Masao; Suzuki, Keiji; Watanabe, Kimiko (Yokohama City Univ. (Japan). Faculty of Medicine); Nakano, Kazushiro

    1991-12-01

    We investigated the in vitro phenotypic transformation of human embryo (HE) cells that were repeatedly irradiated (7.5 cGy once a week) throughout their life-span. Irradiation was repeated until the cells had accumulated 195 cGy (equivalent to the 26th passage). Samples of cells were assayed for survival by colony formation, as well as for mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus and for transformation by focus formation. The life-span (mean number of population doublings) of multiply irradiated cells with a total dose of 97.5 cGy was slightly but significantly prolonged over that of controls. After HE cells had accumulated 195 cGy, the maximum number of divisions increased to 130-160% of the number in non-irradiated control cells. Transformed foci were not observed until cells had accumulated 97.5 cGy, and then increased with the increasing accumulation of radiation. However, no cells showed immortality or expressed a malignant phenotype in vitro. (author).

  16. Absence of specificity in inhibition of DNA repair replication by DNA-binding agents, cocarcinogens, and steroids in human cells

    International Nuclear Information System (INIS)

    Cleaver, J.E.; Painter, R.B.

    1975-01-01

    Although many chemicals, including cocarcinogens, DNA-binding agents, and steroids, inhibit repair replication of ultraviolet-induced damage to DNA in human lymphocytes and proliferating cells in culture, none of these chemicals is specific. Our results show that all the chemicals we tested inhibit normal DNA synthesis as much as or more than they inhibit repair replication. There is thus no evidence in our results to support the hypothesis that cocarcinogens are specific inhibitors of DNA repair or that any of the chemicals studied might be useful adjuncts to tumor therapy merely because of specific inhibition of radiation repair mechanisms

  17. DNA crosslinking and cytotoxicity in normal and transformed human cells treated with antitumor nitrosoureas.

    OpenAIRE

    Erickson, L C; Bradley, M O; Ducore, J M; Ewig, R A; Kohn, K W

    1980-01-01

    Normal (IMR-90) and simian virus 40-transformed (VA-13) human embryo cells were treated with antitumor nitrosoureas, and the effects on cell viability and cell DNA were compared. All six nitrosoureas tested were more toxic to VA-13 cells than to IMR-90 cells as measured by decrease in cell proliferation or in colony formation. The nitrosoureas capable of generating alkylisocyanates produced a smaller difference between the cell types than did derivatives lacking this capacity. DNA damage was ...

  18. Resistance to DNA denaturation in irradiated Chinese hamster V79 fibroblasts is linked to cell shape

    International Nuclear Information System (INIS)

    Olive, P.L.; Vanderbyl, S.; MacPhail, S.H.

    1991-01-01

    Exponentially growing Chinese hamster V79-171b lung fibroblasts seeded at high density on plastic (approximately 7 x 10(3) cells/cm2) flatten, elongate, and produce significant amounts of extracellular fibronectin. When lysed in weak alkali/high salt, the rate of DNA denaturation following exposure to ionizing radiation is exponential. Conversely, cells plated at low density (approximately 7 x 10(2) cells/cm2) on plastic are more rounded 24 h later, produce little extracellular fibronectin, and display unusual DNA denaturation kinetics after X-irradiation. DNA in these cells resists denaturation, as though constraints to DNA unwinding have developed. Cell doubling time and distribution of cells in the growth cycle are identical for both high and low density cultures as is cell survival in response to radiation damage. The connection between DNA conformation and cell shape was examined further in low density cultures grown in conditioned medium. Under these conditions, cells at low density were able to elongate, and DNA denaturation of low density cultures was identical to that of high density cultures. Conversely, cytochalasin D, which interferes with actin polymerization causing cells to round up and release fibronectin, allowed development of constraints in high density cultures. These results suggest that DNA conformation is sensitive to changes in cell shape which result when cells are grown in different environments. However, these changes in DNA conformation detected by the DNA unwinding assay do not appear to play a direct role in radiation-induced cell killing

  19. DNA methylation in states of cell physiology and pathology.

    Directory of Open Access Journals (Sweden)

    Lech Chyczewski

    2007-10-01

    Full Text Available DNA methylation is one of epigenetic mechanisms regulating gene expression. The methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues. In a normal cell, a significant degree of methylation is characteristic for extragenic DNA (cytosine within the CG dinucleotide while CpG islands located in gene promoters are unmethylated, except for inactive genes of the X chromosome and the genes subjected to genomic imprinting. The changes in the methylation pattern, which may appear as the organism age and in early stages of cancerogenesis, may lead to the silencing of over ninety endogenic genes. It has been found, that these disorders consist not only of the methylation of CpG islands, which are normally unmethylated, but also of the methylation of other dinucleotides, e.g. CpA. Such methylation has been observed in non-small cell lung cancer, in three regions of the exon 5 of the p53 gene (so-called "non-CpG" methylation. The knowledge of a normal methylation process and its aberrations appeared to be useful while searching for new markers enabling an early detection of cancer. With the application of the Real-Time PCR technique (using primers for methylated and unmethylated sequences five new genes which are potential biomarkers of lung cancer have been presented.

  20. Involvement of DNA-PK and ATM in radiation- and heat-induced DNA damage recognition and apoptotic cell death

    International Nuclear Information System (INIS)

    Tomita, Masanori

    2010-01-01

    Exposure to ionizing radiation and hyperthermia results in important biological consequences, e.g. cell death, chromosomal aberrations, mutations, and DNA strand breaks. There is good evidence that the nucleus, specifically cellular DNA, is the principal target for radiation-induced cell lethality. DNA double-strand breaks (DSBs) are considered to be the most serious type of DNA damage induced by ionizing radiation. On the other hand, verifiable mechanisms which can lead to heat-induced cell death are damage to the plasma membrane and/or inactivation of heat-labile proteins caused by protein denaturation and subsequent aggregation. Recently, several reports have suggested that DSBs can be induced after hyperthermia because heat-induced phosphorylated histone H2AX (γ-H2AX) foci formation can be observed in several mammalian cell lines. In mammalian cells, DSBs are repaired primarily through two distinct and complementary mechanisms: non-homologous end joining (NHEJ), and homologous recombination (HR) or homology-directed repair (HDR). DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) are key players in the initiation of DSB repair and phosphorylate and/or activate many substrates, including themselves. These phosphorylated substrates have important roles in the functioning of cell cycle checkpoints and in cell death, as well as in DSB repair. Apoptotic cell death is a crucial cell suicide mechanism during development and in the defense of homeostasis. If DSBs are unrepaired or misrepaired, apoptosis is a very important system which can protect an organism against carcinogenesis. This paper reviews recently obtained results and current topics concerning the role of DNA-PK and ATM in heat- or radiation-induced apoptotic cell death. (author)

  1. Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration.

    Science.gov (United States)

    Churchil, R R; Gupta, J; Singh, A; Sharma, D

    2011-06-01

    1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.

  2. Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

    International Nuclear Information System (INIS)

    Mullenders, L.H.F.

    1983-01-01

    The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after iiradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S 1 of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop. (Auth.)

  3. Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

    Energy Technology Data Exchange (ETDEWEB)

    Mullenders, L.H.F. (Rijksuniversiteit Leiden (Netherlands). Lab. voor Stralengenetica en Chemische Mutagenese); Zeeland, A.A. van; Natarajan, A.T. (Cohen (J.A.) Inst. voor Radiopathologie en Stralenbescherming, Leiden (Netherlands))

    1983-09-09

    The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S/sub 1/ of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.

  4. Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols.

    Science.gov (United States)

    Shiwa, Yuh; Hachiya, Tsuyoshi; Furukawa, Ryohei; Ohmomo, Hideki; Ono, Kanako; Kudo, Hisaaki; Hata, Jun; Hozawa, Atsushi; Iwasaki, Motoki; Matsuda, Koichi; Minegishi, Naoko; Satoh, Mamoru; Tanno, Kozo; Yamaji, Taiki; Wakai, Kenji; Hitomi, Jiro; Kiyohara, Yutaka; Kubo, Michiaki; Tanaka, Hideo; Tsugane, Shoichiro; Yamamoto, Masayuki; Sobue, Kenji; Shimizu, Atsushi

    2016-01-01

    Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS) using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03) when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50) when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λ adjusted = 1.14) by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12-1.45) and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λ adjusted = 1.00-1.17). These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.

  5. Adjustment of Cell-Type Composition Minimizes Systematic Bias in Blood DNA Methylation Profiles Derived by DNA Collection Protocols.

    Directory of Open Access Journals (Sweden)

    Yuh Shiwa

    Full Text Available Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03 when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50 when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λ adjusted = 1.14 by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12-1.45 and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λ adjusted = 1.00-1.17. These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.

  6. DNA crosslinking and cytotoxicity in normal and transformed human cells treated with antitumor nitrosoureas.

    Science.gov (United States)

    Erickson, L C; Bradley, M O; Ducore, J M; Ewig, R A; Kohn, K W

    1980-01-01

    Normal (IMR-90) and simian virus 40-transformed (VA-13) human embryo cells were treated with antitumor nitrosoureas, and the effects on cell viability and cell DNA were compared. All six nitrosoureas tested were more toxic to VA-13 cells than to IMR-90 cells as measured by decrease in cell proliferation or in colony formation. The nitrosoureas capable of generating alkylisocyanates produced a smaller difference between the cell types than did derivatives lacking this capacity. DNA damage was measured by alkaline elution in cells treated with four chloroethylnitrosoureas. Whereas VA-13 cells exhibited dose-dependent interstrand crosslinking, little or none was detected in IMR-90 cells. The IMR-90 cells, however, exhibited at least as much DNA-protein crosslinking as did VA-13 cells. The results can be interpreted in terms of a possible difference in DNA repair between the cell lines. PMID:6928639

  7. Effects of DNA polymerase inhibitors on replicative and repair DNA synthesis in ultraviolet-irradiated HeLa cells

    International Nuclear Information System (INIS)

    Morita, T.; Nakamura, H.; Tsutsui, Y.; Nishiyama, Y.; Yoshida, S.

    1982-01-01

    Aphidicolin specifically inhibits eukaryotic DNA polymerase α, while 2',3'-dideoxythymidine 5'-triphosphate (d 2 TTP) inhibits DNA polymerase ν and ν but not α. 1-ν-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) inhibits both DNA polymerase α and ν although to a different extent. Here we measured the effects of these inhibitors on repair DNA synthesis of U.V.-irradiated HeLa cells by two different methods. Firstly, aphidicolin, 1-ν-D-arabinofuranosylcytosine (araC, a precursor of araCTP) and 2',3'-dideoxythimidine (d 2 Thd, a precursor of d 2 TTP) were added directly to the culture medium. In this case, aphidicolin and araC strongly inhibited replicative DNA synthesis of HeLa cells, and they also inhibited repair synthesis after U.V.-irradiation but to a much lesser extent. In contrast, high concentrations of d 2 Thd inhibited repair DNA synthesis to a higher extent than replicative DNA synthesis. Secondly, the active form of inhibitor, d 2 TTP, was microinjected directly into cytoplasm or nuclei or U.V.-irradiated HeLa cells. Microinjection of d 2 TTP effectively inhibited repair synthesis. The microinjection of d 2 TTP, into either cytoplasm or nucleus, strongly inhibited replicative synthesis. These results might indicate that multiple DNA polymerases are involved in repair synthesis as well as in replicative synthesis

  8. DNA Measurement of Overlapping Cell Nuclei in Thick Tissue Sections

    Directory of Open Access Journals (Sweden)

    Liang Ji

    1997-01-01

    Full Text Available The paper describes an improved image analysis procedure for measuring the DNA content of cell nuclei in thick sections of liver tissue by absorption densitometry. Whereas previous methods only permitted the analysis of isolated nuclei, the new technique enables both isolated and overlapping nuclei to be measured. A 3D segmentation procedure determines whether each object is an isolated nucleus or a pair of overlapping nuclei; in the latter case the combined optical density is redistributed to the individual nuclei. A selection procedure ensures that only complete nuclei are measured.

  9. The influence of arachidonic acid metabolites on cell division in the intestinal epithelium and in colonic tumors.

    Science.gov (United States)

    Petry, F M; Tutton, P J; Barkla, D H

    1984-09-01

    Various metabolites of arachidonic acid are now known to influence cell division. In this paper the effects on cell proliferation of arachidonic acid, some inhibitors of arachidonic acid metabolism and some analogs of arachidonic acid metabolites is described. The epithelial cell proliferation rate in the jejunum, in the descending colon and in dimethylhydrazine-induced tumors of rat colon was measured using a stathmokinetic technique. Administration of arachidonic acid resulted in retardation of cell proliferation in each of the tissues examined. A cyclooxygenase inhibitor (Flurbiprofen) prevented this effect of arachidonic acid in the jejunal crypts and in colonic tumors, but not in colonic crypts. In contrast, inhibitors of both cyclooxygenase and lipoxygenase (Benoxaprofen and BW755c) prevented the effect of arachidonic acid in the colonic crypts and reduced its effect on colonic tumours but did not alter its effect on the jejunum. An inhibitor of thromoboxane A2 synthetase (U51,605) was also able to prevent the inhibitory effect of arachidonic acid on colonic tumors. Treatment with 16,16-dimethyl PGE2 inhibited cell proliferation in jejunal crypts and in colonic tumors, as did a thromboxane A2 mimicking agent, U46619. Nafazatrom, an agent that stimulates prostacyclin synthesis and inhibits lypoxygenase, promoted cell proliferation in the jejunal crypts and colonic crypts, but inhibited cell proliferation in colonic tumours.

  10. Induction of Mitochondrial DNA Deletion by Ionizing Radiation in Human Lung Fibroblast IMR-90 Cells

    International Nuclear Information System (INIS)

    Eom, Hyeon Soo; Jung, U Hee; Park, Hae Ran; Jo, Sung Kee

    2009-01-01

    Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging and also contributes to their unfavorable effects in cultured cells and animal tissues. This study was conducted to investigate the effect of ionizing radiation (IR) on mtDNA deletion and the involvement of reactive oxygen species (ROS) in this process in human lung fibroblast (IMR-90) cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated with 137 Cs -rays and the intracellular ROS level was determined by 2',7'-dichlorofluorescein diacetate (DCFH-DA) and mtDNA common deletion (4977bp) was detected by nested PCR. Old cells at PD 55 and H 2 O 2 -treated young cells were compared as the positive control. IR increased the intracellular ROS level and mtDNA 4977 bp deletion in IMR-90 cells dose-dependently. The increases of ROS level and mtDNA deletion were also observed in old cells and H 2 O 2 -treated young cells. To confirm the increased ROS level is essential for mtDNA deletion in irradiated cells, the effects of N-acetylcysteine (NAC) on IRinduced ROS and mtDNA deletion were examined. 5 mM NAC significantly attenuated the IR-induced ROS increase and mtDNA deletion. These results suggest that IR induces the mtDNA deletion and this process is mediated by ROS in IMR-90 cells

  11. Induction of Mitochondrial DNA Deletion by Ionizing Radiation in Human Lung Fibroblast IMR-90 Cells

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Hyeon Soo; Jung, U Hee; Park, Hae Ran; Jo, Sung Kee [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2009-06-15

    Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging and also contributes to their unfavorable effects in cultured cells and animal tissues. This study was conducted to investigate the effect of ionizing radiation (IR) on mtDNA deletion and the involvement of reactive oxygen species (ROS) in this process in human lung fibroblast (IMR-90) cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated with {sup 137}Cs -rays and the intracellular ROS level was determined by 2',7'-dichlorofluorescein diacetate (DCFH-DA) and mtDNA common deletion (4977bp) was detected by nested PCR. Old cells at PD 55 and H{sub 2}O{sub 2}-treated young cells were compared as the positive control. IR increased the intracellular ROS level and mtDNA 4977 bp deletion in IMR-90 cells dose-dependently. The increases of ROS level and mtDNA deletion were also observed in old cells and H{sub 2}O{sub 2}-treated young cells. To confirm the increased ROS level is essential for mtDNA deletion in irradiated cells, the effects of N-acetylcysteine (NAC) on IRinduced ROS and mtDNA deletion were examined. 5 mM NAC significantly attenuated the IR-induced ROS increase and mtDNA deletion. These results suggest that IR induces the mtDNA deletion and this process is mediated by ROS in IMR-90 cells.

  12. Isolating human DNA repair genes using rodent-cell mutants

    International Nuclear Information System (INIS)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-01-01

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab

  13. DNA replication and repair in Tilapia cells. 1. The effect of ultraviolet radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yew, F.H.; Chang, L.M. (National Taiwan Univ., Taipei (China))

    1984-12-01

    The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also effici