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Sample records for cell nuclear transfer-derived

  1. Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor.

    Science.gov (United States)

    Wakayama, Sayaka; Wakayama, Teruhiko

    2010-01-01

    Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.

  2. Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep.

    Science.gov (United States)

    Evans, M J; Gurer, C; Loike, J D; Wilmut, I; Schnieke, A E; Schon, E A

    1999-09-01

    Eukaryotic cells contain two distinct genomes. One is located in the nucleus (nDNA) and is transmitted in a mendelian fashion, whereas the other is located in mitochondria (mtDNA) and is transmitted by maternal inheritance. Cloning of mammals typically has been achieved via nuclear transfer, in which a donor somatic cell is fused by electoporation with a recipient enucleated oocyte. During this whole-cell electrofusion, nDNA as well as mtDNA ought to be transferred to the oocyte. Thus, the cloned progeny should harbour mtDNAs from both the donor and recipient cytoplasms, resulting in heteroplasmy. Although the confirmation of nuclear transfer has been established using somatic cell-specific nDNA markers, no similar analysis of the mtDNA genotype has been reported. We report here the origin of the mtDNA in Dolly, the first animal cloned from an established adult somatic cell line, and in nine other nuclear transfer-derived sheep generated from fetal cells. The mtDNA of each of the ten nuclear-transfer sheep was derived exclusively from recipient enucleated oocytes, with no detectable contribution from the respective somatic donor cells. Thus, although these ten sheep are authentic nuclear clones, they are in fact genetic chimaeras, containing somatic cell-derived nuclear DNA but oocyte-derived mtDNA.

  3. Gamete derivation from embryonic stem cells, induced pluripotent stem cells or somatic cell nuclear transfer-derived embryonic stem cells: state of the art

    Science.gov (United States)

    Easley, Charles A.; Simerly, Calvin R.; Schatten, Gerald

    2015-01-01

    Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and several biomedical rationales. Here, we consider several strategies for deriving gametes from PSCs from mice and primates (human and non-human) and their anticipated strengths, challenges and limitations. Although the ‘Weismann barrier’, which separates the mortal somatic cell lineages from the potentially immortal germline, has long existed, breakthroughs first in mice and now in humans are artificially creating germ cells from somatic cells. Spermatozoa with full reproductive viability establishing multiple generations of seemingly normal offspring have been reported in mice and, in humans, haploid spermatids with correct parent-of-origin imprints have been obtained. Similar progress with making oocytes has been published using mouse PSCs differentiated in vitro into primordial germ cells, which are then cultured after xenografting reconstructed artificial ovaries. Progress in making human oocytes artificially is proving challenging. The usefulness of these artificial gametes, from assessing environmental exposure toxicity to optimising medical treatments to prevent negative off-target effects on fertility, may prove invaluable, as may basic discoveries on the fundamental mechanisms of gametogenesis. PMID:25472048

  4. Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah

    2015-11-01

    Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

  5. Nuclear Mechanics and Stem Cell Differentiation.

    Science.gov (United States)

    Mao, Xinjian; Gavara, Nuria; Song, Guanbin

    2015-12-01

    Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.

  6. Cell fusion and nuclear fusion in plants.

    Science.gov (United States)

    Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

    2016-12-01

    Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

    2009-01-01

    Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

  8. Nuclear Glycogen Inclusions in Canine Parietal Cells.

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    Silvestri, S; Lepri, E; Dall'Aglio, C; Marchesi, M C; Vitellozzi, G

    2017-05-01

    Nuclear glycogen inclusions occur infrequently in pathologic conditions but also in normal human and animal tissues. Their function or significance is unclear. To the best of the authors' knowledge, no reports of nuclear glycogen inclusions in canine parietal cells exist. After initial observations of nuclear inclusions/pseudoinclusions during routine histopathology, the authors retrospectively examined samples of gastric mucosa from dogs presenting with gastrointestinal signs for the presence of intranuclear inclusions/pseudoinclusions and determined their composition using histologic and electron-microscopic methods. In 24 of 108 cases (22%), the authors observed various numbers of intranuclear inclusions/pseudoinclusions within scattered parietal cells. Nuclei were characterized by marked karyomegaly and chromatin margination around a central optically empty or slightly eosinophilic area. The intranuclear inclusions/pseudoinclusions stained positive with periodic acid-Schiff (PAS) and were diastase sensitive, consistent with glycogen. Several PAS-positive/diastase-sensitive sections were further examined by transmission electron microscopy, also using periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining to identify polysaccharides. Ultrastructurally, the nuclear inclusions were composed of electron-dense particles that were not membrane bound, without evidence of nuclear membrane invaginations or cytoplasmic organelles in the nuclei, and positive staining with PA-TCH-SP, confirming a glycogen composition. No cytoplasmic glycogen deposits were observed, suggesting that the intranuclear glycogen inclusions were probably synthesized in loco. Nuclear glycogen inclusions were not associated with gastritis or colonization by Helicobacter-like organisms ( P > .05). Our findings suggest that nuclear glycogen inclusions in canine parietal cells could be an incidental finding. Nevertheless, since nuclear glycogen is present in several pathologic

  9. Human somatic cell nuclear transfer and cloning.

    Science.gov (United States)

    2012-10-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Nuclear microscopy of rat colon epithelial cells

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    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  11. Nuclear microscopy of rat colon epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Ren, M., E-mail: phyrenmq@nus.edu.sg [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Rajendran, Reshmi [Lab of Molecular Imaging, Singapore Bioimaging Consotium, 11 Biopolis Way, 02-02 Helios, Singapore 138667 (Singapore); Ng, Mary [Department of Pharmacology, National University of Singapore (Singapore); Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Jenner, Andrew Michael [Illawara Health and Medical Research Institute (IHMRI), University of Wollongong, NSW 2522 (Australia)

    2011-10-15

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  12. Nuclear size regulation: from single cells to development and disease.

    Science.gov (United States)

    Edens, Lisa J; White, Karen H; Jevtic, Predrag; Li, Xiaoyang; Levy, Daniel L

    2013-04-01

    Cell size varies greatly among different cell types and organisms, especially during early development when cell division is rapid with little overall growth. A fundamental question is how organelle size is regulated relative to cell size. The nucleus exhibits exquisite size scaling during development and between species, and nuclear size is often altered in cancer cells. Recent studies have elucidated mechanisms of nuclear size regulation in a variety of experimental systems, opening the door to future research on how nuclear size impacts upon cell and nuclear function and subnuclear organization. In this review we discuss studies that have clarified nuclear size control mechanisms and how these results have or will contribute to our understanding of the functional significance of nuclear size. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    Science.gov (United States)

    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  14. Transgenesis and nuclear transfer using porcine embryonic germ cells.

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    Ahn, Kwang Sung; Won, Ji Young; Heo, Soon Young; Kang, Jee Hyun; Yang, Hong Seok; Shim, Hosup

    2007-01-01

    Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). Porcine EG cell lines with capacities of both in vitro and in vivo differentiation have been established. Because EG cells can be cultured indefinitely in an undifferentiated state, they may be more suitable for nuclear donor cells in nuclear transfer (NT) than somatic cells that have limited lifespan in primary culture. Use of EG cells could be particularly advantageous to provide an inexhaustible source of transgenic cells for NT. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblasts and EG cells were compared. The rate of development to the blastocyst stage was significantly higher in EG cell NT than somatic cell NT (94 of 518, 18.2% vs. 72 of 501, 14.4%). To investigate if EG cells can be used for transgenesis in pigs, green fluorescent protein (GFP) gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29 of 137, 21.2%) expressing GFP based on observation under fluorescence microscope. The results obtained from the present study suggest that EG cell NT may have advantages over somatic cell NT, and transgenic pigs may be produced using EG cells.

  15. Micronuclei Frequencies and Nuclear Abnormalities in Oral Exfoliated Cells of Nuclear Power Plant Workers

    OpenAIRE

    Sagari, Shitalkumar G; Babannavar, Roopa; Lohra, Abhishek; Kodgi, Ashwin; Bapure, Sunil; Rao, Yogesh; J., Arun; Malghan, Manjunath

    2014-01-01

    Aim: Biomonitoring provides a useful tool to estimate the genetic risk from exposure to genotoxic agents. The aim of this study was to evaluate the frequencies of Micronuclei (MN) and other Nuclear abnormalities (NA) from exfoliated oral mucosal cells in Nuclear Power Station (NPS) workers.

  16. Review of somatic cell nuclear transfer in pig | Muenthaisong ...

    African Journals Online (AJOL)

    It is now more than 8 years, since the first cloned pig from nuclear transfer was reported. Success of somatic cell nuclear transfer (SCNT) in pig is still low compared to that in bovine. Embryonic and neonatal abnormalities of cloned piglets are probably a result of incorrect or incomplete reprogramming of the transferred ...

  17. Joint modeling of cell and nuclear shape variation

    Science.gov (United States)

    Johnson, Gregory R.; Buck, Taraz E.; Sullivan, Devin P.; Rohde, Gustavo K.; Murphy, Robert F.

    2015-01-01

    Modeling cell shape variation is critical to our understanding of cell biology. Previous work has demonstrated the utility of nonrigid image registration methods for the construction of nonparametric nuclear shape models in which pairwise deformation distances are measured between all shapes and are embedded into a low-dimensional shape space. Using these methods, we explore the relationship between cell shape and nuclear shape. We find that these are frequently dependent on each other and use this as the motivation for the development of combined cell and nuclear shape space models, extending nonparametric cell representations to multiple-component three-dimensional cellular shapes and identifying modes of joint shape variation. We learn a first-order dynamics model to predict cell and nuclear shapes, given shapes at a previous time point. We use this to determine the effects of endogenous protein tags or drugs on the shape dynamics of cell lines and show that tagged C1QBP reduces the correlation between cell and nuclear shape. To reduce the computational cost of learning these models, we demonstrate the ability to reconstruct shape spaces using a fraction of computed pairwise distances. The open-source tools provide a powerful basis for future studies of the molecular basis of cell organization. PMID:26354424

  18. Cell fusion through a microslit between adhered cells and observation of their nuclear behavior.

    Science.gov (United States)

    Wada, Ken-Ichi; Hosokawa, Kazuo; Kondo, Eitaro; Ito, Yoshihiro; Maeda, Mizuo

    2014-07-01

    This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had ∼ 100 cell pairing structures (CPSs) making cell pairs through microslits with 2.1 ± 0.3 µm width was fabricated. After trapping NIH3T3 cells with hydrodynamic forces at the CPSs, the cells were fused through the microslit by the Sendai virus envelope method. With following timelapse observation, we discovered that the spread cells were much less susceptible to nuclear migration passing through the microslit compared with round cells, and that cytoplasmic fraction containing mitochondria was transferred through the microslit without nuclear mixing. These findings will provide an effective method for cell fusion without nuclear mixing, and will lead to an efficient method for reprograming and transdifferentiation of target cells toward regenerative medicine. © 2014 Wiley Periodicals, Inc.

  19. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    International Nuclear Information System (INIS)

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.; Wehnert, Manfred; Huebner, Stefan

    2009-01-01

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

  20. Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida

    2011-01-01

    Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-cell...... stage embryos were processed at different points in time post activation (2 hpa, 4 hpa, 8 hpa, and 12 hpa) for detailed nuclear and nucleolar analysis by TEM, and immunofluorescence for visualization of nucleolar proteins related to transcription (UBF) and processing (fibrillarin). Bovine and porcine...... intergeneric SCNT embryos were compared to their parthenogenetic counterparts to assess the effects of the introduced somatic cell. Despite the absence of morphological remodeling (premature chromatin condensation, nuclear envelope breakdown), reconstructed embryos showed nuclear and nucleolar precursor body...

  1. Modeling of Flow in Nuclear Reactor Fuel Cell Outlet

    Directory of Open Access Journals (Sweden)

    František URBAN

    2010-12-01

    Full Text Available Safe and effective load of nuclear reactor fuel cells demands qualitative and quantitative analysis of relations between coolant temperature in fuel cell outlet temperature measured by thermocouple and middle temperature of coolant in thermocouple plane position. In laboratory at Insitute of thermal power engineering of the Slovak University of Technology in Bratislava was installed an experimental physical fuel cell model of VVER 440 nuclear power plant with V 213 nuclear reactors. Objective of measurements on physical model was temperature and velocity profiles analysis in the fuel cell outlet. In this paper the measured temperature and velocity profiles are compared with the results of CFD simulation of fuel cell physical model coolant flow.

  2. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

    Science.gov (United States)

    Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

    2015-01-01

    It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life

  3. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly.

    Science.gov (United States)

    Gil-Ranedo, Jon; Hernando, Eva; Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M

    2015-06-01

    It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life

  4. Induced Pluripotent Stem Cells: Emerging Techniques for Nuclear Reprogramming

    Science.gov (United States)

    Han, Ji Woong

    2011-01-01

    Abstract Introduction of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc, can successfully reprogram somatic cells into embryonic stem (ES)-like cells. These cells, which are referred to as induced pluripotent stem (iPS) cells, closely resemble embryonic stem cells in genomic, cell biologic, and phenotypic characteristics, and the creation of these special cells was a major triumph in cell biology. In contrast to pluripotent stem cells generated by somatic cell nuclear-transfer (SCNT) or ES cells derived from the inner cell mass (ICM) of the blastocyst, direct reprogramming provides a convenient and reliable means of generating pluripotent stem cells. iPS cells have already shown incredible potential for research and for therapeutic applications in regenerative medicine within just a few years of their discovery. In this review, current techniques of generating iPS cells and mechanisms of nuclear reprogramming are reviewed, and the potential for therapeutic applications is discussed. Antioxid. Redox Signal. 15, 1799–1820. PMID:21194386

  5. Cloning animals by somatic cell nuclear transfer – biological factors

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    Enright Brian

    2003-11-01

    Full Text Available Abstract Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other specie, this review will be focused on somatic cell cloning of cattle.

  6. Nuclear microscopy of sperm cell elemental structure

    Energy Technology Data Exchange (ETDEWEB)

    Bench, G.S.; Balhorn, R.; Friz, A.M.; Freeman, S.P.H.T.

    1994-09-28

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility.

  7. Reprogramming of Aged Cells into Pluripotent Stem Cells by Nuclear Transfer.

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    Wu, Dan-Ya; Zhang, Xia; Miao, Yi-Liang

    2018-03-07

    Stem cells have the potential to differentiate into specialized cell types under specific conditions in vivo or in vitro, which are used to cure many diseases related to aging. Somatic cell nuclear transfer (SCNT) can reprogram differential somatic cells into cloned embryos and embryonic stem cells can be derived from these cloned embryos. Recipient oocytes have healthier mitochondria and can improve the metabolism competence, lessen the ROS damage, and rejuvenate mitochondrial function of aged cells during reprogramming. Here, we describe a protocol to isolate aged somatic cells and reprogram them into embryonic stem cells by SCNT. These stem cells can be used to differentiate into regenerative somatic cells and replace the aged cells.

  8. Nuclear DNA Content Varies with Cell Size across Human Cell Types

    Science.gov (United States)

    Gillooly, James F.; Hein, Andrew; Damiani, Rachel

    2015-01-01

    Variation in the size of cells, and the DNA they contain, is a basic feature of multicellular organisms that affects countless aspects of their structure and function. Within humans, cell size is known to vary by several orders of magnitude, and differences in nuclear DNA content among cells have been frequently observed. Using published data, here we describe how the quantity of nuclear DNA across 19 different human cell types increases with cell volume. This observed increase is similar to intraspecific relationships between DNA content and cell volume in other species, and interspecific relationships between diploid genome size and cell volume. Thus, we speculate that the quantity of nuclear DNA content in somatic cells of humans is perhaps best viewed as a distribution of values that reflects cell size distributions, rather than as a single, immutable quantity. PMID:26134319

  9. Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Tonatiuh Barrios-García

    2016-06-01

    Full Text Available Tristetraprolin (TTP is a 34-kDa, zinc finger-containing factor that in mammalian cells acts as a tumor suppressor protein through two different mechanisms. In the cytoplasm TTP promotes the decay of hundreds of mRNAs encoding cell factors involved in inflammation, tissue invasion, and metastasis. In the cell nucleus TTP has been identified as a transcriptional corepressor of the estrogen receptor alpha (ERα, which has been associated to the development and progression of the majority of breast cancer tumors. In this work we report that nuclear TTP modulates the transactivation activity of progesterone receptor (PR, glucocorticoid receptor (GR and androgen receptor (AR. In recent years these steroid nuclear receptors have been shown to be of clinical and therapeutical relevance in breast cancer. The functional association between TTP and steroid nuclear receptors is supported by the finding that TTP physically interacts with ERα, PR, GR and AR in vivo. We also show that TTP overexpression attenuates the transactivation of all the steroid nuclear receptors tested. In contrast, siRNA-mediated reduction of endogenous TTP expression in MCF-7 cells produced an increase in the transcriptional activities of ERα, PR, GR and AR. Taken together, these results suggest that the function of nuclear TTP in breast cancer cells is to act as a corepressor of ERα, PR, GR and AR. We propose that the reduction of TTP expression observed in different types of breast cancer tumors may contribute to the development of this disease by producing a dysregulation of the transactivation activity of multiple steroid nuclear receptors.

  10. A nuclear glutathione cycle within the cell cycle.

    Science.gov (United States)

    Diaz Vivancos, Pedro; Wolff, Tonja; Markovic, Jelena; Pallardó, Federico V; Foyer, Christine H

    2010-10-15

    The complex antioxidant network of plant and animal cells has the thiol tripeptide GSH at its centre to buffer ROS (reactive oxygen species) and facilitate cellular redox signalling which controls growth, development and defence. GSH is found in nearly every compartment of the cell, including the nucleus. Transport between the different intracellular compartments is pivotal to the regulation of cell proliferation. GSH co-localizes with nuclear DNA at the early stages of proliferation in plant and animal cells. Moreover, GSH recruitment and sequestration in the nucleus during the G1- and S-phases of the cell cycle has a profound impact on cellular redox homoeostasis and on gene expression. For example, the abundance of transcripts encoding stress and defence proteins is decreased when GSH is sequestered in the nucleus. The functions of GSHn (nuclear GSH) are considered in the present review in the context of whole-cell redox homoeostasis and signalling, as well as potential mechanisms for GSH transport into the nucleus. We also discuss the possible role of GSHn as a regulator of nuclear proteins such as histones and PARP [poly(ADP-ribose) polymerase] that control genetic and epigenetic events. In this way, a high level of GSH in the nucleus may not only have an immediate effect on gene expression patterns, but also contribute to how cells retain a memory of the cellular redox environment that is transferred through generations.

  11. Cell-fusion method to visualize interphase nuclear pore formation.

    Science.gov (United States)

    Maeshima, Kazuhiro; Funakoshi, Tomoko; Imamoto, Naoko

    2014-01-01

    In eukaryotic cells, the nucleus is a complex and sophisticated organelle that organizes genomic DNA to support essential cellular functions. The nuclear surface contains many nuclear pore complexes (NPCs), channels for macromolecular transport between the cytoplasm and nucleus. It is well known that the number of NPCs almost doubles during interphase in cycling cells. However, the mechanism of NPC formation is poorly understood, presumably because a practical system for analysis does not exist. The most difficult obstacle in the visualization of interphase NPC formation is that NPCs already exist after nuclear envelope formation, and these existing NPCs interfere with the observation of nascent NPCs. To overcome this obstacle, we developed a novel system using the cell-fusion technique (heterokaryon method), previously also used to analyze the shuttling of macromolecules between the cytoplasm and the nucleus, to visualize the newly synthesized interphase NPCs. In addition, we used a photobleaching approach that validated the cell-fusion method. We recently used these methods to demonstrate the role of cyclin-dependent protein kinases and of Pom121 in interphase NPC formation in cycling human cells. Here, we describe the details of the cell-fusion approach and compare the system with other NPC formation visualization methods. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Somatic cell nuclear transfer: pros and cons.

    Science.gov (United States)

    Sumer, Huseyin; Liu, Jun; Tat, Pollyanna; Heffernan, Corey; Jones, Karen L; Verma, Paul J

    2009-01-01

    Even though the technique of mammalian SCNT is just over a decade old it has already resulted in numerous significant advances. Despite the recent advances in the reprogramming field, SCNT remains the bench-mark for the generation of both genetically unmodified autologous pluripotent stem cells for transplantation and for the production of cloned animals. In this review we will discuss the pros and cons of SCNT, drawing comparisons with other reprogramming methods.

  13. Evaluation of porcine stem cells competence for somatic cell nuclear transfer and production of cloned animals

    DEFF Research Database (Denmark)

    Secher, Jan; Liu, Ying; Petkov, Stoyan

    2017-01-01

    Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer tha...

  14. Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Jennifer A Mitchell

    Full Text Available In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.

  15. Nuclear RNA Sequencing of the Mouse Erythroid Cell Transcriptome

    Science.gov (United States)

    Umlauf, David; Chen, Chih-yu; Moir, Catherine A.; Eskiw, Christopher H.; Schoenfelder, Stefan; Chakalova, Lyubomira; Nagano, Takashi; Fraser, Peter

    2012-01-01

    In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq) in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq) of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A)-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs. PMID:23209567

  16. Nuclear myosin I regulates cell membrane tension

    Czech Academy of Sciences Publication Activity Database

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, M.; Hozák, Pavel

    2016-01-01

    Roč. 6, AUG 2 (2016), č. článku 30864. ISSN 2045-2322 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA ČR GAP305/11/2232; GA MŠk(CZ) LO1304 Institutional support: RVO:68378050 Keywords : neuronal growth cone * rna-polymerase-ii * cancer cells * phosphatidylinositol 4,5-bisphosphate * myo1c * actin * transcription * complex * motor * afm Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.259, year: 2016

  17. Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

    International Nuclear Information System (INIS)

    Fang, Zhen F.; Gai, Hui; Huang, You Z.; Li, Shan G.; Chen, Xue J.; Shi, Jian J.; Wu, Li; Liu, Ailian; Xu, Ping; Sheng, Hui Z.

    2006-01-01

    Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines

  18. Nuclear ferritin in corneal epithelial cells: tissue-specific nuclear transport and protection from UV-damage.

    Science.gov (United States)

    Linsenmayer, Thomas F; Cai, Cindy X; Millholland, John M; Beazley, Kelly E; Fitch, John M

    2005-03-01

    We have identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form that is indistinguishable from the cytoplasmic form of ferritin found in other cell types. Thus it most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and other tissues were UV irradiated, and damage to DNA was detected by an in situ 3'-end labeling assay. Consistent with the hypothesis, corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than the other cells types examined. However, when the expression of nuclear ferritin was inhibited the cells now became much more susceptible to UV-induced DNA damage. Since ferritin is normally cytoplasmic, corneal epithelial cells must have a mechanism that effects its nuclear localization. We have determined that this involves a nuclear transport molecule which binds to ferritin and carries it into the nucleus. This transporter, which we have termed ferritoid for its similarity to ferritin, has at least two domains. One domain is ferritin-like and is responsible for binding the ferritin; the other domain contains a nuclear localization signal that is responsible for effecting the nuclear transport. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage. In addition, since ferritoid is structurally similar to ferritin, it may represent an example of a nuclear transporter that evolved from the molecule it transports (i.e., ferritin).

  19. A combined gas cooled nuclear reactor and fuel cell cycle

    Science.gov (United States)

    Palmer, David J.

    Rising oil costs, global warming, national security concerns, economic concerns and escalating energy demands are forcing the engineering communities to explore methods to address these concerns. It is the intention of this thesis to offer a proposal for a novel design of a combined cycle, an advanced nuclear helium reactor/solid oxide fuel cell (SOFC) plant that will help to mitigate some of the above concerns. Moreover, the adoption of this proposal may help to reinvigorate the Nuclear Power industry while providing a practical method to foster the development of a hydrogen economy. Specifically, this thesis concentrates on the importance of the U.S. Nuclear Navy adopting this novel design for its nuclear electric vessels of the future with discussion on efficiency and thermodynamic performance characteristics related to the combined cycle. Thus, the goals and objectives are to develop an innovative combined cycle that provides a solution to the stated concerns and show that it provides superior performance. In order to show performance, it is necessary to develop a rigorous thermodynamic model and computer program to analyze the SOFC in relation with the overall cycle. A large increase in efficiency over the conventional pressurized water reactor cycle is realized. Both sides of the cycle achieve higher efficiencies at partial loads which is extremely important as most naval vessels operate at partial loads as well as the fact that traditional gas turbines operating alone have poor performance at reduced speeds. Furthermore, each side of the cycle provides important benefits to the other side. The high temperature exhaust from the overall exothermic reaction of the fuel cell provides heat for the reheater allowing for an overall increase in power on the nuclear side of the cycle. Likewise, the high temperature helium exiting the nuclear reactor provides a controllable method to stabilize the fuel cell at an optimal temperature band even during transients helping

  20. Daughter-cell-specific modulation of nuclear pore complexes controls cell cycle entry during asymmetric division.

    Science.gov (United States)

    Kumar, Arun; Sharma, Priyanka; Gomar-Alba, Mercè; Shcheprova, Zhanna; Daulny, Anne; Sanmartín, Trinidad; Matucci, Irene; Funaya, Charlotta; Beato, Miguel; Mendoza, Manuel

    2018-04-01

    The acquisition of cellular identity is coupled to changes in the nuclear periphery and nuclear pore complexes (NPCs). Whether and how these changes determine cell fate remain unclear. We have uncovered a mechanism that regulates NPC acetylation to direct cell fate after asymmetric division in budding yeast. The lysine deacetylase Hos3 associates specifically with daughter cell NPCs during mitosis to delay cell cycle entry (Start). Hos3-dependent deacetylation of nuclear basket and central channel nucleoporins establishes daughter-cell-specific nuclear accumulation of the transcriptional repressor Whi5 during anaphase and perinuclear silencing of the G1/S cyclin gene CLN2 in the following G1 phase. Hos3-dependent coordination of both events restrains Start in daughter, but not in mother, cells. We propose that deacetylation modulates transport-dependent and transport-independent functions of NPCs, leading to differential cell cycle progression in mother and daughter cells. Similar mechanisms might regulate NPC functions in specific cell types and/or cell cycle stages in multicellular organisms.

  1. Design of nuclear cells with re linking of trajectories

    International Nuclear Information System (INIS)

    Castillo, A.; Campos S, Y.; Ortiz S, J.J.; Montes, J.L.; Perrusquia, R.; Hernandez, J.L.; Torres, M.

    2006-01-01

    Presently work the results obtained with the Ohtli-RT system obtained when implementing the combinatory optimization technique well-known as Trajectories re linking or Path Re linking in English. The problem to solve is the radial design of nuclear fuel, taking like base nuclear fuel assembles for boiling water reactors (BWR Boiling Water Reactor by its initials in English). To evaluate the objective function used in the system the code in two dimensions Heliums 1.5 was used, which calculates the cross sections of the proposed design. The parameters that were considered for the evaluation of the objective function are the Power peak factor of the bar that generates bigger power in the cell and the Infinite Multiplication Factor. To prove the system its were used assembles 10x10 with 2 water channels. The obtained radial designs of nuclear fuel fulfilled the restrictions imposed to the considered limits, with regard to the involved parameters. (Author)

  2. Nuclear envelope and genome interactions in cell fate

    Science.gov (United States)

    Talamas, Jessica A.; Capelson, Maya

    2015-01-01

    The eukaryotic cell nucleus houses an organism’s genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate. PMID:25852741

  3. Recent advancements in cloning by somatic cell nuclear transfer.

    Science.gov (United States)

    Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

    2013-01-05

    Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

  4. Cloning of ES cells and mice by nuclear transfer.

    Science.gov (United States)

    Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

    2009-01-01

    We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning.

  5. β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

    Science.gov (United States)

    Sui, Lina; Danzl, Nichole; Campbell, Sean R; Viola, Ryan; Williams, Damian; Xing, Yuan; Wang, Yong; Phillips, Neil; Poffenberger, Greg; Johannesson, Bjarki; Oberholzer, Jose; Powers, Alvin C; Leibel, Rudolph L; Chen, Xiaojuan; Sykes, Megan; Egli, Dieter

    2018-01-01

    β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy. © 2017 by the American Diabetes Association.

  6. Coexpression analysis identifies nuclear reprogramming barriers of somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Zuo, Yongchun; Su, Guanghua; Cheng, Lei; Liu, Kun; Feng, Yu; Wei, Zhuying; Bai, Chunling; Cao, Guifang; Li, Guangpeng

    2017-09-12

    The success of cloned animal "Dolly Sheep" demonstrated the somatic cell nuclear transfer (SCNT) technique holds huge potentials for mammalian asexual reproduction. However, the extremely poor development of SCNT embryos indicates their molecular mechanism remain largely unexplored. Deciphering the spatiotemporal patterns of gene expression in SCNT embryos is a crucial step toward understanding the mechanisms associated with nuclear reprogramming. In this study, a valuable transcriptome recourse of SCNT embryos was firstly established, which derived from different inter-/intra donor cells. The gene co-expression analysis identified 26 cell-specific modules, and a series of regulatory pathways related to reprogramming barriers were further enriched. Compared to the intra-SCNT embryos, the inter-SCNT embryos underwent only complete partially reprogramming. As master genome trigger genes, the transcripts related to TFIID subunit, RNA polymerase and mediators were incomplete activated in inter-SCNT embryos. The inter-SCNT embryos only wasted the stored maternal mRNA of master regulators, but failed to activate their self-sustained pathway of RNA polymerases. The KDM family of epigenetic regulator also seriously delayed in inter-SCNT embryo reprogramming process. Our study provided new insight into understanding of the mechanisms of nuclear reprogramming.

  7. Applications of large cell remote handling techniques in nuclear plants

    International Nuclear Information System (INIS)

    Issel, W.; Leister, P.

    1989-01-01

    A comprehensive demonstration project in a special remote handling test facility was performed in parallel to the design of, and the basic engineering work for, the planned reprocessing plant at Wackersdorf. The aim of this project was to demonstrate the feasibility of a completely remote maintenance of the components of the PUREX process. These components were to be arranged as modules in large cells. Remote handling transporters, manipulators and tools (FEMO) for preplanned and unscheduled repair work were constructed and tested. The results of the successful demonstration project are summarized, and potential applications of the remote handling tools in hot cells and other nuclear plants are outlined. (orig./HP) [de

  8. Cloning Endangered Felids by Interspecies Somatic Cell Nuclear Transfer.

    Science.gov (United States)

    Gómez, Martha C; Pope, C Earle

    2015-01-01

    In 2003, the first wild felid was produced by interspecies somatic cell nuclear transfer. Since then other wild felid clone offspring have been produced by using the same technique with minor modifications. This chapter describes detailed protocols used in our laboratory for (1) the isolation, culture, and preparation of fibroblast cells as donor nucleus, and (2) embryo reconstruction with domestic cat enucleated oocytes to produce cloned embryos that develop to the blastocyst stage in vitro and, after transfer into synchronized recipients, establish successful pregnancies.

  9. ERK5 and cell proliferation: nuclear localization is what matters

    Directory of Open Access Journals (Sweden)

    Nestor Gomez

    2016-09-01

    Full Text Available ERK5, the last MAP kinase family member discovered, is activated by the upstream kinase MEK5 in response to growth factors and stress stimulation. MEK5-ERK5 pathway has been associated to different cellular processes, playing a crucial role in cell proliferation in normal and cancer cells by mechanisms that are both dependent and independent of its kinase activity. Thus, nuclear ERK5 activates transcription factors by either direct phosphorylation or acting as co-activator thanks to a unique transcriptional activation TAD domain located at its C-terminal tail. Consequently, ERK5 has been proposed as an interesting target to tackle different cancers, and either inhibitors of ERK5 activity or silencing the protein have shown antiproliferative activity in cancer cells and to block tumour growth in animal models. Here, we review the different mechanisms involved in ERK5 nuclear translocation and their consequences. Inactive ERK5 resides in the cytosol, forming a complex with Hsp90-Cdc37 superchaperone. In a canonical mechanism, MEK5-dependent activation results in ERK5 C-terminal autophosphorylation, Hsp90 dissociation and nuclear translocation. This mechanism integrates signals such as growth factors and stresses that activate the MEK5-ERK5 pathway. Importantly, two other mechanisms, MEK5-independent, have been recently described. These mechanisms allow nuclear shuttling of kinase-inactive forms of ERK5. Although lacking kinase activity, these forms activate transcription by interacting with transcription factors through the TAD domain. Both mechanisms also require Hsp90 dissociation previous to nuclear translocation. One mechanism involves phosphorylation of the C-terminal tail of ERK5 by kinases that are activated during mitosis, such as Cyclin-dependent kinase-1. The second mechanism involves overexpression of chaperone Cdc37, an oncogene that is overexpressed in cancers such as prostate adenocarcinoma, where it collaborates with ERK5 to promote

  10. Nuclear microprobe imaging of gallium nitrate in cancer cells

    International Nuclear Information System (INIS)

    Ortega, Richard; Suda, Asami; Deves, Guillaume

    2003-01-01

    Gallium nitrate is used in clinical oncology as treatment for hypercalcemia and for cancer that has spread to the bone. Its mechanism of antitumor action has not been fully elucidated yet. The knowledge of the intracellular distribution of anticancer drugs is of particular interest in oncology to better understand their cellular pharmacology. In addition, most metal-based anticancer compounds interact with endogenous trace elements in cells, altering their metabolism. The purpose of this experiment was to examine, by use of nuclear microprobe analysis, the cellular distribution of gallium and endogenous trace elements within cancer cells exposed to gallium nitrate. In a majority of cellular analyses, gallium was found homogeneously distributed in cells following the distribution of carbon. In a smaller number of cells, however, gallium appeared concentrated together with P, Ca and Fe within round structures of about 2-5 μm diameter located in the perinuclear region. These intracellular structures are typical of lysosomial material

  11. Nuclear reprogramming of cancer stem cells: Corrupting the epigenetic code of cell identity with oncometabolites.

    Science.gov (United States)

    Menendez, Javier A; Alarcón, Tomás

    2016-01-01

    Generation of cancer stem cell (CSC)-like cells might occur through metabolic corruption of the epigenetic codes that govern cell identity. We recently identified how archetypal oncometabolites, without altering the baseline expression of endogenous stem cell maintenance genes but endowing cells with epigenetic states refractory to differentiation, considerably enhance the global kinetic efficiency of nuclear reprogramming processes that generate CSC-like states de novo . This study highlights that metabolo-epigenetic axes of communication can direct the development and maintenance of CSCs during the natural history of cancer diseases.

  12. Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is Involved in Cell Cycle Progression.

    Science.gov (United States)

    Poli, Alessandro; Fiume, Roberta; Baldanzi, Gianluca; Capello, Daniela; Ratti, Stefano; Gesi, Marco; Manzoli, Lucia; Graziani, Andrea; Suh, Pann-Ghill; Cocco, Lucio; Follo, Matilde Y

    2017-09-01

    Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550-2557, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Nuclear morphometry in canine acanthomatous ameloblastomas and squamous cell carcinomas

    Directory of Open Access Journals (Sweden)

    M Martano

    2009-06-01

    Full Text Available The aim of this study was to evaluate whether morphometrical analysis can be of diagnostic value for canine acanthomatous ameloblastoma. We calculated, by means of an automated image analyser, some morphometric nuclear parameters, in particular: mean nuclear area (MNA, mean nuclear perimeter (MNP, maximum and minimum diameters (MDx and MDm coefficient of variation of the nuclear area (NACV, largest to smallest dimension ratio (LS ratio, and form factor (FF, in 8 canine acanthomatous ameloblastomas, and we compared these morphometric data to those of 13 squamous cell carcinomas of canine gingiva. The results indicated a progressive increase of the MNA, NACV, MNP and MDm proceeding from acanthomatous ameloblastomas (MNA: 42.11±8.74; NACV: 28,36±7,23; MNP: 24.18± 2.68; MDm: 5.69±0.49 to squamous cell carcinomas (MNA:49,69±9,10; NACV: 30,89±7,75; MNP: 25.63±2.54; MDm: 6.64±0.73. On the contrary, the LS ratio and the FF resulted greater in acanthomatous ameloblastomas (LS ratio: 1,63±0,12; FF: 1,13±0,002 than in SCCs (LS ratio: 1,40±0,12; FF:0.91±0.38. Moreover, the MNA, MNP,MDx and MDm resulted similar (MNA: p=0.89; MNP: p=0,65; MDm: p=0,16; MDx: p=0,13 in a subset of four acanthomatous ameloblastomas with cellular atypia (MNA:49,01±6,88; MNP: 26,28±1,99; MDm: 6.08±0.41; MDx: 10.18±0.88 and in squamous cell carcinomas (MNA:49.69±9,10; MNP: 25.63±2.54; MDm: 6.64±0.73; MDx: 9.26±1.05. While the NACV values resulted higher in typical acanthomatous ameloblastoma (29,99±6,06 than in atypical acanthomatous ameloblastoma (26,74±8,84 and similar to those of the SCCs (30,89±7,75. These results seem to confirm that acanthomatous ameloblastoma is a malignant or potentially malignant lesion and emphasizes that nuclear morphometry analysis can be an useful diagnostic and prognostic method in canine oral pathology.

  14. Data mining in the study of nuclear fuel cells

    International Nuclear Information System (INIS)

    Medina P, J. A.; Ortiz S, J. J.; Castillo, A.; Montes T, J. L.; Perusquia, R.

    2015-09-01

    In this paper is presented a study of data mining application in the analysis of fuel cells and their performance within a nuclear boiling water reactor. A decision tree was used to fulfill questions of the type If (condition) and Then (conclusion) to classify if the fuel cells will have good performance. The performance is measured by compliance or not of the cold shutdown margin, the rate of linear heat generation and the average heat generation in a plane of the reactor. It is assumed that the fuel cells are simulated in the reactor under a fuel reload and rod control patterns pre designed. 18125 fuel cells were simulated according to a steady-state calculation. The decision tree works on a target variable which is one of the three mentioned before. To analyze this objective, the decision tree works with a set of attribute variables. In this case, the attributes are characteristics of the cell as number of gadolinium rods, rods number with certain uranium enrichment mixed with a concentration of gadolinium, etc. The found model was able to predict the execution or not of the shutdown margin with a precision of around 95%. However, the other two variables showed lower percentages due to few learning cases of the model in which these variables were or were not achieved. Even with this inconvenience, the model is quite reliable and can be used in way coupled in optimization systems of fuel cells. (Author)

  15. Nuclear Reprogramming in Mouse Primordial Germ Cells: Epigenetic Contribution

    Directory of Open Access Journals (Sweden)

    Massimo De Felici

    2011-01-01

    Full Text Available The unique capability of germ cells to give rise to a new organism, allowing the transmission of primary genetic information from generation to generation, depends on their epigenetic reprogramming ability and underlying genomic totipotency. Recent studies have shown that genome-wide epigenetic modifications, referred to as “epigenetic reprogramming”, occur during the development of the gamete precursors termed primordial germ cells (PGCs in the embryo. This reprogramming is likely to be critical for the germ line development itself and necessary to erase the parental imprinting and setting the base for totipotency intrinsic to this cell lineage. The status of genome acquired during reprogramming and the associated expression of key pluripotency genes render PGCs susceptible to transform into pluripotent stem cells. This may occur in vivo under still undefined condition, and it is likely at the origin of the formation of germ cell tumors. The phenomenon appears to be reproduced under partly defined in vitro culture conditions, when PGCs are transformed into embryonic germ (EG cells. In the present paper, I will try to summarize the contribution that epigenetic modifications give to nuclear reprogramming in mouse PGCs.

  16. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  17. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    International Nuclear Information System (INIS)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    2015-01-01

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  18. Characterization of Aes nuclear foci in colorectal cancer cells

    Science.gov (United States)

    Itatani, Yoshiro; Sonoshita, Masahiro; Kakizaki, Fumihiko; Okawa, Katsuya; Stifani, Stefano; Itoh, Hideaki; Sakai, Yoshiharu; Taketo, M. Mark

    2016-01-01

    Amino-terminal enhancer of split (Aes) is a member of Groucho/Transducin-like enhancer (TLE) family. Aes is a recently found metastasis suppressor of colorectal cancer (CRC) that inhibits Notch signalling, and forms nuclear foci together with TLE1. Although some Notch-associated proteins are known to form subnuclear bodies, little is known regarding the dynamics or functions of these structures. Here, we show that Aes nuclear foci in CRC observed under an electron microscope are in a rather amorphous structure, lacking surrounding membrane. Investigation of their behaviour during the cell cycle by time-lapse cinematography showed that Aes nuclear foci dissolve during mitosis and reassemble after completion of cytokinesis. We have also found that heat shock cognate 70 (HSC70) is an essential component of Aes foci. Pharmacological inhibition of the HSC70 ATPase activity with VER155008 reduces Aes focus formation. These results provide insight into the understanding of Aes-mediated inhibition of Notch signalling. PMID:26229111

  19. Inverted nuclear architecture and its development during differentiation of mouse rod photoreceptor cells: a new model to study nuclear architecture.

    Science.gov (United States)

    Solovei, I; Joffe, B

    2010-09-01

    Interphase nuclei have a conserved architecture: heterochromatin occupies the nuclear periphery, whereas euchromatin resides in the nuclear interior. It has recently been found that rod photoreceptor cells of nocturnal mammals have an inverted architecture, which transforms these nuclei in microlenses and supposedly facilitates a reduction in photon loss in the retina. This unique deviation from the nearly universal pattern throws a new light on the nuclear organization. In the article we discuss the implications of the studies of the inverted nuclei for understanding the role of the spatial organization of the nucleus in nuclear functions.

  20. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium from ...

  1. Nuclear import of glucokinase in pancreatic beta-cells is mediated by a nuclear localization signal and modulated by SUMOylation.

    Science.gov (United States)

    Johansson, Bente Berg; Fjeld, Karianne; Solheim, Marie Holm; Shirakawa, Jun; Zhang, Enming; Keindl, Magdalena; Hu, Jiang; Lindqvist, Andreas; Døskeland, Anne; Mellgren, Gunnar; Flatmark, Torgeir; Njølstad, Pål Rasmus; Kulkarni, Rohit N; Wierup, Nils; Aukrust, Ingvild; Bjørkhaug, Lise

    2017-10-15

    The localization of glucokinase in pancreatic beta-cell nuclei is a controversial issue. Although previous reports suggest such a localization, the mechanism for its import has so far not been identified. Using immunofluorescence, subcellular fractionation and mass spectrometry, we present evidence in support of glucokinase localization in beta-cell nuclei of human and mouse pancreatic sections, as well as in human and mouse isolated islets, and murine MIN6 cells. We have identified a conserved, seven-residue nuclear localization signal ( 30 LKKVMRR 36 ) in the human enzyme. Substituting the residues KK 31,32 and RR 35,36 with AA led to a loss of its nuclear localization in transfected cells. Furthermore, our data indicates that SUMOylation of glucokinase modulates its nuclear import, while high glucose concentrations do not significantly alter the enzyme nuclear/cytosolic ratio. Thus, for the first time, we provide data in support of a nuclear import of glucokinase mediated by a redundant mechanism, involving a nuclear localization signal, and which is modulated by its SUMOylation. These findings add new knowledge to the functional role of glucokinase in the pancreatic beta-cell. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Identification of nuclear τ isoforms in human neuroblastoma cells

    International Nuclear Information System (INIS)

    Loomis, P.A.; Howard, T.H.; Castleberry, R.P.; Binder, L.I.

    1990-01-01

    The τ proteins have been reported only in association with microtubules and with ribosomes in situ, in the normal central nervous system. In addition, τ has been shown to be an integral component of paired helical filaments, the principal constituent of the neurofibrillary tangles found in brains of patients with Alzheimer's disease and of most aged individuals with Down syndrome (trisomy 21). The authors report here the localization of the well-characterized Tau-1 monoclonal antibody to the nucleolar organizer regions of the acrocentric chromosomes and to their interphase counterpart, the fibrillar component of the nucleolus, in human neuroblastoma cells. Similar localization to the nucleolar organizer regions was also observed in other human cell lines and in one monkey kidney cell line but was not seen in non-primate species. Immunochemically, they further demonstrated the existence of the entire τ molecule in the isolated nuclei of neuroblastoma cells. Nuclear τ proteins, like the τ proteins of the paired helical filaments, cannot be extracted in standard SDS-containing electrophoresis sample buffer but require pretreatment with formic acid prior to immunoblot analysis. This work indicates that τ may function in processes not directly associated with microtubules and that highly insoluble complexes of τ may also play a role in normal cellular physiology

  3. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    Science.gov (United States)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Telomere Elongation and Naive Pluripotent Stem Cells Achieved from Telomerase Haplo-Insufficient Cells by Somatic Cell Nuclear Transfer

    Directory of Open Access Journals (Sweden)

    Li-Ying Sung

    2014-12-01

    Full Text Available Summary: Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs have been efficiently achieved by somatic cell nuclear transfer (SCNT. We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/− mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/− cells exhibit naive pluripotency as evidenced by generation of Terc+/− ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells. : Sung et al. demonstrate in a mouse model that telomeres of telomerase haplo-insufficient cells can be elongated by somatic cell nuclear transfer. Moreover, ntESCs derived from Terc+/− cells exhibit pluripotency evidenced by generation of Terc+/−ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency.

  5. Global Reorganization of the Nuclear Landscape in Senescent Cells

    Directory of Open Access Journals (Sweden)

    Tamir Chandra

    2015-02-01

    Full Text Available Cellular senescence has been implicated in tumor suppression, development, and aging and is accompanied by large-scale chromatin rearrangements, forming senescence-associated heterochromatic foci (SAHF. However, how the chromatin is reorganized during SAHF formation is poorly understood. Furthermore, heterochromatin formation in senescence appears to contrast with loss of heterochromatin in Hutchinson-Gilford progeria. We mapped architectural changes in genome organization in cellular senescence using Hi-C. Unexpectedly, we find a dramatic sequence- and lamin-dependent loss of local interactions in heterochromatin. This change in local connectivity resolves the paradox of opposing chromatin changes in senescence and progeria. In addition, we observe a senescence-specific spatial clustering of heterochromatic regions, suggesting a unique second step required for SAHF formation. Comparison of embryonic stem cells (ESCs, somatic cells, and senescent cells shows a unidirectional loss in local chromatin connectivity, suggesting that senescence is an endpoint of the continuous nuclear remodelling process during differentiation.

  6. Global reorganization of the nuclear landscape in senescent cells.

    Science.gov (United States)

    Chandra, Tamir; Ewels, Philip Andrew; Schoenfelder, Stefan; Furlan-Magaril, Mayra; Wingett, Steven William; Kirschner, Kristina; Thuret, Jean-Yves; Andrews, Simon; Fraser, Peter; Reik, Wolf

    2015-02-03

    Cellular senescence has been implicated in tumor suppression, development, and aging and is accompanied by large-scale chromatin rearrangements, forming senescence-associated heterochromatic foci (SAHF). However, how the chromatin is reorganized during SAHF formation is poorly understood. Furthermore, heterochromatin formation in senescence appears to contrast with loss of heterochromatin in Hutchinson-Gilford progeria. We mapped architectural changes in genome organization in cellular senescence using Hi-C. Unexpectedly, we find a dramatic sequence- and lamin-dependent loss of local interactions in heterochromatin. This change in local connectivity resolves the paradox of opposing chromatin changes in senescence and progeria. In addition, we observe a senescence-specific spatial clustering of heterochromatic regions, suggesting a unique second step required for SAHF formation. Comparison of embryonic stem cells (ESCs), somatic cells, and senescent cells shows a unidirectional loss in local chromatin connectivity, suggesting that senescence is an endpoint of the continuous nuclear remodelling process during differentiation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  7. A Comprehensive Nuclear Receptor Network for Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ralf Kittler

    2013-02-01

    Full Text Available In breast cancer, nuclear receptors (NRs play a prominent role in governing gene expression, have prognostic utility, and are therapeutic targets. We built a regulatory map for 24 NRs, six chromatin state markers, and 14 breast-cancer-associated transcription factors (TFs that are expressed in the breast cancer cell line MCF-7. The resulting network reveals a highly interconnected regulatory matrix where extensive crosstalk occurs among NRs and other breast -cancer-associated TFs. We show that large numbers of factors are coordinately bound to highly occupied target regions throughout the genome, and these regions are associated with active chromatin state and hormone-responsive gene expression. This network also provides a framework for stratifying and predicting patient outcomes, and we use it to show that the peroxisome proliferator-activated receptor delta binds to a set of genes also regulated by the retinoic acid receptors and whose expression is associated with poor prognosis in breast cancer.

  8. 3D nuclear architecture reveals coupled cell cycle dynamics of chromatin and nuclear pores in the malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Weiner, Allon; Dahan-Pasternak, Noa; Shimoni, Eyal; Shinder, Vera; von Huth, Palle; Elbaum, Michael; Dzikowski, Ron

    2011-07-01

    The deadliest form of human malaria is caused by the protozoan parasite Plasmodium falciparum. The complex life cycle of this parasite is associated with tight transcriptional regulation of gene expression. Nuclear positioning and chromatin dynamics may play an important role in regulating P. falciparum virulence genes. We have applied an emerging technique of electron microscopy to construct a 3D model of the parasite nucleus at distinct stages of development within the infected red blood cell. We have followed the distribution of nuclear pores and chromatin throughout the intra-erythrocytic cycle, and have found a striking coupling between the distributions of nuclear pores and chromatin organization. Pore dynamics involve clustering, biogenesis, and division among daughter cells, while chromatin undergoes stage-dependent changes in packaging. Dramatic changes in heterochromatin distribution coincide with a previously identified transition in gene expression and nucleosome positioning during the mid-to-late schizont phase. We also found a correlation between euchromatin positioning at the nuclear envelope and the local distribution of nuclear pores, as well as a dynamic nuclear polarity during schizogony. These results suggest that cyclic patterns in gene expression during parasite development correlate with gross changes in cellular and nuclear architecture. © 2011 Blackwell Publishing Ltd.

  9. 48 CFR 952.225-70 - Subcontracting for nuclear hot cell services.

    Science.gov (United States)

    2010-10-01

    ... nuclear power plants, generally referred to in the industry as “hot cells.” Nuclear waste, as used in this... hot cell services. 952.225-70 Section 952.225-70 Federal Acquisition Regulations System DEPARTMENT OF ENERGY CLAUSES AND FORMS SOLICITATION PROVISIONS AND CONTRACT CLAUSES Text of Provisions and Clauses 952...

  10. Propagation of elite rescue dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Oh, Hyun Ju; Choi, Jin; Kim, Min Jung; Kim, Geon A; Jo, Young Kwang; Choi, Yoo Bin; Lee, Byeong Chun

    2016-01-01

    The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6-dimethylaminopyridine (DMAP) (SOF-DMAP) or porcine zygote medium (PZM-5) with 1.9 mmol/L DMAP (PZM-DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM-DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF-DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6-DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies. © 2015 Japanese Society of Animal Science.

  11. Somatic cell nuclear transfer cloning: practical applications and current legislation.

    Science.gov (United States)

    Niemann, H; Lucas-Hahn, A

    2012-08-01

    Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved. © 2012 Blackwell Verlag GmbH.

  12. Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Rebekka Müller

    Full Text Available Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM. Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.

  13. Scyl1 Facilitates Nuclear tRNA Export in Mammalian Cells by Acting at the Nuclear Pore Complex

    Science.gov (United States)

    Chafe, Shawn C.

    2010-01-01

    Scyl1 is an evolutionarily conserved N-terminal protein kinase-like domain protein that plays a role in COP1-mediated retrograde protein trafficking in mammalian cells. Furthermore, loss of Scyl1 function has been shown to result in neurodegenerative disorders in mice. Here, we report that Scyl1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery. Like exportin-t, overexpression of Scyl1 restored export of a nuclear export-defective serine amber suppressor tRNA mutant in COS-7 cells. Scyl1 binds tRNA saturably, and associates with the nuclear pore complex by interacting, in part, with Nup98. Scyl1 copurifies with the nuclear tRNA export receptors exportin-t and exportin-5, the RanGTPase, and the eukaryotic elongation factor eEF-1A, which transports aminoacyl-tRNAs to the ribosomes. Scyl1 interacts directly with exportin-t and RanGTP but not with eEF-1A or RanGDP in vitro. Moreover, exportin-t containing tRNA, Scyl1, and RanGTP form a quaternary complex in vitro. Biochemical characterization also suggests that the nuclear aminoacylation-dependent pathway is primarily responsible for tRNA export in mammalian cells. These findings together suggest that Scyl1 participates in the nuclear aminoacylation-dependent tRNA export pathway and may unload aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to eEF-1A. PMID:20505071

  14. The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

    Directory of Open Access Journals (Sweden)

    Vahid Mansouri

    2015-06-01

    Full Text Available Nuclear transfer embryonic stem cells (ntESCs show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB in EB culture medium supplemented with bone morphogenetic protein 4(BMP4 as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.

  15. A Stem Cell-rescue Operation in a Nuclear Devastation

    Directory of Open Access Journals (Sweden)

    Editorial

    2011-01-01

    Full Text Available Dear friends, The aftermath of one of the most devastating post earthquake- tsunamis in the history of mankind, the March 2011 post-earthquake-tsunami-hitnuclear accident in Fukushima, Japan, has brought to fore several questions throughout the world on the safety of nuclearinstallations. Such Calamities,which are far beyond our comprehension, has left all nations with such installations and those planning to expand their nuclear power plant programmes in a dilemma. However, the recent news that the Japanese authorities are considering to harvest and store bone marrow stem cells for transfusion to radiation-exposed workers has ushered in a new ray of hope. The hematopoietic stem cells will be harvested before exposure and in case of bone marrow depletion after radiation exposure; these cells can be immediately retrieved from storage and transfused to the radiation-exposed worker so that they have the potential to re-constitute the entire bone marrow in the patient. This will be a great life-saver and game-changer as bone marrow depletion is one of the major consequences of high levels of radiation exposure. This strategy can be prescribed as a routine procedure in all the countries with such nuclear installations, especially for workers at very high risk of radiation exposure. In future, such preventive strategies should be considered for implementation to not only the workers in the facility, but also the rescue team and the vulnerable population in the radiation zone.The regenerative potential of hematopoietic stem cell (HSC is the key factor to be considered now. This is high time that the studies on optimal in vitro expansion of HSCs and ways of successful engraftment of the transfused cells to a niche of radiation-exposed host environment should be attempted in all earnestness by the regenerative medicine community at present. Natural calamities like this help man to realise that the millions of scientific discoveries and inventions can

  16. Automated analysis of cell migration and nuclear envelope rupture in confined environments.

    Science.gov (United States)

    Elacqua, Joshua J; McGregor, Alexandra L; Lammerding, Jan

    2018-01-01

    Recent in vitro and in vivo studies have highlighted the importance of the cell nucleus in governing migration through confined environments. Microfluidic devices that mimic the narrow interstitial spaces of tissues have emerged as important tools to study cellular dynamics during confined migration, including the consequences of nuclear deformation and nuclear envelope rupture. However, while image acquisition can be automated on motorized microscopes, the analysis of the corresponding time-lapse sequences for nuclear transit through the pores and events such as nuclear envelope rupture currently requires manual analysis. In addition to being highly time-consuming, such manual analysis is susceptible to person-to-person variability. Studies that compare large numbers of cell types and conditions therefore require automated image analysis to achieve sufficiently high throughput. Here, we present an automated image analysis program to register microfluidic constrictions and perform image segmentation to detect individual cell nuclei. The MATLAB program tracks nuclear migration over time and records constriction-transit events, transit times, transit success rates, and nuclear envelope rupture. Such automation reduces the time required to analyze migration experiments from weeks to hours, and removes the variability that arises from different human analysts. Comparison with manual analysis confirmed that both constriction transit and nuclear envelope rupture were detected correctly and reliably, and the automated analysis results closely matched a manual analysis gold standard. Applying the program to specific biological examples, we demonstrate its ability to detect differences in nuclear transit time between cells with different levels of the nuclear envelope proteins lamin A/C, which govern nuclear deformability, and to detect an increase in nuclear envelope rupture duration in cells in which CHMP7, a protein involved in nuclear envelope repair, had been depleted

  17. Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig

    Directory of Open Access Journals (Sweden)

    Richter Anne

    2012-11-01

    Full Text Available Abstract Background Somatic cell nuclear transfer (SCNT is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination. Results PKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs and porcine ear fibroblasts (PEFs could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the NucleofectorTM technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT. Conclusion The high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT.

  18. Nanosecond pulsed electric field induced cytoskeleton, nuclear membrane and telomere damage adversely impact cell survival.

    Science.gov (United States)

    Stacey, M; Fox, P; Buescher, S; Kolb, J

    2011-10-01

    We investigated the effects of nanosecond pulsed electric fields (nsPEF) on three human cell lines and demonstrated cell shrinkage, breakdown of the cytoskeleton, nuclear membrane and chromosomal telomere damage. There was a differential response between cell types coinciding with cell survival. Jurkat cells showed cytoskeleton, nuclear membrane and telomere damage that severely impacted cell survival compared to two adherent cell lines. Interestingly, disruption of the actin cytoskeleton in adherent cells prior to nsPEF exposure significantly reduced cell survival. We conclude that nsPEF applications are able to induce damage to the cytoskeleton and nuclear membrane. Telomere sequences, regions that tether and stabilize DNA to the nuclear membrane, are severely compromised as measured by a pan-telomere probe. Internal pore formation following nsPEF applications has been described as a factor in induced cell death. Here we suggest that nsPEF induced physical changes to the cell in addition to pore formation need to be considered as an alternative method of cell death. We suggest nsPEF electrochemical induced depolymerization of actin filaments may account for cytoskeleton and nuclear membrane anomalies leading to sensitization. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Birth of Beagle dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Hossein, Mohammad Shamim; Jeong, Yeon Woo; Park, Sun Woo; Kim, Joung Joo; Lee, Eugine; Ko, Kyeong Hee; Hyuk, Park; Hoon, Song Seung; Kim, Yeun Wook; Hyun, Sang Hwan; Shin, Taeyoung; Hwang, Woo Suk

    2009-09-01

    The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 micros electrical stimulation, chemically activated after 1h of fusion using 10 microM calcium ionophore for 4 min and cultured 4h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4-10, 11-25 and 26-40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P>0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4-10 embryos (n=7) and 26-40 embryos (n=12) while pregnancy was detected in 23.53% recipients received a group of 11-25 embryos (n=17). Among them, five (1.76%) live puppies were born (P<0.05). These data show an increase in the overall efficiency of SCNT in canine species.

  20. [Program optimization before enucleation on ovine somatic cell nuclear transfer].

    Science.gov (United States)

    Guo, Yanhua; Zhang, Yiyuan; Wang, Limin; Tang, Hong; Li, Yingli; Zhou, Ping

    2017-05-25

    Ovine somatic cell nuclear transfer (SCNT) efficiency remains lower. Therefore, we optimized the program before oocyte enucleation on ovine SCNT. Four experiments were done including exposure duration of ovaries (3 h or 3 to 5 h), duration of oocytes maturation (18 h and 24 h), rate of donor adherent and enucleation time of maturate oocyte. The maturation rates of oocyte, fusion rates and cleavation rates of cloned embryos were used to assess the efficiency of different procedures. The maturation rates of ovaries with 3 h exposure was higher than that of 3 to 5 h (60.18% vs 52.50%) (P0.05). The maturation rates were significantly different between group18 h and 24 h (53.81% vs 89.06%, P0.05); fusion rates of donor adherent 30% group was higher than that of 10% group. Embryonic development competence had no significant difference (P>0.05). Different adherent donor characterizes the difference in plateau phase. The cleavation rates of 18 hpm group was higher than that of 16 hpm group. Embryonic development competence had no significant difference (P>0.05), the enucleation of 16 hpm group obtained one clone fetus, we got four clone fetus to repeat the 16 hpm group. Five microsatellite was analyzed by PAGE, the bands indicated that fingerprint of cloned fetus were completely the same as those of donor cells. Our data therefore suggests program optimization before enucleation assurance quality of material which be able to improve the quantity and quality of clone embryos, and optimized scheme can obtain clone sheep offspring.

  1. [Program optimization for bovine somatic cells nuclear transfer].

    Science.gov (United States)

    Lei, Anmin; Ma, Xiaoling; Gao, Zhimin; Hu, Yongce; Sui, Jinqiang; Huang, Weiwei; Zan, Linsen; Dou, Zhongying

    2009-09-01

    To optimize program of bovine somatic nuclear transfer, we used two different enucleation procedures (by Spindle-view system & Hoechst 33342 staining), two different procedures to introduce donor nuclei (by ooplasm microinjection & electrofusion), and three different group electrofusion parameters (group 1: 1.9 kV/cm, 10 micros, two; group 2: 1.5 kV/cm, 25 micros, two; group 3: 0.6 kV/cm, 100 micros, one) to reconstruct bovine cloned embryos. The cleavation rates and blastocyst development rates of cloned embryos were used to assess the efficiency of different operational procedure. Finally, the best combination of operational procedure, that the spindle-viewer system was used for oocytes enucleating, and donor cell was electrofused into ooplasm by electrical pulse (1.9 kV/cm, 10 micros, two) to reconstruct bovine cloned embryos. Then the excellent blastocysts were transferred to fosters for producing cloned cattle 80 high-quality cloned blastocysts were transferred into 33 fosters, two cloned calves were produced. According to the results, the optimized program could be used to produce cloned cattle.

  2. Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer.

    Science.gov (United States)

    Wu, Zhenfang; Xu, Zhiqian; Zou, Xian; Zeng, Fang; Shi, Junsong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Li, Zicong

    2013-12-01

    The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2% of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.

  3. Nuclear architecture of rod photoreceptor cells adapts to vision in mammalian evolution.

    Science.gov (United States)

    Solovei, Irina; Kreysing, Moritz; Lanctôt, Christian; Kösem, Süleyman; Peichl, Leo; Cremer, Thomas; Guck, Jochen; Joffe, Boris

    2009-04-17

    We show that the nuclear architecture of rod photoreceptor cells differs fundamentally in nocturnal and diurnal mammals. The rods of diurnal retinas possess the conventional architecture found in nearly all eukaryotic cells, with most heterochromatin situated at the nuclear periphery and euchromatin residing toward the nuclear interior. The rods of nocturnal retinas have a unique inverted pattern, where heterochromatin localizes in the nuclear center, whereas euchromatin, as well as nascent transcripts and splicing machinery, line the nuclear border. The inverted pattern forms by remodeling of the conventional one during terminal differentiation of rods. The inverted rod nuclei act as collecting lenses, and computer simulations indicate that columns of such nuclei channel light efficiently toward the light-sensing rod outer segments. Comparison of the two patterns suggests that the conventional architecture prevails in eukaryotic nuclei because it results in more flexible chromosome arrangements, facilitating positional regulation of nuclear functions.

  4. SUMOylation regulates the nuclear mobility of CREB binding protein and its association with nuclear bodies in live cells

    International Nuclear Information System (INIS)

    Ryan, Colm M.; Kindle, Karin B.; Collins, Hilary M.; Heery, David M.

    2010-01-01

    The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP-CBPΔ998-1087 had a retarded recovery time in the nucleus, as compared to YFP-CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.

  5. SUMOylation regulates the nuclear mobility of CREB binding protein and its association with nuclear bodies in live cells

    Energy Technology Data Exchange (ETDEWEB)

    Ryan, Colm M.; Kindle, Karin B.; Collins, Hilary M. [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom); Heery, David M., E-mail: david.heery@nottingham.ac.uk [Gene Regulation Group, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom)

    2010-01-01

    The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP-CBP{Delta}998-1087 had a retarded recovery time in the nucleus, as compared to YFP-CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.

  6. Extensive Nuclear Reprogramming Underlies Lineage Conversion into Functional Trophoblast Stem-like Cells

    NARCIS (Netherlands)

    Benchetrit, Hana; Herman, Shay; van Wietmarschen, Niek; Wu, Tao; Makedonski, Kirill; Maoz, Noam; Tov, Nataly Yom; Stave, Danielle; Lasry, Rachel; Zayat, Valery; Xiao, Andrew; Lansdorp, Peter M.; Sebban, Shulamit; Buganim, Yosef

    2015-01-01

    Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often

  7. Multi-tasking Sulf1/Sulf2 enzymes do not only facilitate extracellular cell signalling but also participate in cell cycle related nuclear events.

    Science.gov (United States)

    Krishnakumar, Kavithanjali; Chakravorty, Ishani; Foy, Wendy; Allen, Steve; Justo, Tiago; Mukherjee, Abir; Dhoot, Gurtej K

    2018-03-01

    This study demonstrates highly dynamic spatial and temporal pattern of SULF1/SULF2 expression in a number of neuronal cell types growing in normal culture medium that included their transient nuclear mobilisation. Their nuclear translocation became particularly apparent during cell proliferation as both SULF1/SULF2 demonstrated not only cell membrane associated expression, their known site of function but also transient nuclear mobilisation during nuclear cell division. Nuclear localisation was apparent not only by immunocytochemical staining but also confirmed by immunoblotting staining of isolated nuclear fractions of C6, U87 and N2A cells. Immunocytochemical analysis demonstrated rapid nuclear exit of both SULF1/SULF2 following cell division that was slightly delayed but not blocked in a fraction of the polyploid cells observed in C6 cells. The overexpression of both Sulf1 and Sulf2 genes in C6 and U87 cells markedly promoted in vitro growth of these cells accompanied by nuclear mobilisation while inhibition of both these genes inhibited cell proliferation with little or no nuclear SULF1/SULF2 mobilisation. SULF1/SULF2 activity in these cells thus demonstrated a clear co-ordination of extracellular cell signalling with nuclear events related to cell proliferation. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

  8. Nuclear thioredoxin-1 is required to suppress cisplatin-mediated apoptosis of MCF-7 cells

    International Nuclear Information System (INIS)

    Chen, Xiao-Ping; Liu, Shou; Tang, Wen-Xin; Chen, Zheng-Wang

    2007-01-01

    Different cell line with increased thioredoxin-1 (Trx-1) showed a decreased or increased sensitivity to cell killing by cisplatin. Recently, several studies found that the subcellular localization of Trx-1 is closely associated with its functions. In this study, we explored the association of the nuclear Trx-1 with the cisplatin-mediated apoptosis of breast cancer cells MCF-7. Firstly, we found that higher total Trx-1 accompanied by no change of nuclear Trx-1 can not influence apoptosis induced by cisplatin in MCF-7 cells transferred with Trx-1 cDNA. Secondly, higher nuclear Trx-1 accompanied by no change of total Trx-1 can protect cells from apoptosis induced by cisplatin. Thirdly, high nuclear Trx-1 involves in the cisplatin-resistance in cisplatin-resistive cells. Meanwhile, we found that the mRNA level of p53 is closely correlated with the level of nuclear Trx-1. In summary, we concluded that the nuclear Trx-1 is required to resist apoptosis of MCF-7 cells induced by cisplatin, probably through up-regulating the anti-apoptotic gene, p53

  9. The depletion of nuclear glutathione impairs cell proliferation in 3t3 fibroblasts.

    Directory of Open Access Journals (Sweden)

    Jelena Markovic

    2009-07-01

    Full Text Available Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM and buthionine sulfoximine (BSO, and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.

  10. DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Gao, Fei; Li, Shengting; Lin, Lin

    2011-01-01

    To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development...... in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls....... In the result, we found that globally there was no significant difference of DNA methylation patterns between the two groups. Locus-specifically, some genes involved in embryonic development presented a generally increased level of methylation. Our findings suggest that in cloned pigs with normal phenotypes...

  11. Nuclear organization during in vitro differentiation of porcine mesenchymal stem cells (MSCs) into adipocytes.

    Science.gov (United States)

    Stachecka, Joanna; Walczak, Agnieszka; Kociucka, Beata; Ruszczycki, Błażej; Wilczyński, Grzegorz; Szczerbal, Izabela

    2018-02-01

    Differentiation of progenitor cells into adipocytes is accompanied by remarkable changes in cell morphology, cytoskeletal organization, and gene expression profile. Mature adipocytes are filled with a large lipid droplet and the nucleus tends to move to the cell periphery. It was hypothesized that the differentiation process is also associated with changes of nuclear organization. The aim of this study was to determine the number and distribution of selected components of nuclear architecture during porcine in vitro adipogenesis. The pig is an important animal model sharing many similarities to humans at the anatomical, physiological, and genetic levels and has been recognized as a good model for human obesity. Thus, understanding how cellular structures important for fundamental nuclear processes may be altered during adipocyte differentiation is of great importance. Mesenchymal stem cells (MSCs) were derived from bone marrow (BM-MSCs) and adipose tissue (AD-MSCs) and were cultured for 7 days in the adipogenic medium. A variable differentiation potential of these cell populations towards adipogenic lineage was observed, and for further study, a comparative characteristic of the nuclear organization in BM-MSCs and AD-MSCs was performed. Nuclear substructures were visualized by indirect immunofluorescence (nucleoli, nuclear speckles, PML bodies, lamins, and HP1α) or fluorescence in situ hybridization (telomeres) on fixed cells at 0, 3, 5, and 7 days of differentiation. Comprehensive characterization of these structures, in terms of their number, size, dynamics, and arrangement in three-dimensional space of the nucleus, was performed. It was found that during differentiation of porcine MSCs into adipocytes, changes of nuclear organization occurred and concerned: (1) the nuclear size and shape; (2) reduced lamin A/C expression; and (3) reorganization of chromocenters. Other elements of nuclear architecture such as nucleoli, SC-35 nuclear speckles, and telomeres

  12. Emergence of nuclear heparanase induces differentiation of human mammary cancer cells

    International Nuclear Information System (INIS)

    Nobuhisa, Tetsuji; Naomoto, Yoshio; Takaoka, Munenori; Tabuchi, Yoko; Ookawa, Keizou; Kitamoto, Dai; Gunduz, Esra; Gunduz, Mehmet; Nagatsuka, Hitoshi; Haisa, Minoru; Matsuoka, Junji; Nakajima, Motowo; Tanaka, Noriaki

    2005-01-01

    The study of epithelial differentiation touches upon many modern aspects of biology. The epithelium is in constant dialogue with the underlying mesenchyme to control stem cell activity, proliferation in transit-amplifying compartments, lineage commitment, terminal differentiation and, ultimately, cell death. There are spatially distinct compartments dedicated to each of these events. Recently we reported that heparanase is expressed in nucleus as well as in the cytoplasm and that nuclear heparanase seems to be related to cell differentiation. In this study, we investigated the role of nuclear heparanase in differentiation by transducing human mammary epithelial cancer cells with heparanase which was delivered specifically into nucleus. We observed that expression of nuclear heparanase allowed the cells to differentiate with the appearance of lipid droplets. This finding supports the idea that heparanase plays a novel role in epithelial cell differentiation apart from its known enzymatic function

  13. Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

    Energy Technology Data Exchange (ETDEWEB)

    Saad, Fawzy A. [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Torres, Marie [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Wang, Hao [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Graham, Lila, E-mail: lilagraham@cs.com [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

  14. Design of nuclear cells with re linking of trajectories; Diseno de celdas nucleares con re-encadenamiento de trayectorias

    Energy Technology Data Exchange (ETDEWEB)

    Castillo, A.; Campos S, Y.; Ortiz S, J.J.; Montes, J.L.; Perrusquia, R.; Hernandez, J.L.; Torres, M. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    2006-07-01

    ently work the results obtained with the Ohtli-RT system obtained when implementing the combinatory optimization technique well-known as Trajectories re linking or Path Re linking in English. The problem to solve is the radial design of nuclear fuel, taking like base nuclear fuel assembles for boiling water reactors (BWR Boiling Water Reactor by its initials in English). To evaluate the objective function used in the system the code in two dimensions Heliums 1.5 was used, which calculates the cross sections of the proposed design. The parameters that were considered for the evaluation of the objective function are the Power peak factor of the bar that generates bigger power in the cell and the Infinite Multiplication Factor. To prove the system its were used assembles 10x10 with 2 water channels. The obtained radial designs of nuclear fuel fulfilled the restrictions imposed to the considered limits, with regard to the involved parameters. (Author)

  15. Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation.

    Science.gov (United States)

    Strzalka, Wojciech; Ziemienowicz, Alicja

    2011-05-01

    PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome. This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution. Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.

  16. Applications of nuclear microprobe analysis in cancer cell biology and pharmacology

    International Nuclear Information System (INIS)

    Ortega, R.

    1999-01-01

    Nuclear microprobe analysis studies in cancer cell pharmacology and biology carried out at Bordeaux-Gradignan are reported. The cellular pharmacology of two anticancer agents, cis-diammine-dichloroplatinum(II), and 4'-iodo-4'-deoxy-doxorubicin, were investigated, as well as the role of iron in neuroblastoma carcinogenesis, and chromium(III) in trans-generation carcinogenesis. Nuclear microprobe analysis, using PIXE and particle backscattering microanalysis, was able to reveal intracellular and tissue distributions of the elements under investigation. Moreover, the fully quantitative and multi-elemental character of nuclear microprobe analysis offered information on possible mechanisms of drug action, metal carcinogenesis, and interactions with endogenous trace elements in cancer cells. (author)

  17. Assessing mRNA nuclear export in mammalian cells by microinjection.

    Science.gov (United States)

    Lee, Eliza S; Palazzo, Alexander F

    2017-08-15

    The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Primordial germ cell differentiation of nuclear transfer embryonic stem cells using surface modified electroconductive scaffolds.

    Science.gov (United States)

    Eslami-Arshaghi, Tarlan; Vakilian, Saeid; Seyedjafari, Ehsan; Ardeshirylajimi, Abdolreza; Soleimani, Masoud; Salehi, Mohammad

    2017-04-01

    A combination of nanotopographical cues and surface modification of collagen and fibronectin is a potential platform in primordial germ cells (PGCs) differentiation. In the present study, the synergistic effect of nanotopography and surface modification on differentiation of nuclear transfer embryonic stem cells (nt-ESCs) toward PGC lineage was investigated. In order to achieve this goal, poly-anyline (PANi) was mix within poly-L-lactic acid (PLLA). Afterward, the random composite mats were fabricated using PLLA and PANi mix solution. The nanofiber topography notably upregulated the expressions of prdm14, mvh and c-kit compared with tissue culture polystyrene (TCP). Moreover, the combination of nanofiber topography and surface modification resulted in more enhancement of PGCs differentiation compared with non-modified nanofibrous scaffold. Additionally, gene expression results showed that mvh and c-kit were expressed at higher intensity in cells exposed to collagen and fibronectin rather than collagen or fibronectin solitary. These results demonstrated the importance of combined effect of collagen and fibronectin in order to develop a functional extracellular matrix (ECM) mimic in directing stem cell fate and the potential of such biofunctional scaffolds for treatment of infertility.

  19. Alterations in the nuclear proteome of HIV-1 infected T-cells

    Energy Technology Data Exchange (ETDEWEB)

    DeBoer, Jason [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Jagadish, Teena; Haverland, Nicole A. [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Ciborowski, Pawel [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States)

    2014-11-15

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.

  20. Alterations in the nuclear proteome of HIV-1 infected T-cells

    International Nuclear Information System (INIS)

    DeBoer, Jason; Jagadish, Teena; Haverland, Nicole A.; Madson, Christian J.; Ciborowski, Pawel; Belshan, Michael

    2014-01-01

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines

  1. High throughput sequencing identifies an imprinted gene, Grb10, associated with the pluripotency state in nuclear transfer embryonic stem cells.

    Science.gov (United States)

    Li, Hui; Gao, Shuai; Huang, Hua; Liu, Wenqiang; Huang, Huanwei; Liu, Xiaoyu; Gao, Yawei; Le, Rongrong; Kou, Xiaochen; Zhao, Yanhong; Kou, Zhaohui; Li, Jia; Wang, Hong; Zhang, Yu; Wang, Hailin; Cai, Tao; Sun, Qingyuan; Gao, Shaorong; Han, Zhiming

    2017-07-18

    Somatic cell nuclear transfer and transcription factor mediated reprogramming are two widely used techniques for somatic cell reprogramming. Both fully reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells hold potential for regenerative medicine, and evaluation of the stem cell pluripotency state is crucial for these applications. Previous reports have shown that the Dlk1-Dio3 region is associated with pluripotency in induced pluripotent stem cells and the incomplete somatic cell reprogramming causes abnormally elevated levels of genomic 5-methylcytosine in induced pluripotent stem cells compared to nuclear transfer embryonic stem cells and embryonic stem cells. In this study, we compared pluripotency associated genes Rian and Gtl2 in the Dlk1-Dio3 region in exactly syngeneic nuclear transfer embryonic stem cells and induced pluripotent stem cells with same genomic insertion. We also assessed 5-methylcytosine and 5-hydroxymethylcytosine levels and performed high-throughput sequencing in these cells. Our results showed that Rian and Gtl2 in the Dlk1-Dio3 region related to pluripotency in induced pluripotent stem cells did not correlate with the genes in nuclear transfer embryonic stem cells, and no significant difference in 5-methylcytosine and 5-hydroxymethylcytosine levels were observed between fully and partially reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells. Through syngeneic comparison, our study identifies for the first time that Grb10 is associated with the pluripotency state in nuclear transfer embryonic stem cells.

  2. Cell-mediated transgenesis in rabbits: chimeric and nuclear transfer animals.

    Science.gov (United States)

    Zakhartchenko, V; Flisikowska, T; Li, S; Richter, T; Wieland, H; Durkovic, M; Rottmann, O; Kessler, B; Gungor, T; Brem, G; Kind, A; Wolf, E; Schnieke, A

    2011-02-01

    The ability to perform precise genetic engineering such as gene targeting in rabbits would benefit biomedical research by enabling, for example, the generation of genetically defined rabbit models of human diseases. This has so far not been possible because of the lack of functional rabbit embryonic stem cells and the high fetal and perinatal mortality associated with rabbit somatic cell nuclear transfer. We examined cultured pluripotent and multipotent cells for their ability to support the production of viable animals. Rabbit putative embryonic stem (ES) cells were derived and shown capable of in vitro and in vivo pluripotent differentiation. We report the first live born ES-derived rabbit chimera. Rabbit mesenchymal stem cells (MSCs) were derived from bone marrow, and multipotent differentiation was demonstrated in vitro. Nuclear transfer was carried out with both cell types, and embryo development was assessed in vitro and in vivo. Rabbit MSCs were markedly more successful than ES cells as nuclear donors. MSCs were transfected with fluorescent reporter gene constructs and assessed for nuclear transfer competence. Transfected MSCs supported development with similar efficiency as normal MSCs and resulted in the first live cloned rabbits from genetically manipulated MSCs. Reactivation of fluorescence reporter gene expression in reconstructed embryos was investigated as a means of identifying viable embryos in vitro but was not a reliable predictor. We also examined serial nuclear transfer as a means of rescuing dead animals.

  3. Stereological estimates of nuclear volume in squamous cell carcinoma of the uterine cervix and its precursors

    DEFF Research Database (Denmark)

    Sørensen, Flemming Brandt; Bichel, P; Jakobsen, A

    1991-01-01

    Using modern stereology, this study was carried out to obtain base-line data concerning three-dimensional, mean nuclear size in precancerous and invasive lesions of the uterine cervix. Unbiased estimates of the volume-weighted mean nuclear volume (nuclear vv) were obtained by point-sampling of nu......Using modern stereology, this study was carried out to obtain base-line data concerning three-dimensional, mean nuclear size in precancerous and invasive lesions of the uterine cervix. Unbiased estimates of the volume-weighted mean nuclear volume (nuclear vv) were obtained by point......-sampling of nuclear intercepts in 51 pre-treatment biopsies from patients with invasive squamous cell carcinomas (SCC). Vertical sections from 27 specimens with cervical intraepithelial neoplasia (CIN) grades I through III were also investigated, along with 10 CIN III associated with microinvasion (CIN III + M......). On average, nuclear vv was larger in SCC than in CIN III and CIN III + M together (2 P = 8.9 . 10(-5). A conspicuous overlap of nuclear vv existed between all investigated lesional groups. The reproducibility of estimates of nuclear vv in biopsies with SCC was acceptable (r = 0.85 and r = 0.84 in intra...

  4. Cell cycle-dependent alteration in NAC1 nuclear body dynamics and morphology

    International Nuclear Information System (INIS)

    Wu, Pei-Hsun; Hung, Shen-Hsiu; Ren, Tina; Tseng, Yiider; Shih, Ie-Ming

    2011-01-01

    NAC1, a BTB/POZ family member, has been suggested to participate in maintaining the stemness of embryonic stem cells and has been implicated in the pathogenesis of human cancer. In ovarian cancer, NAC1 upregulation is associated with disease aggressiveness and with the development of chemoresistance. Like other BTB/POZ proteins, NAC1 forms discrete nuclear bodies in non-dividing cells. To investigate the biological role of NAC1 nuclear bodies, we characterized the expression dynamics of NAC1 nuclear bodies during different phases of the cell cycle. Fluorescence recovery after photobleaching assays revealed that NAC1 was rapidly exchanged between the nucleoplasm and NAC1 nuclear bodies in interphase cells. The number of NAC1 bodies significantly increased and their size decreased in the S phase as compared to the G 0 /G 1 and G 2 phases. NAC1 nuclear bodies disappeared and NAC1 became diffuse during mitosis. NAC1 nuclear bodies reappeared immediately after completion of mitosis. These results indicate that a cell cycle-dependent regulatory mechanism controls NAC1 body formation in the nucleus and suggest that NAC1 body dynamics are associated with mitosis or cytokinesis

  5. Statistical analysis in the design of nuclear fuel cells; Analisis estadistico en el diseno de celdas de combustible nuclear

    Energy Technology Data Exchange (ETDEWEB)

    Castillo M, J. A.; Ortiz S, J. J.; Montes T, J. L.; Perusquia del Cueto, R., E-mail: alejandro.castillo@inin.gob.mx [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2012-10-15

    This work presents the preliminary results of a statistical analysis carried out for the design of nuclear fuel cells. The analysis consists in verifying the behavior of a cell, related with the frequency of the pines used for its design. In this preliminary study was analyzed the behavior of infinite multiplication factor and the peak factor of local power. On the other hand, the mentioned analysis was carried out using a pines group of enriched uranium previously established, for which varies the pines frequency used in the design. To carry out the study, the CASMO-IV code was used. The obtained designs are for the different axial areas of a fuel assembly. A balance cycle of the unit 1 of the nuclear power plant of Laguna Verde was used like reference. To obtain the result of the present work, systems that are already had and in which have already been implemented the heuristic techniques of ant colonies, neural networks and a hybrid between the dispersed search and the trajectories re-chaining. The results show that is possible to design nuclear fuel cells with a good performance, if is considered a statistical behavior in the frequency of the used pines, in a same way. (Author)

  6. Nuclear Wiskott–Aldrich syndrome protein co-regulates T cell factor 1-mediated transcription in T cells

    Directory of Open Access Journals (Sweden)

    Nikolai V. Kuznetsov

    2017-10-01

    Full Text Available Abstract Background The Wiskott–Aldrich syndrome protein (WASp family of actin-nucleating factors are present in the cytoplasm and in the nucleus. The role of nuclear WASp for T cell development remains incompletely defined. Methods We performed WASp chromatin immunoprecipitation and deep sequencing (ChIP-seq in thymocytes and spleen CD4+ T cells. Results WASp was enriched at genic and intergenic regions and associated with the transcription start sites of protein-coding genes. Thymocytes and spleen CD4+ T cells showed 15 common WASp-interacting genes, including the gene encoding T cell factor (TCF12. WASp KO thymocytes had reduced nuclear TCF12 whereas thymocytes expressing constitutively active WASpL272P and WASpI296T had increased nuclear TCF12, suggesting that regulated WASp activity controlled nuclear TCF12. We identify a putative DNA element enriched in WASp ChIP-seq samples identical to a TCF1-binding site and we show that WASp directly interacted with TCF1 in the nucleus. Conclusions These data place nuclear WASp in proximity with TCF1 and TCF12, essential factors for T cell development.

  7. Nuclear location of tumor suppressor protein maspin inhibits proliferation of breast cancer cells without affecting proliferation of normal epithelial cells

    International Nuclear Information System (INIS)

    Machowska, Magdalena; Wachowicz, Katarzyna; Sopel, Mirosław; Rzepecki, Ryszard

    2014-01-01

    Maspin, which is classified as a tumor suppressor protein, is downregulated in many types of cancer. Several studies have suggested potential anti-proliferative activity of maspin as well as sensitizing activity of maspin for therapeutic cytotoxic agents in breast cancer tissue culture and animal models. All of the experimental data gathered so far have been based on studies with maspin localized cytoplasmically, while maspin in breast cancer tumor cells may be located in the cytoplasm, nucleus or both. In this study, the effect of maspin cytoplasmic and nuclear location and expression level on breast cancer proliferation and patient survival was studied. Tissue sections from 166 patients with invasive ductal breast cancer were stained by immunohistochemistry for maspin and Ki-67 protein. The localization and expression level of maspin were correlated with estimated patient overall survival and percent of Ki-67-positive cells. In further studies, we created constructs for transient transfection of maspin into breast cancer cells with targeted cytoplasmic and nuclear location. We analyzed the effect of maspin location in normal epithelial cell line MCF10A and three breast cancer cell lines - MCF-7, MDA-MB-231 and SKBR-3 - by immunofluorescence and proliferation assay. We observed a strong positive correlation between moderate and high nuclear maspin level and survival of patients. Moreover, a statistically significant negative relationship was observed between nuclear maspin and Ki-67 expression in patients with invasive ductal breast cancer. Spearman’s correlation analysis showed a negative correlation between level of maspin localized in nucleus and percentage of Ki-67 positive cells. No such differences were observed in cells with cytoplasmic maspin. We found a strong correlation between nuclear maspin and loss of Ki-67 protein in breast cancer cell lines, while there was no effect in normal epithelial cells from breast. The anti-proliferative effect of nuclear

  8. Nuclear location of tumor suppressor protein maspin inhibits proliferation of breast cancer cells without affecting proliferation of normal epithelial cells

    Science.gov (United States)

    2014-01-01

    Background Maspin, which is classified as a tumor suppressor protein, is downregulated in many types of cancer. Several studies have suggested potential anti-proliferative activity of maspin as well as sensitizing activity of maspin for therapeutic cytotoxic agents in breast cancer tissue culture and animal models. All of the experimental data gathered so far have been based on studies with maspin localized cytoplasmically, while maspin in breast cancer tumor cells may be located in the cytoplasm, nucleus or both. In this study, the effect of maspin cytoplasmic and nuclear location and expression level on breast cancer proliferation and patient survival was studied. Methods Tissue sections from 166 patients with invasive ductal breast cancer were stained by immunohistochemistry for maspin and Ki-67 protein. The localization and expression level of maspin were correlated with estimated patient overall survival and percent of Ki-67-positive cells. In further studies, we created constructs for transient transfection of maspin into breast cancer cells with targeted cytoplasmic and nuclear location. We analyzed the effect of maspin location in normal epithelial cell line MCF10A and three breast cancer cell lines - MCF-7, MDA-MB-231 and SKBR-3 - by immunofluorescence and proliferation assay. Results We observed a strong positive correlation between moderate and high nuclear maspin level and survival of patients. Moreover, a statistically significant negative relationship was observed between nuclear maspin and Ki-67 expression in patients with invasive ductal breast cancer. Spearman’s correlation analysis showed a negative correlation between level of maspin localized in nucleus and percentage of Ki-67 positive cells. No such differences were observed in cells with cytoplasmic maspin. We found a strong correlation between nuclear maspin and loss of Ki-67 protein in breast cancer cell lines, while there was no effect in normal epithelial cells from breast. The anti

  9. 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.

  10. Vorinostat differentially alters 3D nuclear structure of cancer and non-cancerous esophageal cells.

    Science.gov (United States)

    Nandakumar, Vivek; Hansen, Nanna; Glenn, Honor L; Han, Jessica H; Helland, Stephanie; Hernandez, Kathryn; Senechal, Patti; Johnson, Roger H; Bussey, Kimberly J; Meldrum, Deirdre R

    2016-08-09

    The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an 'epigenetic' drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue. Using a combination of novel optical computed tomography (CT)-based quantitative 3D absorption microscopy and conventional confocal fluorescence microscopy, we show that subjecting malignant cells to vorinostat preferentially alters their 3D nuclear architecture relative to non-cancerous cells. Optical CT (cell CT) imaging of fixed single cells showed that drug-treated cancer cells exhibit significant alterations in nuclear morphometry. Confocal microscopy revealed that vorinostat caused changes in the distribution of H3K9ac-marked euchromatin and H3K9me3-marked constitutive heterochromatin. Additionally, 3D immuno-FISH showed that drug-induced expression of the DNA repair gene MGMT was accompanied by spatial relocation toward the center of the nucleus in the nuclei of metaplastic but not in non-neoplastic cells. Our data suggest that vorinostat's differential modulation of 3D nuclear architecture in normal and abnormal cells could play a functional role in its anti-cancer action.

  11. Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells.

    Science.gov (United States)

    Gautier, Violette; Cayrol, Corinne; Farache, Dorian; Roga, Stéphane; Monsarrat, Bernard; Burlet-Schiltz, Odile; Gonzalez de Peredo, Anne; Girard, Jean-Philippe

    2016-10-03

    IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. Extracellular IL-33 activates a growing number of target cells, including group 2 innate lymphoid cells, mast cells and regulatory T cells, but it remains unclear whether intracellular nuclear IL-33 has additional functions in the nucleus. Here, we used a global proteomic approach based on high-resolution mass spectrometry to compare the extracellular and intracellular roles of IL-33 in primary human endothelial cells, a major source of IL-33 protein in human tissues. We found that exogenous extracellular IL-33 cytokine induced expression of a distinct set of proteins associated with inflammatory responses in endothelial cells. In contrast, knockdown of endogenous nuclear IL-33 expression using two independent RNA silencing strategies had no reproducible effect on the endothelial cell proteome. These results suggest that IL-33 acts as a cytokine but not as a nuclear factor regulating gene expression in endothelial cells.

  12. Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction.

    Science.gov (United States)

    Poleshko, Andrey; Shah, Parisha P; Gupta, Mudit; Babu, Apoorva; Morley, Michael P; Manderfield, Lauren J; Ifkovits, Jamie L; Calderon, Damelys; Aghajanian, Haig; Sierra-Pagán, Javier E; Sun, Zheng; Wang, Qiaohong; Li, Li; Dubois, Nicole C; Morrisey, Edward E; Lazar, Mitchell A; Smith, Cheryl L; Epstein, Jonathan A; Jain, Rajan

    2017-10-19

    Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin, and independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Nuclear Phosphatidylcholine and Sphingomyelin Metabolism of Thyroid Cells Changes during Stratospheric Balloon Flight

    Directory of Open Access Journals (Sweden)

    Elisabetta Albi

    2009-01-01

    Full Text Available Nuclear sphingomyelin and phosphatidylcholine metabolism is involved in the response to ultraviolet radiation treatment in different ways related to the physiological state of cells. To evaluate the effects of low levels of radiation from the stratosphere on thyroid cells, proliferating and quiescent FRTL-5 cells were flown in a stratospheric balloon (BIRBA mission. After recovery, the activity of neutral sphingomyelinase, phosphatidylcholine-specific phospholipase C, sphingomyelin synthase, and reverse sphingomyelin synthase was assayed in purified nuclei and the nuclei-free fraction. In proliferating FRTL-5, space radiation stimulate nuclear neutral sphingomyelinase and reverse sphingomyelin synthase activity, whereas phosphatidylcholine-specific phospholipase C and sphingomyelin synthase were inhibited, thus inducing sphingomyelin degradation and phosphatidylcholine synthesis. This effect was lower in quiescent cells. The possible role of nuclear lipid metabolism in the thyroid damage induced by space radiations is discussed.

  14. Multidimensional profiling of cell surface proteins and nuclear markers

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Helen Barcellos-Hoff, Mary; Parvin, Bahram

    2009-01-30

    Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables.

  15. Application of Nuclear Volume Measurements to Comprehend the Cell Cycle in Root-Knot Nematode-Induced Giant Cells

    Directory of Open Access Journals (Sweden)

    José Dijair Antonino de Souza Junior

    2017-06-01

    Full Text Available Root-knot nematodes induce galls that contain giant-feeding cells harboring multiple enlarged nuclei within the roots of host plants. It is recognized that the cell cycle plays an essential role in the set-up of a peculiar nuclear organization that seemingly steers nematode feeding site induction and development. Functional studies of a large set of cell cycle genes in transgenic lines of the model host Arabidopsis thaliana have contributed to better understand the role of the cell cycle components and their implication in the establishment of functional galls. Mitotic activity mainly occurs during the initial stages of gall development and is followed by an intense endoreduplication phase imperative to produce giant-feeding cells, essential to form vigorous galls. Transgenic lines overexpressing particular cell cycle genes can provoke severe nuclei phenotype changes mainly at later stages of feeding site development. This can result in chaotic nuclear phenotypes affecting their volume. These aberrant nuclear organizations are hampering gall development and nematode maturation. Herein we report on two nuclear volume assessment methods which provide information on the complex changes occurring in nuclei during giant cell development. Although we observed that the data obtained with AMIRA tend to be more detailed than Volumest (Image J, both approaches proved to be highly versatile, allowing to access 3D morphological changes in nuclei of complex tissues and organs. The protocol presented here is based on standard confocal optical sectioning and 3-D image analysis and can be applied to study any volume and shape of cellular organelles in various complex biological specimens. Our results suggest that an increase in giant cell nuclear volume is not solely linked to increasing ploidy levels, but might result from the accumulation of mitotic defects.

  16. Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Kwang Sung; Won, Ji Young [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Park, Jin-Ki [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Sorrell, Alice M. [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Heo, Soon Young; Kang, Jee Hyun [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Woo, Jae-Seok [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Choi, Bong-Hwan [Genomics and Bioinformatics Division, National Institute of Animal Science, Suwon (Korea, Republic of); Chang, Won-Kyong [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of)

    2010-10-01

    Research highlights: {yields} Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. {yields} hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. {yields} hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

  17. Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

    International Nuclear Information System (INIS)

    Ahn, Kwang Sung; Won, Ji Young; Park, Jin-Ki; Sorrell, Alice M.; Heo, Soon Young; Kang, Jee Hyun; Woo, Jae-Seok; Choi, Bong-Hwan; Chang, Won-Kyong; Shim, Hosup

    2010-01-01

    Research highlights: → Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. → hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. → hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

  18. Nuclear receptor 4a3 (nr4a3 regulates murine mast cell responses and granule content.

    Directory of Open Access Journals (Sweden)

    Gianni Garcia-Faroldi

    Full Text Available Nuclear receptor 4a3 (Nr4a3 is a transcription factor implicated in various settings such as vascular biology and inflammation. We have recently shown that mast cells dramatically upregulate Nuclear receptor 4a3 upon activation, and here we investigated the functional impact of Nuclear receptor 4a3 on mast cell responses. We show that Nuclear receptor 4a3 is involved in the regulation of cytokine/chemokine secretion in mast cells following activation via the high affinity IgE receptor. Moreover, Nuclear receptor 4a3 negatively affects the transcript and protein levels of mast cell tryptase as well as the mast cell's responsiveness to allergen. Together, these findings identify Nuclear receptor 4a3 as a novel regulator of mast cell function.

  19. Nuclear morphometry in columnar cell lesions of the breast: is it useful?

    Science.gov (United States)

    Lim, C N; Ho, B C S; Bay, B H; Yip, G; Tan, P H

    2006-12-01

    To evaluate the nuclear morphometric features of breast columnar cell lesions (CCLs) observed on mammotome core biopsies, to determine if there are significant measurable differences between those with atypia and those without. Correlation with follow-up open excision specimens was made. Mammotome core biopsies performed on patients that contained CCLs were derived from the departmental case files. Histological material was reviewed and foci of CCLs demarcated for nuclear morphometric assessment, which was accomplished using an imaging system. Nuclear parameters studied were nuclear area and perimeter, circularity factor and feret's diameter. Statistical analysis used the GraphPad Prism software, with pparameter in the assessment of nuclear atypia. Whether it can be potentially used as an adjunctive tool depends on the establishment of appropriate cut-offs.

  20. Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID.

    Directory of Open Access Journals (Sweden)

    Quy Le

    2015-09-01

    Full Text Available AID (Activation Induced Deaminase deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.

  1. Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells.

    Science.gov (United States)

    Lachaud, Christophe; Da Silva, Daniel; Cotelle, Valérie; Thuleau, Patrice; Xiong, Tou Cheu; Jauneau, Alain; Brière, Christian; Graziana, Annick; Bellec, Yannick; Faure, Jean-Denis; Ranjeva, Raoul; Mazars, Christian

    2010-01-01

    Studies performed in animals have highlighted the major role of sphingolipids in regulating the balance between cell proliferation and cell death. Sphingolipids have also been shown to induce cell death in plants via calcium-based signalling pathways but the contribution of free cytosolic and/or nuclear calcium in the overall process has never been evaluated. Here, we show that increase in tobacco BY-2 cells of the endogenous content of Long Chain Bases (LCBs) caused by external application of d-erythro-sphinganine (DHS) is followed by immediate dose-dependent elevations of cellular free calcium concentration within the first minute in the cytosol and 10min later in the nucleus. Cells challenged with DHS enter a death process through apoptotic-like mechanisms. Lanthanum chloride, a general blocker of calcium entry, suppresses the cellular calcium variations and the PCD induced by DHS. Interestingly, dl-2-amino-5-phosphopentanoic acid (AP5) and [(+)-dizocilpine] (MK801), two inhibitors of animal and plant ionotropic glutamate receptors, suppress DHS-induced cell death symptoms by selectively inhibiting the variations of nuclear calcium concentration. The selective action of these compounds demonstrates the crucial role of nuclear calcium signature in controlling DHS-induced cell death in tobacco cells. 2009 Elsevier Ltd. All rights reserved.

  2. Bio-chemo-mechanical models for nuclear deformation in adherent eukaryotic cells.

    Science.gov (United States)

    Nava, Michele M; Raimondi, Manuela T; Pietrabissa, Riccardo

    2014-10-01

    Adherent eukaryotic cells are subjected to a broad variety of extracellular and intracellular stimuli regulating their behaviour. These stimuli can be either purely chemical, for example soluble factors binding to the cell membrane, or mechano-chemical, for example integrin-based adhesion complexes stretching the cell cytoskeleton. Here, we focus on mechano-chemical stimuli such as extracellular forces (interstitial flow, pressurization) and intracellular forces (due to cell adhesion), which may combine generating stress-strain states in the cytoskeleton. These states are transferred to the nucleus to influence the transcription of specific genes, likely by changing the chromatin organization and by altering the permeability of the nuclear membrane. While there exists increasing experimental evidence of the mechanosensing role of the cell nucleus, both the underlying molecular mechanisms involved, and the nuclear structural behaviour in response to forces, are still poorly understood. Here, we review the existing literature on computational models developed to investigate the chemo-mechanical behaviour of adherent eukaryotic cells. We analyse two main classes of models of single-cell mechanics, based either on the discrete or on the continuum approaches. We focus on the bio-chemo-mechanical model and modelling techniques accounting for the nuclear body. The modelling techniques are discussed highlighting their ability in predicting cytoskeletal contractility states and nuclear stress-strain states.

  3. Evaluating cytoplasmic and nuclear levels of inflammatory cytokines in cancer cells by western blotting.

    Science.gov (United States)

    Gatla, Himavanth R; Singha, Bipradeb; Persaud, Valerie; Vancurova, Ivana

    2014-01-01

    Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian cancer cells by western blotting. IL-8 is localized in the cytoplasm in both cell types, and its protein levels are significantly increased by BZ. In contrast, HMGB1 is localized in the nucleus, and BZ increases its nuclear levels only in ovarian cancer cells. The protocol includes isolation of cytoplasmic and nuclear extracts, followed by SDS electrophoresis and western blotting, and can be easily modified to analyze the cytoplasmic and nuclear cytokine levels in other cell types.

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

    Science.gov (United States)

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

    2016-01-01

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

  5. Nuclear transplantation, the conservation of the genome, and prospects for cell replacement.

    Science.gov (United States)

    Gurdon, J B

    2017-01-01

    Initial nuclear transplantation experiments in Xenopus eggs provided the first evidence for the conservation of the genome after cellular differentiation. This Discovery-in-Context Review recounts the early experiments that led to successful nuclear transfer in amphibians and the establishment of totipotency of a differentiated cell and shows how these discoveries paved the way for similar cloning experiments in other organisms. © 2016 Federation of European Biochemical Societies.

  6. Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential

    Energy Technology Data Exchange (ETDEWEB)

    Song, Kai [Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Qingdao University, Shandong Province (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Song, Yong [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Department of Stomatology, Liu Zhou People' s Hospital, Guangxi (China); Zhao, Xiao-Ping; Shen, Hui; Wang, Meng; Yan, Ting-lin [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Liu, Ke, E-mail: liuke.1999@aliyun.com [Department of Oral and Maxillofacial-Head and Neck oncology, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Shang, Zheng-jun, E-mail: shangzhengjun@hotmail.com [Department of Oral and Maxillofacial-Head and Neck oncology, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China)

    2014-10-15

    Most previous studies have linked cancer–macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression. - Highlights: • The fusion events between oral cancer and endothelial cells undergo nuclear fusion. • The resulting hybrid cells acquire a new property of drug resistance. • The resulting hybrid cells express the markers of both parental cells (i.e. vimentin and cytokeratin 18). • The hybrid cells contribute to tumor repopulation in vivo.

  7. Nuclear structure and gene activity in human differentiated cells

    Czech Academy of Sciences Publication Activity Database

    Bártová, Eva; Kozubek, Stanislav; Jirsová, Pavla; Kozubek, Michal; Gajová, H.; Lukášová, Emilie; Skalníková, M.; Cafourková, Alena; Koutná, I.; Hausmann, M.

    2002-01-01

    Roč. 139, č. 2 (2002), s. 76-89 ISSN 1047-8477 R&D Projects: GA AV ČR IBS5004010; GA AV ČR IAB5004102; GA ČR GA301/01/0186 Institutional research plan: CEZ:AV0Z5004920 Keywords : nuclear compartments * gene expression * FISH Subject RIV: BO - Biophysics Impact factor: 4.194, year: 2002

  8. Phenotype Clustering of Breast Epithelial Cells in Confocal Imagesbased on Nuclear Protein Distribution Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Long, Fuhui; Peng, Hanchuan; Sudar, Damir; Levievre, Sophie A.; Knowles, David W.

    2006-09-05

    Background: The distribution of the chromatin-associatedproteins plays a key role in directing nuclear function. Previously, wedeveloped an image-based method to quantify the nuclear distributions ofproteins and showed that these distributions depended on the phenotype ofhuman mammary epithelial cells. Here we describe a method that creates ahierarchical tree of the given cell phenotypes and calculates thestatistical significance between them, based on the clustering analysisof nuclear protein distributions. Results: Nuclear distributions ofnuclear mitotic apparatus protein were previously obtained fornon-neoplastic S1 and malignant T4-2 human mammary epithelial cellscultured for up to 12 days. Cell phenotype was defined as S1 or T4-2 andthe number of days in cultured. A probabilistic ensemble approach wasused to define a set of consensus clusters from the results of multipletraditional cluster analysis techniques applied to the nucleardistribution data. Cluster histograms were constructed to show how cellsin any one phenotype were distributed across the consensus clusters.Grouping various phenotypes allowed us to build phenotype trees andcalculate the statistical difference between each group. The resultsshowed that non-neoplastic S1 cells could be distinguished from malignantT4-2 cells with 94.19 percent accuracy; that proliferating S1 cells couldbe distinguished from differentiated S1 cells with 92.86 percentaccuracy; and showed no significant difference between the variousphenotypes of T4-2 cells corresponding to increasing tumor sizes.Conclusion: This work presents a cluster analysis method that canidentify significant cell phenotypes, based on the nuclear distributionof specific proteins, with high accuracy.

  9. Genotoxic Effects of Tobacco on Buccal Epithelium: Cell Nuclear Anomalies as Biomarker

    Directory of Open Access Journals (Sweden)

    Sohini Das Biswas

    2014-12-01

    Full Text Available Background: Tobacco use has toxic effects on different organs. This study was carried out to assess the effect of indigenous tobacco both in smoking (bidi and smokeless (gutkha, zarda and khaini forms on buccal cells at chromosomal level, through assessment of different nuclear anomalies as biomarker. Methods:This study was done on people living in Durgapur and its adjacent areas, West Bengal, India during January to July 2011. The samples were collected from 50 smokers (case group, 50 smokeless tobacco consumers or chewers (case group and 50 non-tobacco consumers (control group. Micronucleus assay was used to assess buccal cell nuclear changes. Buccal smears collected from study subjects were prepared on a grease free slide. Prepared slides were observed under light microscope and 2 to 5 fields were observed randomly for counting the different anomalies. In each field, the frequency of each anomaly was assessed in 100 cells and reported with percentage. Results:Chewers had significantly the highest frequency of all nuclear anomalies compared to smokers and healthy controls (HCs. Smokers also had significantly more anomalies compared to HCs. Condensed chromatin (CC, karyolysis (KL and bi-nucleation (BN in chewers and CC, pyknosis and BN in smokers were the most frequent anomalies. KL was significantly more frequent in chewers compared to smokers (59.8 ± 6.4 vs. 24.2 ± 12.4%, P < 0.001, however, the frequency of other nuclear anomalies were not significantly different in these two study groups. Presence of each nuclear anomaly was significantly greater in older ages in all study groups. Conclusion:Tobacco can cause and increase the rate of nuclear anomalies in both smoking and smokeless forms compared to HCs. The genotoxic effects of tobacco on buccal cells are partly age-related. Cell nuclear anomalies in buccal tissue can be used as biomarker indicating the detrimental effects of tobacco.

  10. Opposing microtubule motors drive robust nuclear dynamics in developing muscle cells.

    Science.gov (United States)

    Wilson, Meredith H; Holzbaur, Erika L F

    2012-09-01

    Dynamic interactions with the cytoskeleton drive the movement and positioning of nuclei in many cell types. During muscle cell development, myoblasts fuse to form syncytial myofibers with nuclei positioned regularly along the length of the cell. Nuclear translocation in developing myotubes requires microtubules, but the mechanisms involved have not been elucidated. We find that as nuclei actively translocate through the cell, they rotate in three dimensions. The nuclear envelope, nucleoli and chromocenters within the nucleus rotate together as a unit. Both translocation and rotation require an intact microtubule cytoskeleton, which forms a dynamic bipolar network around nuclei. The plus- and minus-end-directed microtubule motor proteins, kinesin-1 and dynein, localize to the nuclear envelope in myotubes. Kinesin-1 localization is mediated at least in part by interaction with klarsicht/ANC-1/Syne homology (KASH) proteins. Depletion of kinesin-1 abolishes nuclear rotation and significantly inhibits nuclear translocation, resulting in the abnormal aggregation of nuclei at the midline of the myotube. Dynein depletion also inhibits nuclear dynamics, but to a lesser extent, leading to altered spacing between adjacent nuclei. Thus, oppositely directed motors acting from the surface of the nucleus drive nuclear motility in myotubes. The variable dynamics observed for individual nuclei within a single myotube are likely to result from the stochastic activity of competing motors interacting with a complex bipolar microtubule cytoskeleton that is also continuously remodeled as the nuclei move. The three-dimensional rotation of myotube nuclei may facilitate their motility through the complex and crowded cellular environment of the developing muscle cell, allowing for proper myonuclear positioning.

  11. Quantitative mapping of RNA-mediated nuclear estrogen receptor β interactome in human breast cancer cells

    Science.gov (United States)

    Giurato, Giorgio; Nassa, Giovanni; Salvati, Annamaria; Alexandrova, Elena; Rizzo, Francesca; Nyman, Tuula A.; Weisz, Alessandro; Tarallo, Roberta

    2018-03-01

    The nuclear receptor estrogen receptor 2 (ESR2, ERβ) modulates cancer cell proliferation and tumor growth, exerting an oncosuppressive role in breast cancer (BC). Interaction proteomics by tandem affinity purification coupled to mass spectrometry was previously applied in BC cells to identify proteins acting in concert with ERβ to control key cellular functions, including gene transcription, RNA splicing and post-transcriptional mRNA regulation. These studies revealed an involvement of RNA in ERβ interactome assembly and functions. By applying native protein complex purification followed by nano LC-MS/MS before and after in vitro RNA removal, we generated a large dataset of newly identified nuclear ERβ interactors, including a subset associating with the receptor via RNA bridging. These datasets will be useful to investigate further the role of ERβ, nuclear RNAs and the other proteins identified here in BC and other cell types.

  12. Data mining in the study of nuclear fuel cells; Mineria de datos en el estudio de celdas de combustible nuclear

    Energy Technology Data Exchange (ETDEWEB)

    Medina P, J. A. [Universidad Autonoma de Campeche, Av. Agustin Melgar s/n, Col. Buenavista, 24039 San Francisco de Campeche, Campeche (Mexico); Ortiz S, J. J.; Castillo, A.; Montes T, J. L.; Perusquia, R., E-mail: j.angel.mp@hotmail.com [ININ, Departamento de Sistemas Nucleares, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2015-09-15

    In this paper is presented a study of data mining application in the analysis of fuel cells and their performance within a nuclear boiling water reactor. A decision tree was used to fulfill questions of the type If (condition) and Then (conclusion) to classify if the fuel cells will have good performance. The performance is measured by compliance or not of the cold shutdown margin, the rate of linear heat generation and the average heat generation in a plane of the reactor. It is assumed that the fuel cells are simulated in the reactor under a fuel reload and rod control patterns pre designed. 18125 fuel cells were simulated according to a steady-state calculation. The decision tree works on a target variable which is one of the three mentioned before. To analyze this objective, the decision tree works with a set of attribute variables. In this case, the attributes are characteristics of the cell as number of gadolinium rods, rods number with certain uranium enrichment mixed with a concentration of gadolinium, etc. The found model was able to predict the execution or not of the shutdown margin with a precision of around 95%. However, the other two variables showed lower percentages due to few learning cases of the model in which these variables were or were not achieved. Even with this inconvenience, the model is quite reliable and can be used in way coupled in optimization systems of fuel cells. (Author)

  13. Nuclear reprogramming: the strategy used in normal development is also used in somatic cell nuclear transfer and parthenogenesis.

    Science.gov (United States)

    Gao, Tianlong; Zheng, Junke; Xing, Fengying; Fang, Haiyan; Sun, Feng; Yan, Ayong; Gong, Xun; Ding, Hui; Tang, Fan; Sheng, Hui Z

    2007-02-01

    Somatic cell nuclear transfer (SCNT) and parthenogenesis are alternative forms of reproduction and development, building new life cycles on differentiated somatic cell nuclei and duplicated maternal chromatin, respectively. In the preceding paper (Sun F, et al., Cell Res 2007; 17:117-134.), we showed that an "erase-and-rebuild" strategy is used in normal development to transform the maternal gene expression profile to a zygotic one. Here, we investigate if the same strategy also applies to SCNT and parthenogenesis. The relationship between chromatin and chromatin factors (CFs) during SCNT and parthenogenesis was examined using immunochemical and GFP-fusion protein assays. Results from these studies indicated that soon after nuclear transfer, a majority of CFs dissociated from somatic nuclei and were redistributed to the cytoplasm of the egg. The erasure process in oogenesis is recaptured during the initial phase in SCNT. Most CFs entered pseudo-pronuclei shortly after their formation. In parthenogenesis, all parthenogenotes underwent normal oogenesis, and thus had removed most CFs from chromosomes before the initiation of development. The CFs were subsequently re-associated with female pronuclei in time and sequence similar to that in fertilized embryos. Based on these data, we conclude that the "erase-and-rebuild" process observed in normal development also occurs in SCNT and in parthenogenesis, albeit in altered fashions. The process is responsible for transcription reprogramming in these procedures. The "erase" process in SCNT is compressed and the efficiency is compromised, which likely contribute to the developmental defects often observed in nuclear transfer (nt) embryos. Furthermore, results from this study indicated that the cytoplasm of an egg contains most, if not all, essential components for assembling the zygotic program and can assemble them onto appropriate diploid chromatin of distinct origins.

  14. Progress in molecular nuclear medicine imaging of pancreatic beta cells

    International Nuclear Information System (INIS)

    Wu Haifei; Yin Hongyan; Liu Shuai; Zhang Yifan

    2010-01-01

    Diabetes mellitus is a common and frequently occurring disease which seriously threaten the health of human beings. Type 1 and type 2 diabetes respectively results from being destroyed and insufficient beta-cell mass. The associated symptoms appear until 50%-60% decrease of beta-cell mass. Because pancreas is deeply located in the body, with few beta-cell mass, the current methods of clinical diagnosis are invasive and late. So diagnosis of metabolism disease of beta-cell early non-invasively becomes more and more popular, imaging diagnosis of diabetes mellitus becomes the focus of researches, but how to estimate the mass of beta-cell still an important subject in imaging technology. (authors)

  15. Nuclear donor cell lines considerably influence cloning efficiency and the incidence of large offspring syndrome in bovine somatic cell nuclear transfer.

    Science.gov (United States)

    Liu, J; Wang, Y; Su, J; Luo, Y; Quan, F; Zhang, Y

    2013-08-01

    Total five ear skin fibroblast lines (named F1, F2, F3, F4 and F5) from different newborn Holstein cows have been used as nuclear donor cells for producing cloned cows by somatic cell nuclear transfer (SCNT). The effects of these cell lines on both in vitro and in vivo developmental rates of cloned embryos, post-natal survivability and incidence of large offspring syndrome (LOS) were examined in this study. We found that the different cell lines possessed the same capacity to support pre-implantation development of cloned embryos, the cleavage and blastocyst formation rates ranged from 80.2 ± 0.9 to 84.5 ± 2.5% and 28.5 ± 0.9 to 33.3 ± 1.4%, respectively. However, their capacities to support the in vivo development of SCNT embryos showed significant differences (p cloning efficiency was significantly higher in group F5 than those in group F1, F2, F3 and F4 (9.3% vs 4.1%, 1.2%, 2.0% and 5.0%, respectively, p cloned offspring from cell line F1, F2, F3 and F4 showed LOS and gestation length delay, while all cloned offspring from F5 showed normal birthweight and gestation length. We concluded that the nuclear donor cell lines have significant impact on the in vivo development of cloned embryos and the incidence of LOS in cloned calves. © 2013 Blackwell Verlag GmbH.

  16. Association of the nuclear matrix component NuMA with the Cajal body and nuclear speckle compartments during transitions in transcriptional activity in lens cell differentiation.

    Science.gov (United States)

    Gribbon, Chris; Dahm, Ralf; Prescott, Alan R; Quinlan, Roy A

    2002-10-01

    The transcriptional status of cells can be deduced from the staining pattern of various nuclear markers such as the Cajal body, nucleolus and nuclear speckles. In this study we have used these markers to correlate transcriptional status with cell differentiation in the lens. As a closed system with no cell loss and with each stage being spatially preserved, it is particularly well suited to such studies. To confirm that the nuclear markers in lens cells follow the same trends as in other cells, primary bovine lens epithelial cells were cultured and then treated with actinomycin D to inhibit transcription. This reduced the Cajal body markers to one or two foci per nucleus and the nucleoli became compacted as revealed by fibrillarin staining. The nuclear speckles, containing snRNPs (e.g. Sm) and the splicing factor, SC35, also became larger and more numerous while the signal for trimethylguanine (TMG) decreased suggesting a role hierarchy for the various speckle factors during transcriptional shutdown. The signal for survival of motor neurones gene product (SMN) also decreased at this point. In the lens epithelium, postmitotic cells near the equatorial region had one or two Cajal bodies per nucleus, indicating these cells had only basal levels of transcription. Sm was also present as large foci in these cells. Interestingly, both the speckles and Cajal bodies were NuMA-positive in these post-mitotic cells. At the epithelial-fibre cell transition, Cajal body number increased, while their size decreased indicative of increased transcriptional activity. Fibrillarin adopted the open floret pattern indicating increased transcriptional activity. The nuclear speckles adopted a more diffuse nucleoplasmic pattern, although some spots were still observed. All NuMA colocalisation with the Cajal bodies and nuclear speckles was lost at this stage of lens cell differentiation. Transcriptional shutdown occurs at a later stage in fibre cell differentiation, prior to programmed

  17. Somatic cell nuclear transfer: origins, the present position and future opportunities.

    Science.gov (United States)

    Wilmut, Ian; Bai, Yu; Taylor, Jane

    2015-10-19

    Nuclear transfer that involves the transfer of the nucleus from a donor cell into an oocyte or early embryo from which the chromosomes have been removed was considered first as a means of assessing changes during development in the ability of the nucleus to control development. In mammals, development of embryos produced by nuclear transfer depends upon coordination of the cell cycles of donor and recipient cells. Our analysis of nuclear potential was completed in 1996 when a nucleus from an adult ewe mammary gland cell controlled development to term of Dolly the sheep. The new procedure has been used to target the first precise genetic modification into livestock; however, the greatest inheritance of the Dolly experiment was to make biologists think differently. If unknown factors in the recipient oocyte could reprogramme the nucleus to a stage very early in development then there must be other ways of making that change. Within 10 years, two laboratories working independently established protocols by which the introduction of selected transcription factors changes a small proportion of the treated cells to pluripotent stem cells. This ability to produce 'induced pluripotent stem cells' is providing revolutionary new opportunities in research and cell therapy. © 2015 The Author(s).

  18. Color-coded Live Imaging of Heterokaryon Formation and Nuclear Fusion of Hybridizing Cancer Cells.

    Science.gov (United States)

    Suetsugu, Atsushi; Matsumoto, Takuro; Hasegawa, Kosuke; Nakamura, Miki; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Bouvet, Michael; Hoffman, Robert M

    2016-08-01

    Fusion of cancer cells has been studied for over half a century. However, the steps involved after initial fusion between cells, such as heterokaryon formation and nuclear fusion, have been difficult to observe in real time. In order to be able to visualize these steps, we have established cancer-cell sublines from the human HT-1080 fibrosarcoma, one expressing green fluorescent protein (GFP) linked to histone H2B in the nucleus and a red fluorescent protein (RFP) in the cytoplasm and the other subline expressing RFP in the nucleus (mCherry) linked to histone H2B and GFP in the cytoplasm. The two reciprocal color-coded sublines of HT-1080 cells were fused using the Sendai virus. The fused cells were cultured on plastic and observed using an Olympus FV1000 confocal microscope. Multi-nucleate (heterokaryotic) cancer cells, in addition to hybrid cancer cells with single-or multiple-fused nuclei, including fused mitotic nuclei, were observed among the fused cells. Heterokaryons with red, green, orange and yellow nuclei were observed by confocal imaging, even in single hybrid cells. The orange and yellow nuclei indicate nuclear fusion. Red and green nuclei remained unfused. Cell fusion with heterokaryon formation and subsequent nuclear fusion resulting in hybridization may be an important natural phenomenon between cancer cells that may make them more malignant. The ability to image the complex processes following cell fusion using reciprocal color-coded cancer cells will allow greater understanding of the genetic basis of malignancy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  19. Nuclear respiratory factor-1 and bioenergetics in tamoxifen-resistant breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Radde, Brandie N.; Ivanova, Margarita M.; Mai, Huy Xuan; Alizadeh-Rad, Negin; Piell, Kellianne; Van Hoose, Patrick; Cole, Marsha P.; Muluhngwi, Penn; Kalbfleisch, Ted S. [Department of Biochemistry & Molecular Genetics, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, KY 40292 (United States); Rouchka, Eric C. [Bioinformatics and Biomedical Computing Laboratory, Department of Computer Engineering and Computer Science, University of Louisville, Louisville, KY 40292 (United States); Hill, Bradford G. [Department of Medicine, University of Louisville School of Medicine, Louisville, KY 40292 (United States); Klinge, Carolyn M., E-mail: carolyn.klinge@louisville.edu [Department of Biochemistry & Molecular Genetics, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, KY 40292 (United States)

    2016-09-10

    Acquired tamoxifen (TAM) resistance is a significant clinical problem in treating patients with estrogen receptor α (ERα)+ breast cancer. We reported that ERα increases nuclear respiratory factor-1 (NRF-1), which regulates nuclear-encoded mitochondrial gene transcription, in MCF-7 breast cancer cells and NRF-1 knockdown stimulates apoptosis. Whether NRF-1 and target gene expression is altered in endocrine resistant breast cancer cells is unknown. We measured NRF-1and metabolic features in a cell model of progressive TAM-resistance. NRF-1 and its target mitochondrial transcription factor A (TFAM) were higher in TAM-resistant LCC2 and LCC9 cells than TAM-sensitive MCF-7 cells. Using extracellular flux assays we observed that LCC1, LCC2, and LCC9 cells showed similar oxygen consumption rate (OCR), but lower mitochondrial reserve capacity which was correlated with lower Succinate Dehydrogenase Complex, Subunit B in LCC1 and LCC2 cells. Complex III activity was lower in LCC9 than MCF-7 cells. LCC1, LCC2, and LCC9 cells had higher basal extracellular acidification (ECAR), indicating higher aerobic glycolysis, relative to MCF-7 cells. Mitochondrial bioenergetic responses to estradiol and 4-hydroxytamoxifen were reduced in the endocrine-resistant cells compared to MCF-7 cells. These results suggest the acquisition of altered metabolic phenotypes in response to long term antiestrogen treatment may increase vulnerability to metabolic stress. - Highlights: • NRF-1 and TFAM expression are higher in endocrine-resistant breast cancer cells. • Oxygen consumption rate is similar in endocrine-sensitive and resistant cells. • Mitochondrial reserve capacity is lower in endocrine-resistant cells. • Endocrine-resistant breast cancer cells have increased glycolysis. • Bioenergetic responses to E2 and tamoxifen are lower in endocrine-resistant cells.

  20. Extensive Nuclear Reprogramming Underlies Lineage Conversion into Functional Trophoblast Stem-like Cells.

    Science.gov (United States)

    Benchetrit, Hana; Herman, Shay; van Wietmarschen, Niek; Wu, Tao; Makedonski, Kirill; Maoz, Noam; Yom Tov, Nataly; Stave, Danielle; Lasry, Rachel; Zayat, Valery; Xiao, Andrew; Lansdorp, Peter M; Sebban, Shulamit; Buganim, Yosef

    2015-11-05

    Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. 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

  2. DOT1L inhibitor improves early development of porcine somatic cell nuclear transfer embryos

    DEFF Research Database (Denmark)

    Tao, Jia; Zhang, Yu; Zuo, Xiaoyuan

    2017-01-01

    inhibitor EPZ004777 (EPZ), significantly improved reprogramming efficiency during the generation of mouse induced pluripotent stem cells. However, the roles of DOT1L in porcine nuclear transfer-mediated cellular reprogramming are not yet known. Here we showed that DOT1L inhibition via 0.5 nM EPZ treatment......Incomplete epigenetic reprogramming of the genome of donor cells causes poor early and full-term developmental efficiency of somatic cell nuclear transfer (SCNT) embryos. Previous research indicate that inhibition of the histone H3 K79 methyltransferase DOT1L, using a selective pharmacological...... for 12 or 24 h significantly enhanced the blastocyst rate of SCNT embryos and dramatically reduced the level of H3K79me2 during SCNT 1-cell embryonic development. Additionally, H3K79me2 level in the EPZ-treated SCNT embryos was similar to that in in vitro fertilized embryos, suggesting that DOT1L...

  3. Nuclear receptors expression chart in peripheral blood mononuclear cells identifies patients with Metabolic Syndrome.

    Science.gov (United States)

    D'Amore, Simona; Vacca, Michele; Graziano, Giusi; D'Orazio, Andria; Cariello, Marica; Martelli, Nicola; Di Tullio, Giuseppe; Salvia, Roberto; Grandaliano, Giuseppe; Belfiore, Anna; Pellegrini, Fabio; Palasciano, Giuseppe; Moschetta, Antonio

    2013-12-01

    Nuclear receptors are a class of 48 ligand-activated transcription factors identified as key players of metabolic and developmental processes. Most of these receptors are potential targets for pharmacological strategies in the Metabolic Syndrome. In the present study, we analyzed changes in the mRNA expression of nuclear receptors in the peripheral blood mononuclear cells of patients with Metabolic Syndrome, in order to identify novel biomarkers of disease and candidate targets for putative therapeutical approaches. We enrolled thirty healthy controls (14 M:16 F) and thirty naïve patients (16 M: 14 F; >3 criteria for Metabolic Syndrome upon Adult Treatment Panel III) without organ damage. Using quantitative real-time PCR, we assessed the expression patterns of nuclear receptors in peripheral blood mononuclear cells. 33/48 nuclear receptors were expressed in peripheral blood mononuclear cells. In patients with Metabolic Syndrome, we found a significant down-regulation of the entire PPAR, NR4A and RAR families, together with a repression of RXRα, VDR, and Rev-Erbα. Furthermore, we performed a novel statistical analysis with classification trees, which allowed us to depict a predictive core of nuclear receptor expression patterns characterizing subjects with Metabolic Syndrome. Random Forest Analysis identified NOR1 and PPARδ, which were both reduced in peripheral blood mononuclear cells and specifically in CD14(+) cells (mostly monocytes), as classifiers of Metabolic Syndrome, with high specificity and sensitivity. Our results point to the use of PPAR and NR4A mRNA levels in the overall peripheral blood mononuclear cells as biomarkers of Metabolic Syndrome and bona fide putative targets of pharmacological therapy. © 2013.

  4. Nuclear DAMP complex-mediated RAGE-dependent macrophage cell death

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ruochan [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Fu, Sha; Fan, Xue-Gong [Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Lotze, Michael T.; Zeh, Herbert J. [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Tang, Daolin, E-mail: tangd2@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Kang, Rui, E-mail: kangr@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States)

    2015-03-13

    High mobility group box 1 (HMGB1), histone, and DNA are essential nuclear components involved in the regulation of chromosome structure and function. In addition to their nuclear function, these molecules act as damage-associated molecular patterns (DAMPs) alone or together when released extracellularly. The synergistic effect of these nuclear DNA-HMGB1-histone complexes as DAMP complexes (nDCs) on immune cells remains largely unexplored. Here, we demonstrate that nDCs limit survival of macrophages (e.g., RAW264.7 and peritoneal macrophages) but not cancer cells (e.g., HCT116, HepG2 and Hepa1-6). nDCs promote production of inflammatory tumor necrosis factor α (TNFα) release, triggering reactive oxygen species-dependent apoptosis and necrosis. Moreover, the receptor for advanced glycation end products (RAGE), but not toll-like receptor (TLR)-4 and TLR-2, was required for Akt-dependent TNFα release and subsequent cell death following treatment with nDCs. Genetic depletion of RAGE by RNAi, antioxidant N-Acetyl-L-cysteine, and TNFα neutralizing antibody significantly attenuated nDC-induced cell death. These findings provide evidence supporting novel signaling mechanisms linking nDCs and inflammation in macrophage cell death. - Highlights: • Nuclear DAMP complexes (nDCs) selectively induce cell death in macrophages, but not cancer cells. • TNFα-mediated oxidative stress is required for nDC-induced death. • RAGE-mediated Akt activation is required for nDC-induced TNFα release. • Blocking RAGE and TNFα inhibits nDC-induced macrophage cell death.

  5. Monodisperse Magnetite Nanoparticles Coupled with Nuclear Localization Signal Peptide for Cell-Nucleus Targeting

    OpenAIRE

    Xu, Chenjie; Xie, Jin; Kohler, Nathan; Walsh, Edward G.; Chin, Y. Eugene; Sun, Shouheng

    2008-01-01

    Functionalization of monodisperse superparamagnetic magnetite (Fe3O4) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe3O4 nanoparticles functionalized with protein and nuclear locali...

  6. Emerging role for nuclear rotation and orientation in cell migration

    Czech Academy of Sciences Publication Activity Database

    Maninová, Miloslava; Iwanicki, M. P.; Vomastek, Tomáš

    2014-01-01

    Roč. 8, č. 1 (2014), s. 42-48 ISSN 1933-6918 R&D Projects: GA ČR GA204/09/0614 Grant - others:Marie Cúrie EU FP7(BE) 231086 Institutional support: RVO:61388971 Keywords : cell polarity * actin * migration * microtubules Subject RIV: EE - Microbiology, Virology Impact factor: 4.505, year: 2014

  7. Nuclear thyroid hormone receptor binding in human mononuclear blood cells after goitre resection

    DEFF Research Database (Denmark)

    Kvetny, J; Matzen, L E; Blichert-Toft, M

    1989-01-01

    Nuclear thyroxine and triiodothyronine receptor-binding in human mononuclear blood cells were examined in 14 euthyroid persons prior to and 1, 6, 24 and 53 weeks after goitre resection. One week after resection decreased serum T3 from 1.47 nmol/l to 1.14 nmol/l (P less than 0.05), FT4I from 103 a...

  8. The Role of the Nuclear Envelope Protein MAN1 in Mesenchymal Stem Cell Differentiation

    DEFF Research Database (Denmark)

    Bermeo, Sandra; Al-Saedi, Ahmed; Kassem, Moustapha

    2017-01-01

    Mutations in MAN1, a protein of the nuclear envelope, cause bone phenotypes characterized by hyperostosis. The mechanism of this pro-osteogenic phenotype remains unknown. We increased and decreased MAN1 expression in mesenchymal stem cells (MSC) upon which standard osteogenic and adipogenic...

  9. Structure of the nuclear pore complex in mammalian cells. Two annular components.

    NARCIS (Netherlands)

    J.H.J. Hoeijmakers (Jan); J.H.N. Schel; F. Wanka

    1974-01-01

    textabstractThe ultrastructure of the nuclear pore complex has been investigated in isolated nuclei of an in vitro cultured bovine liver cell line. In shadow-cast replicas of the surface of nuclei isolated in Tris buffer containing low K+ and Mg2+ concentrations (RSB) the rims of the pores appeared

  10. Cell and nuclear enlargement of SW480 cells induced by a plant lignan, arctigenin: evaluation of cellular DNA content using fluorescence microscopy and flow cytometry.

    Science.gov (United States)

    Kang, Kyungsu; Lee, Hee Ju; Yoo, Ji-Hye; Jho, Eun Hye; Kim, Chul Young; Kim, Minkyun; Nho, Chu Won

    2011-08-01

    Arctigenin is a natural plant lignan previously shown to induce G(2)/M arrest in SW480 human colon cancer cells as well as AGS human gastric cancer cells, suggesting its use as a possible cancer chemopreventive agent. Changes in cell and nuclear size often correlate with the functionality of cancer-treating agents. Here, we report that arctigenin induces cell and nuclear enlargement of SW480 cells. Arctigenin clearly induced the formation of giant nuclear shapes in SW480, as demonstrated by fluorescence microscopic observation and quantitative determination of nuclear size. Cell and nuclear size were further assessed by flow cytometric analysis of light scattering and fluorescence pulse width after propidium iodide staining. FSC-H and FL2-W values (parameters referring to cell and nuclear size, respectively) significantly increased after arctigenin treatment; the mean values of FSC-H and FL2-W in arctigenin-treated SW480 cells were 572.6 and 275.1, respectively, whereas those of control cells were 482.0 and 220.7, respectively. Our approach may provide insights into the mechanism behind phytochemical-induced cell and nuclear enlargement as well as functional studies on cancer-treating agents.

  11. Targeting cytosolic proliferating cell nuclear antigen (PCNA in neutrophil-dominated inflammation

    Directory of Open Access Journals (Sweden)

    Alessia eDe Chiara

    2012-10-01

    Full Text Available New therapeutic approaches that can accelerate neutrophil apoptosis under inflammatory conditions to enhance the resolution of inflammation are now under study. Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA, a nuclear factor involved in DNA replication and repair of proliferating cells is a key regulator of neutrophil survival. In this review, we will try to put into perspective the physiologic relevance of PCNA in neutrophils. We will discuss key issues such as molecular structure, post-translational modifications, based on our knowledge of nuclear PCNA, assuming that similar principles governing its function are conserved between nuclear and cytosolic PCNA. The example of cystic fibrosis that features one of the most intense neutrophil-dominated pulmonary inflammation will be discussed. We believe that through an intimate comprehension of the cytosolic PCNA scaffold based on nuclear PCNA knowledge, novel pathways regulating neutrophil survival can be unraveled and innovative agents can be developed to dampen inflammation where it proves detrimental.

  12. Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Kara G Lassen

    2006-07-01

    Full Text Available HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART. We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

  13. Development of interspecies nuclear transfer embryos reconstructed with argali (Ovis ammon) somatic cells and sheep ooplasm.

    Science.gov (United States)

    Pan, Xiaoyan; Zhang, Yanli; Guo, Zhiqin; Wang, Feng

    2014-02-01

    Interspecies nuclear transfer has already achieved success in several species, which shows great potential in recovery and conservation of endangered animals. The study was conducted to establish an efficient system for in vitro argali (Ovis ammon)-sheep embryo reconstruction via interspecies somatic cell nuclear transfer (iSCNT). The competence of domestic sheep cytoplasts to reprogram the adult argali fibroblast nuclei was evaluated, and the effects of enucleation methods and donor cell passage and cell state on the in vitro development of argali-sheep cloned embryos were also examined. Sheep oocytes could support argali and sheep fibroblast cell nuclei transfer and develop to blastocysts in vitro. Oocytes matured for 21–23 h and enucleated by chemically assisted enucleation (CAE) had a higher enucleation rate than blind enucleation (BE), but the development rate of iSCNTembryos was the same (P>0.05). Moreover, passage numbers of fibroblast cells embryos. Thus sheep cytoplasm successfully supports argali nucleus development to blastocyst stage after optimising the nuclear transfer procedure, which indicates that iSCNT can be used to conserve endangered argali in the near future.

  14. Ethics and embryonic stem cell research: altered nuclear transfer as a way forward.

    Science.gov (United States)

    Hurlbut, William B

    2007-01-01

    Ethical controversy in stem cell research arises because current methods to produce embryonic stem cell lines require the destruction of living human embryos. For this reason, there is increasing interest in developing alternative, non-embryonic sources of pluripotent stem cells. This effort is especially important in the US due to the prevailing policy against federal funding of embryo-destructive research. Altered nuclear transfer (ANT) is one of several potential methods to develop alternative sources of pluripotent stem cells. This approach employs the technique of somatic cell nuclear transfer, but the somatic cell nucleus or egg cytoplasm (or both) are first altered before the somatic cell nucleus is transferred into the oocyte. This alteration precludes the coordinated organization and developmental potential that is necessary for the resulting biological entity to be an embryo, but it still allows the entity to generate pluripotent stem cells. Proof-of-principle for one variant of ANT has been established in mice by silencing the functional expression of the gene Cdx2 in the somatic cell nucleus prior to its transfer into an enucleated egg. From the resulting non-embryonic laboratory construct, fully functional pluripotent stem cells were procured. Other more recent studies have suggested the possibility of achieving the same results by preemptively silencing maternally derived Cdx2 messenger RNA in the egg before the act of nuclear transfer. The procedure would produce the equivalent of a tissue culture of pluripotent stem cells. In contrast to the use of embryos 'left over' from clinical in vitro fertilization, ANT could produce pluripotent stem cell lines with an unlimited range of specifically selected and controlled genotypes. Such flexibility would greatly facilitate the study of disease, drug development, and toxicology testing, and may allow the production of therapeutically useful pluripotent stem cells that are immune-compatible. If developed to

  15. Cell for receipting and dismantling nuclear fuel assembly

    International Nuclear Information System (INIS)

    Beneck, J.A.; Quayre, C.

    1989-01-01

    The cell has a vertical structure with a right section corresponding at that of the assembly to receive, a mechanism for keeping fuel pins at their nominal separation in the form of at least two combs and mechanisms of holding grids and bottom nozzle. The comb arrangements are moved into position by hydraulic actuators so that they cross each other to form a lattice round the fuel pins. The mechanism for holding grid assemblies consist of joints that articulate from a free position to a position where the joints press of the grid on all sides [fr

  16. Proliferating cell nuclear antigen immunostaining in breast carcinoma and its relationship to clinical and pathological variables.

    Science.gov (United States)

    Agarwal, S; Jain, R; Rusia, U; Gupta, R L

    1997-01-01

    Tumour proliferative activity of 74 breast lesions was assessed by determining mitotic index and immunostaining for proliferative cell nuclear antigen using Peroxidase antiperoxidase method. The indices were correlated with histomorphology and clinical stage of the disease. Positively stained nuclei and mitotic figures were counted per 1000 cells to calculate Proliferating Cell Nuclear Antigen (PCNA) and mitotic index respectively. Sixty four cases stained positive for PCNA. The index ranged between 0 to 98. PCNA index was significantly low in benign lesions as compared to malignant lesions (p < 0.0002). There was a linear correlation between the mitotic index and PCNA index. PCNA index also showed significant correlation with tumour size and histologic grade; however, it had no correlation with axillary lymph node status.

  17. A hybrid approach to solving the problem of design of nuclear fuel cells

    International Nuclear Information System (INIS)

    Montes T, J. L.; Perusquia del C, R.; Ortiz S, J. J.; Castillo, A.

    2015-09-01

    An approach to solving the problem of fuel cell design for BWR power reactor is presented. For this purpose the hybridization of a method based in heuristic knowledge rules called S15 and the advantages of a meta-heuristic method is proposed. The synergy of potentialities of both techniques allows finding solutions of more quality. The quality of each solution is obtained through a multi-objective function formed from the main cell parameters that are provided or obtained during the simulation with the CASMO-4 code. To evaluate this alternative of solution nuclear fuel cells of reference of nuclear power plant of Laguna Verde were used. The results show that in a systematic way the results improve when both methods are coupled. As a result of the hybridization process of the mentioned techniques an improvement is achieved in a range of 2% with regard to the achieved results in an independent way by the S15 method. (Author)

  18. Parthenogenesis and somatic cell nuclear transfer in sheep oocytes using Polscope.

    Science.gov (United States)

    Nandedkar, Pandit; Chohan, Parul; Patwardhan, Archana; Gaikwad, Santosh; Bhartiya, Deepa

    2009-07-01

    Parthenogenesis and Somatic cell nuclear transfer (SCNT) techniques, offer a unique approach to manipulate the genetic composition of derived human embryonic stem cells - an essential step if the full opportunities for disease modeling, drug discovery or individualized stem cell therapy are to be realized. The present study describes the use of sheep oocytes to acquire expertise and establish methods to reconstruct embryos for obtaining blastocysts before venturing into human SCNT where the oocytes are a very precious starting material. Maturation of sheep eggs in vitro for 20-24 hr resulted in 65% metaphase II (MII) eggs which were either parthenogenetically activated using calcium ionomycin or ethanol or subjected to SCNT using cumulus cell as somatic cell. Sixteen blastocysts were produced by parthenogenetic activation of 350 eggs whereas reconstructed embryos, after SCNT carried out in 139 eggs, progressed only up to morula stage. The procedure of parthenogenesis and SCNT will be useful to generate autologous ES cells using human eggs.

  19. Numerical and experimental calibration of a calorimetric sample cell dedicated to nuclear heating measurements

    International Nuclear Information System (INIS)

    Brun, J.; Reynard-Carette, C.; Merroun, O.; Carette, M.; Janulyte, A.; Zerega, Y.; Andre, J.; Lyoussi, A.; Bignan, G.; Chauvin, J.P.; Fourmentel, D.; Gonnier, C.; Guimbal, P.; Malo, J.Y.; Villard, J.F.

    2012-01-01

    Online nuclear measurements inside experimental channels of material testing reactors (MTRs) are needed for experimental works (to design mock-ups) and for numerical works (input data) in order to better understanding complex phenomena occurring during the accelerated ageing of materials and the irradiation of nuclear fuels. In this paper, we focus only on one kind of measurements: nuclear heating performed by means of a radiometric calorimeter. The aims of numerical and experimental works are firstly to optimize the sensor response: in particular the sensitivity for new energy deposit ranges (new lower nuclear heating level in the reflector), and then to miniaturize and adapt this sensor for irradiation conditions in the Jules Horowitz Reactor (JHR). A calorimeter, developed previously by the CEA, is studied. It corresponds to a graphite differential calorimeter. It is used with a nonadiabatic mode called heat flow mode too. Experimental calibration of the sample cell is presented. In that case, energy deposit is simulated by Joule effect and the sample cell is inserted into a bath at a regulated temperature and controlled flow. The response of the sensor is discussed versus electrical power imposed for two flow rates. Numerical works show the influence of the gas conductivity and of specific dimensions on the cell sensitivity. (authors)

  20. Induction of polyploidy by nuclear fusion mechanism upon decreased expression of the nuclear envelope protein LAP2β in the human osteosarcoma cell line U2OS.

    Science.gov (United States)

    Ben-Shoshan, Shirley Oren; Simon, Amos J; Jacob-Hirsch, Jasmine; Shaklai, Sigal; Paz-Yaacov, Nurit; Amariglio, Ninette; Rechavi, Gideon; Trakhtenbrot, Luba

    2014-01-28

    Polyploidy has been recognized for many years as an important hallmark of cancer cells. Polyploid cells can arise through cell fusion, endoreplication and abortive cell cycle. The inner nuclear membrane protein LAP2β plays key roles in nuclear envelope breakdown and reassembly during mitosis, initiation of replication and transcriptional repression. Here we studied the function of LAP2β in the maintenance of cell ploidy state, a role which has not yet been assigned to this protein. By knocking down the expression of LAP2β, using both viral and non-viral RNAi approaches in osteosarcoma derived U2OS cells, we detected enlarged nuclear size, nearly doubling of DNA content and chromosomal duplications, as analyzed by fluorescent in situ hybridization and spectral karyotyping methodologies. Spectral karyotyping analyses revealed that near-hexaploid karyotypes of LAP2β knocked down cells consisted of not only seven duplicated chromosomal markers, as could be anticipated by genome duplication mechanism, but also of four single chromosomal markers. Furthermore, spectral karyotyping analysis revealed that both of two near-triploid U2OS sub-clones contained the seven markers that were duplicated in LAP2β knocked down cells, whereas the four single chromosomal markers were detected only in one of them. Gene expression profiling of LAP2β knocked down cells revealed that up to a third of the genes exhibiting significant changes in their expression are involved in cancer progression. Our results suggest that nuclear fusion mechanism underlies the polyploidization induction upon LAP2β reduced expression. Our study implies on a novel role of LAP2β in the maintenance of cell ploidy status. LAP2β depleted U2OS cells can serve as a model to investigate polyploidy and aneuploidy formation by nuclear fusion mechanism and its involvement in cancerogenesis.

  1. Nuclear bodies in the maturing egg cell of a fern, Pteridium aquilinum.

    Science.gov (United States)

    Bell, P R

    1983-03-01

    Nuclear bodies about 250 nm in diameter, and with a strong affinity for uranium and acriflavine, appear in the nuclei of maturing egg cells of Pteridium. Many enter well-defined evaginations of the nucleus. The nuclear bodies are almost wholly digested by Pronase, but are resistant to ribonuclease and deoxyribonuclease. Radioactive labelling gives no evidence of the presence of nucleic acids, but X-ray microprobe analysis indicates phosphorus. It is concluded that the bodies consist entirely of acidic protein, possibly phosphorylated. This protein may be a structural component of the nucleus, temporarily displaced and aggregated as a consequence of the fine dispersal of the chromatin.

  2. Hydrogen Gas Production from Nuclear Power Plant in Relation to Hydrogen Fuel Cell Technologies Nowadays

    International Nuclear Information System (INIS)

    Yusibani, Elin; Kamil, Insan; Suud, Zaki

    2010-01-01

    Recently, world has been confused by issues of energy resourcing, including fossil fuel use, global warming, and sustainable energy generation. Hydrogen may become the choice for future fuel of combustion engine. Hydrogen is an environmentally clean source of energy to end-users, particularly in transportation applications because without release of pollutants at the point of end use. Hydrogen may be produced from water using the process of electrolysis. One of the GEN-IV reactors nuclear projects (HTGRs, HTR, VHTR) is also can produce hydrogen from the process. In the present study, hydrogen gas production from nuclear power plant is reviewed in relation to commercialization of hydrogen fuel cell technologies nowadays.

  3. Perspective for special Gurdon issue for differentiation: can cell fusion inform nuclear reprogramming?

    Science.gov (United States)

    Burns, David; Blau, Helen M

    2014-07-01

    Nuclear reprogramming was first shown to be possible by Sir John Gurdon over a half century ago. The process has been revolutionized by the production of induced pluripotent cells by overexpression of the four transcription factors discovered by Shinya Yamanaka, which now enables mammalian applications. Yet, reprogramming by a few transcription factors remains incomplete and inefficient, whether to pluripotent or differentiated cells. We propose that a better understanding of mechanistic insights based on developmental principles gained from heterokaryon studies may inform the process of directing cell fate, fundamentally and clinically. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  4. Surface localization of the nuclear receptor CAR in influenza A virus-infected cells

    International Nuclear Information System (INIS)

    Takahashi, Tadanobu; Moriyama, Yusuke; Ikari, Akira; Sugatani, Junko; Suzuki, Takashi; Miwa, Masao

    2008-01-01

    Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism

  5. Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer

    OpenAIRE

    Li, Honghui; Wang, Gui; Hao, Zhiqiang; Zhang, Guozhong; Qing, Yubo; Liu, Shuanghui; Qing, Lili; Pan, Weirong; Chen, Lei; Liu, Guichun; Zhao, Ruoping; Jia, Baoyu; Zeng, Luyao; Guo, Jianxiong; Zhao, Lixiao

    2016-01-01

    Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were...

  6. E-cadherin expression increases cell proliferation by regulating energy metabolism through nuclear factor-κB in AGS cells.

    Science.gov (United States)

    Park, Song Yi; Shin, Jee-Hye; Kee, Sun-Ho

    2017-09-01

    β-Catenin is a central player in Wnt signaling, and activation of Wnt signaling is associated with cancer development. E-cadherin in complex with β-catenin mediates cell-cell adhesion, which suppresses β-catenin-dependent Wnt signaling. Recently, a tumor-suppressive role for E-cadherin has been reconsidered, as re-expression of E-cadherin was reported to enhance the metastatic potential of malignant tumors. To explore the role of E-cadherin, we established an E-cadherin-expressing cell line, EC96, from AGS cells that featured undetectable E-cadherin expression and a high level of Wnt signaling. In EC96 cells, E-cadherin re-expression enhanced cell proliferation, although Wnt signaling activity was reduced. Subsequent analysis revealed that nuclear factor-κB (NF-κB) activation and consequent c-myc expression might be involved in E-cadherin expression-mediated cell proliferation. To facilitate rapid proliferation, EC96 cells enhance glucose uptake and produce ATP using both mitochondria oxidative phosphorylation and glycolysis, whereas AGS cells use these mechanisms less efficiently. These events appeared to be mediated by NF-κB activation. Therefore, E-cadherin re-expression and subsequent induction of NF-κB signaling likely enhance energy production and cell proliferation. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  7. Evaluation of the Cell Proliferation Process of Ovarian Follicles in Hypothyroid Rats by Proliferation Cell Nuclear Antigen Immunohistochemical Technique

    Directory of Open Access Journals (Sweden)

    M. Moghaddam Dorafshani

    2012-10-01

    Full Text Available Introduction & Objective: The normal females reproductive function , needs hypothalamus-hypophysis-ovarian extensive hormonal messages. Primary hypothyroidism is characterized by reduced production and secretion of thyroid hormones. During follicular growth PCNA (Proliferating Cell Nuclear Antigen and cycklin D complex play an important role in regulating cell proliferation .This study aimed to determine the cell proliferation index and how this process changes induced by thyroid hormone decreased in rat ovarian follicles.Materials & Methods: In this experimental study, 20 Wistar female rats were divided into experimental and control groups. Experimental group was chemically thyroidectomized by administering propylthiouracil (PTU (500 mg per liter of drinking water. The control group received normal drinking water. After three weeks rats were killed and their ovaries dissected and fixed for the histological preparation. Cell proliferation was determined by PCNA and stereological methods were used for counting cells.Results: Cell proliferation index showed a significant decrease in the frequency of follicular growth from prenatal to graafian follicles in hypothyroidism groups(P0.05 . PCNA expression determined that Primary follicle growth begins earlier. Positive PCNA cells were not observed in primordial follicles of the groups.Conclusion: According to the results of our study, this hypothesis is raised that granulosa cells in growing follicles may be increased by follicle adjacent cells in ovarian stroma . Hormonal changes following the reduction of thyroid hormones may greatly affect the cell proliferation index and lead to faster follicle degeneration.(Sci J Hamadan Univ Med Sci 2012; 19 (3:5-15

  8. Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells.

    Science.gov (United States)

    Sundaresan, Sinju; Meininger, Cameron A; Kang, Anthony J; Photenhauer, Amanda L; Hayes, Michael M; Sahoo, Nirakar; Grembecka, Jolanta; Cierpicki, Tomasz; Ding, Lin; Giordano, Thomas J; Else, Tobias; Madrigal, David J; Low, Malcolm J; Campbell, Fiona; Baker, Ann-Marie; Xu, Haoxing; Wright, Nicholas A; Merchant, Juanita L

    2017-12-01

    The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. Primary enteric glial cultures were generated from the VillinCre:Men1 FL/FL :Sst -/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. MEN1-associated

  9. STK35L1 associates with nuclear actin and regulates cell cycle and migration of endothelial cells.

    Directory of Open Access Journals (Sweden)

    Pankaj Goyal

    Full Text Available BACKGROUND: Migration and proliferation of vascular endothelial cells are essential for repair of injured endothelium and angiogenesis. Cyclins, cyclin-dependent kinases (CDKs, and cyclin-dependent kinase inhibitors play an important role in vascular tissue injury and wound healing. Previous studies suggest a link between the cell cycle and cell migration: cells present in the G(1 phase have the highest potential to migrate. The molecular mechanism linking these two processes is not understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we explored the function of STK35L1, a novel Ser/Thr kinase, localized in the nucleus and nucleolus of endothelial cells. Molecular biological analysis identified a bipartite nuclear localization signal, and nucleolar localization sequences in the N-terminal part of STK35L1. Nuclear actin was identified as a novel binding partner of STK35L1. A class III PDZ binding domains motif was identified in STK35L1 that mediated its interaction with actin. Depletion of STK35L1 by siRNA lead to an accelerated G(1 to S phase transition after serum-stimulation of endothelial cells indicating an inhibitory role of the kinase in G(1 to S phase progression. Cell cycle specific genes array analysis revealed that one gene was prominently downregulated (8.8 fold in STK35L1 silenced cells: CDKN2A alpha transcript, which codes for p16(INK4a leading to G(1 arrest by inhibition of CDK4/6. Moreover in endothelial cells seeded on Matrigel, STK35L1 expression was rapidly upregulated, and silencing of STK35L1 drastically inhibited endothelial sprouting that is required for angiogenesis. Furthermore, STK35L1 depletion profoundly impaired endothelial cell migration in two wound healing assays. CONCLUSION/SIGNIFICANCE: The results indicate that by regulating CDKN2A and inhibiting G1- to S-phase transition STK35L1 may act as a central kinase linking the cell cycle and migration of endothelial cells. The interaction of STK35L1 with nuclear

  10. Potential of adipose-derived mesenchymal stem cells and skeletal muscle-derived satellite cells for somatic cell nuclear transfer mediated transgenesis in Arbas Cashmere goats.

    Directory of Open Access Journals (Sweden)

    Yu Ren

    Full Text Available Somatic cell nuclear transfer is used to generate genetic models for research and new, genetically modified livestock varieties. Goat fetal fibroblast cells (gFFCs are the predominant nuclear donors in Cashmere goat transgenic cloning, but have disadvantages. We evaluated the potential of goat adipose-derived mesenchymal stem cells (gADSCs and goat skeletal muscle-derived satellite cells (gMDSCs for somatic cell nuclear transfer, evaluating their proliferation, pluripotency, transfection efficiency and capacity to support full term development of embryos after additive gene transfer or homologous recombination. gADSCs and gMDSCs were isolated by enzyme digestion and differentiated into neurocytes, myotube cells and insulin-producing cells. Neuron-specific enolase, fast muscle myosin and insulin expression were determined by immunohistochemistry. Following somatic cell nuclear transfer with donor cells derived from gADSCs, gMDSCs and gFFCs, transfection and cloning efficiencies were compared. Red fluorescent protein levels were determined by quantitative PCR and western blotting. 5-Methylcytosine, H4K5, H4K12 and H3K18 were determined immunohistochemically. gADSCs and gMDSCs were maintained in culture for up to 65 passages, whereas gFFCs could be passaged barely more than 15 times. gADSCs and gMDSCs had higher fluorescent colony forming efficiency and greater convergence (20% and cleavage (10% rates than gFFCs, and exhibited differing H4K5 histone modification patterns after somatic cell nuclear transfer and in vitro cultivation. After transfection with a pDsRed2-1 expression plasmid, the integrated exogenous genes did not influence the pluripotency of gADSCs-pDsRed2-1 or gMDSCs-pDsRed2-1. DsRed2 mRNA expression by cloned embryos derived from gADSCs-pDsRed2-1 or gMDSCs-pDsRed2-1 was more than twice that of gFFCs-pDsRed2-1 embryos (P<0.01. Pregnancy rates of gADSCs-pDsRed2-1 and gMDSCs-pDsRed2-1 recipients were higher than those of gFFCs-pDsRed2

  11. Potential of adipose-derived mesenchymal stem cells and skeletal muscle-derived satellite cells for somatic cell nuclear transfer mediated transgenesis in Arbas Cashmere goats.

    Science.gov (United States)

    Ren, Yu; Wu, Haiqing; Ma, Yuzhen; Yuan, Jianlong; Liang, Hao; Liu, Dongjun

    2014-01-01

    Somatic cell nuclear transfer is used to generate genetic models for research and new, genetically modified livestock varieties. Goat fetal fibroblast cells (gFFCs) are the predominant nuclear donors in Cashmere goat transgenic cloning, but have disadvantages. We evaluated the potential of goat adipose-derived mesenchymal stem cells (gADSCs) and goat skeletal muscle-derived satellite cells (gMDSCs) for somatic cell nuclear transfer, evaluating their proliferation, pluripotency, transfection efficiency and capacity to support full term development of embryos after additive gene transfer or homologous recombination. gADSCs and gMDSCs were isolated by enzyme digestion and differentiated into neurocytes, myotube cells and insulin-producing cells. Neuron-specific enolase, fast muscle myosin and insulin expression were determined by immunohistochemistry. Following somatic cell nuclear transfer with donor cells derived from gADSCs, gMDSCs and gFFCs, transfection and cloning efficiencies were compared. Red fluorescent protein levels were determined by quantitative PCR and western blotting. 5-Methylcytosine, H4K5, H4K12 and H3K18 were determined immunohistochemically. gADSCs and gMDSCs were maintained in culture for up to 65 passages, whereas gFFCs could be passaged barely more than 15 times. gADSCs and gMDSCs had higher fluorescent colony forming efficiency and greater convergence (20%) and cleavage (10%) rates than gFFCs, and exhibited differing H4K5 histone modification patterns after somatic cell nuclear transfer and in vitro cultivation. After transfection with a pDsRed2-1 expression plasmid, the integrated exogenous genes did not influence the pluripotency of gADSCs-pDsRed2-1 or gMDSCs-pDsRed2-1. DsRed2 mRNA expression by cloned embryos derived from gADSCs-pDsRed2-1 or gMDSCs-pDsRed2-1 was more than twice that of gFFCs-pDsRed2-1 embryos (P<0.01). Pregnancy rates of gADSCs-pDsRed2-1 and gMDSCs-pDsRed2-1 recipients were higher than those of gFFCs-pDsRed2-1 recipients (P

  12. Automation in nuclear hot cells (Paper No. 020)

    International Nuclear Information System (INIS)

    Pal, B.C.; Chougule, A.S.; Radke, M.G.; Ramaswamy, N.V.; Ramkumar, M.S.

    1987-02-01

    Bhabha Atomic Research Centre (BARC) in Trombay produces radioactive sources for a wide variety of uses in industry, agriculture and medicine, both within the country and abroad. The production and delivery of the radioactive sources in a form, ready for the end use, entails a number of operations, most of which are to be done with remote handling facilities, to maintain the prescribed biological shield to protect the operators. One of the repetitive operation among these which has to be done inside a concrete shielded hot-cell is the picking up of tiny radioactive wafers of iridium as small as 2.5mm dia., 0.3mm thick placing them in the required numbers to make up the total activity inside a capsule, closing the capsule with a top lid and finally welding the capsule. For doing this job remotely, recourse had to be taken to the use of master-slave manipulators (MSM), needing highly skilled operators to handle it for such delicate jobs repetitively. The operations for this repetitive job unlike most of other hot-cell operations, can be structured for machine operation and also fully automated. An automated system synthesising electromechanical, pneumatic and welding operations developed by the Division of Remote Handling and Robotics, BARC is described here. This relieves the operator of a number of jobs, to be repetitively done by MSM which would be strenous and taxing on account of the extremely small sizes of the pellets and wafers when they are to be handled, by remote indirect means with reliance on the master slave manipulators. A description of the automated system is given. (author). 3 figs

  13. Nuclear localization of the mitochondrial ncRNAs in normal and cancer cells.

    Science.gov (United States)

    Landerer, Eduardo; Villegas, Jaime; Burzio, Veronica A; Oliveira, Luciana; Villota, Claudio; Lopez, Constanza; Restovic, Franko; Martinez, Ronny; Castillo, Octavio; Burzio, Luis O

    2011-08-01

    We have previously shown a differential expression of a family of mitochondrial ncRNAs in normal and cancer cells. Normal proliferating cells and cancer cells express the sense mitochondrial ncRNA (SncmtRNA). In addition, while normal proliferating cells express two antisense mitochondrial ncRNAs (ASncmtRNAs-1 and -2), these transcripts seem to be universally down-regulated in cancer cells. In situ hybridization (ISH) of some normal and cancer tissues reveals nuclear localization of these transcripts suggesting that they are exported from mitochondria. FISH and confocal microscopy, in situ digestion with RNase previous to ISH and electron microscopy ISH was employed to confirm the extra-mitochondrial localization of the SncmtRNA and the ASncmtRNAs in normal proliferating and cancer cells of human and mouse. In normal human kidney and mouse testis the SncmtRNA and the ASncmtRNAs were found outside the organelle and especially localized in the nucleus associated to heterochromatin. In cancer cells, only the SncmtRNA was expressed and was found associated to heterochromatin and nucleoli. The ubiquitous localization of these mitochondrial transcripts in the nucleus suggests that they are new players in the mitochondrial-nuclear communication pathway or retrograde signaling. Down regulation of the ASncmtRNAs seems to be an important step on neoplastic transformation and cancer progression.

  14. Nuclear trafficking of secreted factors and cell-surface receptors: new pathways to regulate cell proliferation and differentiation, and involvement in cancers

    Directory of Open Access Journals (Sweden)

    Planque Nathalie

    2006-10-01

    Full Text Available Abstract Secreted factors and cell surface receptors can be internalized by endocytosis and translocated to the cytoplasm. Instead of being recycled or proteolysed, they sometimes translocate to the nucleus. Nuclear import generally involves a nuclear localization signal contained either in the secreted factor or its transmembrane receptor, that is recognized by the importins machinery. In the nucleus, these molecules regulate transcription of specific target genes by direct binding to transcription factors or general coregulators. In addition to the transcription regulation, nuclear secreted proteins and receptors seem to be involved in other important processes for cell life and cellular integrity such as DNA replication, DNA repair and RNA metabolism. Nuclear secreted proteins and transmembrane receptors now appear to induce new signaling pathways to regulate cell proliferation and differentiation. Their nuclear localization is often transient, appearing only during certain phases of the cell cycle. Nuclear secreted and transmembrane molecules regulate the proliferation and differentiation of a large panel of cell types during embryogenesis and adulthood and are also potentially involved in wound healing. Secreted factors such as CCN proteins, EGF, FGFs and their receptors are often detected in the nucleus of cancer cells. Nuclear localization of these molecules has been correlated with tumor progression and poor prognosis for patient survival. Nuclear growth factors and receptors may be responsible for resistance to radiotherapy.

  15. Insertion of a nuclear factor kappa B DNA nuclear-targeting sequence potentiates suicide gene therapy efficacy in lung cancer cell lines

    DEFF Research Database (Denmark)

    Cramer, F; Christensen, C L; Poulsen, T T

    2012-01-01

    Lung cancer currently causes the majority of cancer-related deaths worldwide and new treatments are in high demand. Gene therapy could be a promising treatment but currently lacks sufficient efficiency for clinical use, primarily due to limited cellular and nuclear DNA delivery. In the present...... study, we investigated whether it was possible to exploit the endogenous nuclear-shuttling activity by the nuclear factor kappa B (NFκB) system, which is highly prominent in many cancers as well as lung cancer. We observed that insertion of a DNA nuclear-targeting sequence (DTS) recognized by NFκB could...... improve plasmid nuclear delivery and enhance the therapeutic effect of a validated transcriptionally cancer-targeted suicide gene therapy system. A clear correlation between the number of inserted NFκB-binding sites and the therapeutic effect of the suicide system was observed in both small cell lung...

  16. Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, Edith E., E-mail: ed.mueller@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Mayr, Johannes A., E-mail: h.mayr@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Zimmermann, Franz A., E-mail: f.zimmermann@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Feichtinger, Rene G., E-mail: r.feichtinger@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Stanger, Olaf, E-mail: o.stanger@rbht.nhs.uk [Department of Cardiac Surgery, Paracelsus Medical University, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Sperl, Wolfgang, E-mail: w.sperl@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria); Kofler, Barbara, E-mail: b.kofler@salk.at [Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Muellner Hauptstrasse 48, 5020 Salzburg (Austria)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complex II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.

  17. Role of nesprin-1 in nuclear deformation in endothelial cells under static and uniaxial stretching conditions

    International Nuclear Information System (INIS)

    Anno, Toshiro; Sakamoto, Naoya; Sato, Masaaki

    2012-01-01

    Highlights: ► Nesprin-1 knockdown decreases widths of nuclei in ECs under static condition. ► Nuclear strain caused by stretching is increased by nesprin-1 knockdown in ECs. ► We model mechanical interactions of F-actin with the nucleus in stretched cells. ► F-actin bound to nesprin-1 may cause sustainable force transmission to the nucleus. -- Abstract: The linker of nucleus and cytoskeleton (LINC) complex, including nesprin-1, has been suggested to be crucial for many biological processes. Previous studies have shown that mutations in nesprin-1 cause abnormal cellular functions and diseases, possibly because of insufficient force transmission to the nucleus through actin filaments (F-actin) bound to nesprin-1. However, little is known regarding the mechanical interaction between the nucleus and F-actin through nesprin-1. In this study, we examined nuclear deformation behavior in nesprin-1 knocked-down endothelial cells (ECs) subjected to uniaxial stretching by evaluating nuclear strain from lateral cross-sectional images. The widths of nuclei in nesprin-1 knocked-down ECs were smaller than those in wild-type cells. In addition, nuclear strain in nesprin-1 knocked-down cells, which is considered to be compressed by the actin cortical layer, increased compared with that in wild-type cells under stretching condition. These results indicate that nesprin-1 knockdown releases the nucleus from the tension of F-actin bound to the nucleus, thereby increasing allowance for deformation before stretching, and that F-actin bound to the nucleus through nesprin-1 causes sustainable force transmission to the nucleus.

  18. Nuclear expression of claudin-3 in human colorectal adenocarcinoma cell lines and tissues.

    Science.gov (United States)

    Tokuhara, Yasunori; Morinishi, Tatsuya; Matsunaga, Toru; Sakai, Manabu; Sakai, Takayoshi; Ohsaki, Hiroyuki; Kadota, Kyuichi; Kushida, Yoshio; Haba, Reiji; Hirakawa, Eiichiro

    2018-01-01

    Claudins are members of a large family of transmembrane proteins, which are essential for the formation of tight junctions and have a significant effect on the biological behavior of tumor progression. Previous studies have demonstrated that several claudins show aberrant expression patterns in numerous types of cancer. The present study investigated the expression and localization of claudin-3 and claudin-7 in human colorectal adenocarcinoma cell lines and tissues. The protein expression levels of claudin-3 and claudin-7 were determined using immunocytochemical and immunohistochemical staining. Claudin-3, but not claudin-7, exhibited nuclear localization in the human colorectal adenocarcinoma Caco-2 and SW620 cell lines. Surgically resected colorectal adenocarcinoma tissue specimens were obtained, and the associations between the expression of claudin-3 or claudin-7 and various clinicopathological parameters were analyzed. The membranous expression rates of claudin-3 and claudin-7 were 58.0 and 50.0%, while their nuclear expression rates were 22.0 and 2.0%, respectively. The membranous expression of claudin-3 and claudin-7 was not associated with any clinicopathological factors, whereas the nuclear expression of claudin-3 was associated with histological type and was significantly increased in colorectal mucinous adenocarcinomas compared with that in well- to moderately-differentiated colorectal adenocarcinomas (P<0.01). However, no associations were observed between the nuclear expression of claudin-7 and any clinicopathological parameter. In conclusion, the nuclear expression of claudin-3 in colorectal mucinous adenocarcinoma may be involved in the biological transformation of tumors. The results from the present study indicated that claudin-3 is an important protein associated with histological type and has potential as a prognostic marker. Although the mechanisms underlying the nuclear localization of claudin-3 in tumorigenesis have not yet been elucidated in

  19. Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

    Science.gov (United States)

    Iuso, Domenico; Czernik, Marta; Di Egidio, Fiorella; Sampino, Silvestre; Zacchini, Federica; Bochenek, Michal; Smorag, Zdzislaw; Modlinski, Jacek A; Ptak, Grazyna; Loi, Pasqualino

    2013-01-01

    The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

  20. Nuclear microanalysis of platinum and trace elements in cisplatin-resistant human ovarian adenocarcinoma cells

    Science.gov (United States)

    Moretto, P.; Ortega, R.; Llabador, Y.; Simonoff, M.; Bénard, J.; Moretto, Ph.

    1995-09-01

    Macro-and Micro-PIXE analysis were applied to study the mechanisms of cellular resistance to cisplatin, a chemotherapeutic agent, widely used nowadays for the treatment of ovarian cancer. Two cultured cell lines, a cisplatin-sensitive and a resistant one, were compared for their trace elements content and platinum accumulation following in vitro exposure to the drug. Bulk analysis revealed significant differences in copper and iron content between the two lines. Subsequent individual cell microanalysis permitted us to characterize the response of the different morphological cell types of the resistant line. This study showed that the metabolism of some trace metals in cisplatin-resistant cells could be affected but the exact relationship with the resistant phenotype remains to be determined. From a technical point of view, this experiment demonstrated that an accurate measurement of trace elements could be derived from nuclear microprobe analysis of individual cell.

  1. Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Domenico Iuso

    Full Text Available The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT. Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

  2. CYP3A5 regulates prostate cancer cell growth by facilitating nuclear translocation of AR.

    Science.gov (United States)

    Mitra, Ranjana; Goodman, Oscar B

    2015-04-01

    The central role of androgen receptor (AR) signaling is established in prostate cancer growth and progression. We propose CYP3A5 is part of a feedback loop that modulates the sensitivity of AR to androgen exposure. The purpose of this study is to elucidate the mechanism of regulation of AR expression by CYP3A5. To identify the role of CYP3A5 in regulating AR signaling, CYP3A5 protein expression was inhibited using CYP3A5 siRNA and azamulin. Both cell fractionation and immunocytochemical approaches in combination with dihydrotestosterone (DHT) and R1881 treatment were used to evaluate changes in AR nuclear translocation. CYP3A5 siRNA blocked growth of LNCaP and C4-2 cells by 30-60% (P ≤ 0.005). Azamulin, a CYP3A pharmacologic inhibitor, reduced the growth of LNCaP, C4-2 and 22RV1 lines by ∼ 40% (P ≤ 0.005). CYP3A5 siRNA inhibited growth in response to DHT and R1881 treatment in LNCaP and C4-2 by decreasing nuclear AR localization and resulting in diminished PSA and TMPRSS2 expression. Decreased AR nuclear localization resulting from CYP3A5 inhibition resulted in growth inhibition comparable to IC60 and IC40 of bicalutamide in LNCaP and C4-2 cell lines. Conversely, the CYP3A inducer rifampicin enhanced AR nuclear localization. As CYP3A5 regulates the nuclear translocation of AR; co-targeting CYP3A5 may provide a novel strategy for enhancing the efficacy of androgen deprivation therapy. Consequentially, these data suggest that concomitant medications may impact androgen deprivation therapy's efficacy. © 2015 Wiley Periodicals, Inc.

  3. Nuclear expression of Rac1 in cervical premalignant lesions and cervical cancer cells

    International Nuclear Information System (INIS)

    Mendoza-Catalán, Miguel A; Castañeda-Saucedo, Eduardo; Cristóbal-Mondragón, Gema R; Adame-Gómez, Jesús; Valle-Flores, Heidi N del; Coppe, José Fco; Sierra-López, Laura; Romero-Hernández, Mirna A; Carmen Alarcón-Romero, Luz del; Illades-Aguiar, Berenice

    2012-01-01

    Abnormal expression of Rho-GTPases has been reported in several human cancers. However, the expression of these proteins in cervical cancer has been poorly investigated. In this study we analyzed the expression of the GTPases Rac1, RhoA, Cdc42, and the Rho-GEFs, Tiam1 and beta-Pix, in cervical pre-malignant lesions and cervical cancer cell lines. Protein expression was analyzed by immunochemistry on 102 cervical paraffin-embedded biopsies: 20 without Squamous Intraepithelial Lesions (SIL), 51 Low- grade SIL, and 31 High-grade SIL; and in cervical cancer cell lines C33A and SiHa, and non-tumorigenic HaCat cells. Nuclear localization of Rac1 in HaCat, C33A and SiHa cells was assessed by cellular fractionation and Western blotting, in the presence or not of a chemical Rac1 inhibitor (NSC23766). Immunoreacivity for Rac1, RhoA, Tiam1 and beta-Pix was stronger in L-SIL and H-SIL, compared to samples without SIL, and it was significantly associated with the histological diagnosis. Nuclear expression of Rac1 was observed in 52.9% L-SIL and 48.4% H-SIL, but not in samples without SIL. Rac1 was found in the nucleus of C33A and SiHa cells but not in HaCat cells. Chemical inhibition of Rac1 resulted in reduced cell proliferation in HaCat, C33A and SiHa cells. Rac1 is expressed in the nucleus of epithelial cells in SILs and cervical cancer cell lines, and chemical inhibition of Rac1 reduces cellular proliferation. Further studies are needed to better understand the role of Rho-GTPases in cervical cancer progression

  4. Distinct roles for classical nuclear import receptors in the growth of multinucleated muscle cells.

    Science.gov (United States)

    Hall, Monica N; Griffin, Christine A; Simionescu, Adriana; Corbett, Anita H; Pavlath, Grace K

    2011-09-01

    Proper muscle function is dependent on spatial and temporal control of gene expression in myofibers. Myofibers are multinucleated cells that are formed, repaired and maintained by the process of myogenesis in which progenitor myoblasts proliferate, differentiate and fuse. Gene expression is dependent upon proteins that require facilitated nuclear import, however little is known about the regulation of nucleocytoplasmic transport during the formation of myofibers. We analyzed the role of karyopherin alpha (KPNA), a key classical nuclear import receptor, during myogenesis. We established that five karyopherin alpha paralogs are expressed by primary mouse myoblasts in vitro and that their steady-state levels increase in multinucleated myotubes, suggesting a global increase in demand for classical nuclear import during myogenesis. We used siRNA-mediated knockdown to identify paralog-specific roles for KPNA1 and KPNA2 during myogenesis. KPNA1 knockdown increased myoblast proliferation, whereas KPNA2 knockdown decreased proliferation. In contrast, no proliferation defect was observed with KPNA4 knockdown. Only knockdown of KPNA2 decreased myotube growth. These results identify distinct pathways involved in myoblast proliferation and myotube growth that rely on specific nuclear import receptors suggesting that regulation of classical nuclear import pathways likely plays a critical role in controlling gene expression in skeletal muscle. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Transcriptional activation of nuclear-related factor 2 by FK506 in Jurkat T cells.

    Science.gov (United States)

    Chung, Y W; Jeong, Y M; Chung, M W; Choi, S K; Choi, S J N; Chung, S Y

    2015-04-01

    We investigated the effect of FK506 in transcriptional activation of nuclear factor (erythroid-derived 2)-like2 (Nrf2) in human Jurkat T cells. FK506 treatment increased the generation of reactive oxygen species and reactive nitrogen species in Jurkat cells in a dose-dependent manner. Generation of nitric oxide was also increased after treatment with FK506 in Jurkat cells. Peak levels of endothelial nitricoxide synthase expression occurred at 24 hours and then decreased after 48 hours. We found that a marked dissociation of Nrf 2 from Kelch-like ECH-associated protein-1 and subsequently Nrf 2 nuclear translocation occurred in Jurkat cells treated with FK506 during 48 hours. Immunohistochemistry and Western blot analysis data revealed that the FK506 treatment increased expression of heme oxygenase-1 (HO-1) in Jurkat cells in a dose-dependent manner. HO-1 expression was induced after 6 hours of treatment of FK506 to Jurkat cells, peaked at 24 hours, and then decreased after 48 hours. These results suggest that FK506 induces Nrf 2-driven transcriptional activation of the antioxidant response element by activating HO-1 and free radicals such as reactive oxygen species and nitric oxide. Copyright © 2015. Published by Elsevier Inc.

  6. Design of nuclear fuel cells by means of a statistical analysis and a sensibility study

    International Nuclear Information System (INIS)

    Jauregui C, V.; Castillo M, J. A.; Ortiz S, J. J.; Montes T, J. L.; Perusquia del C, R.

    2013-10-01

    This work contains the results of the statistical analysis realized to study the nuclear fuel cells performance, considering the frequencies for the election of fuel bars used in the design of the same ones. The election of the bars used for the cells design are of 3 types, the first election shows that to the plotting the respective frequency is similar to a normal distribution, in the second case the frequencies graph is of type inverted square X 2 and the last election is when the bars are chosen in aleatory form. The heuristic techniques used for the cells design were the neural networks, the ant colonies and a hybrid between the dispersed search and the trajectories re-linkage. To carry out the statistical analysis in the cells design were considered the local power peak factor and the neutron infinite multiplication factor (k∞) of this. On the other hand, the performance of the designed cells was analyzed when verifying the position of the bars containing gadolinium. The results show that is possible to design cells of nuclear fuel with a good performance, when considering the frequency of the bars used in their design. (Author)

  7. Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential.

    Science.gov (United States)

    Song, Kai; Song, Yong; Zhao, Xiao-Ping; Shen, Hui; Wang, Meng; Yan, Ting-Lin; Liu, Ke; Shang, Zheng-Jun

    2014-10-15

    Most previous studies have linked cancer-macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Bone Marrow Mesenchymal Stem Cells Are an Attractive Donor Cell Type for Production of Cloned Pigs As Well As Genetically Modified Cloned Pigs by Somatic Cell Nuclear Transfer

    Science.gov (United States)

    Li, Zicong; He, Xiaoyan; Chen, Liwen; Shi, Junsong; Zhou, Rong; Xu, Weihua

    2013-01-01

    Abstract The somatic cell nuclear transfer (SCNT) technique has been widely applied to clone pigs or to produce genetically modified pigs. Currently, this technique relies mainly on using terminally differentiated fibroblasts as donor cells. To improve cloning efficiency, only partially differentiated multipotent mesenchymal stem cells (MSCs), thought to be more easily reprogrammed to a pluripotent state, have been used as nuclear donors in pig SCNT. Although in vitro–cultured embryos cloned from porcine MSCs (MSCs-embryos) were shown to have higher preimplantation developmental ability than cloned embryos reconstructed from fibroblasts (Fs-embryos), the difference in in vivo full-term developmental rate between porcine MSCs-embryos and Fs-embryos has not been investigated so far. In this study, we demonstrated that blastocyst total cell number and full-term survival abilities of MSCs-embryos were significantly higher than those of Fs-embryos cloned from the same donor pig. The enhanced developmental potential of MSCs-embryos may be associated with their nuclear donors' DNA methylation profile, because we found that the methylation level of imprinting genes and repeat sequences differed between MSCs and fibroblasts. In addition, we showed that use of transgenic porcine MSCs generated from transgene plasmid transfection as donor cells for SCNT can produce live transgenic cloned pigs. These results strongly suggest that porcine bone marrow MSCs are a desirable donor cell type for production of cloned pigs and genetically modified cloned pigs via SCNT. PMID:24033142

  9. Nuclear organization of PML bodies in leukaemic and multiple myeloma cells

    Czech Academy of Sciences Publication Activity Database

    Kroupová, Jana; Harničarová, Andrea; Kůrová, Jana; Uhlířová, Radka; Kozubek, Stanislav; Legartová, Soňa; Hájek, R.; Bártová, Eva

    2008-01-01

    Roč. 32, č. 12 (2008), s. 1866-1877 ISSN 0145-2126 R&D Projects: GA ČR(CZ) GA204/06/0978; GA MŠk(CZ) LC06027 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : PML bodies * nuclear arrangement * leukaemic cells Subject RIV: BO - Biophysics Impact factor: 2.390, year: 2008

  10. Identification of abnormal gene expression in bovine transgenic somatic cell nuclear transfer embryos

    OpenAIRE

    Cho, Jongki; Kang, Sungkeun; Lee, Byeong Chun

    2014-01-01

    This study was conducted to investigate the expression of three genes related to early embryonic development in bovine transgenic cloned embryos. To accomplish this, development of bovine transgenic somatic cell nuclear transfer (SCNT) embryos was compared with non-transgenic embryos. Next, mRNA transcription of three specific genes (DNMT1, Hsp 70.1, and Mash2) related to early embryo development in transgenic SCNT embryos was compared between transgenic and non-transgenic SCNTs, parthenogene...

  11. Analysis and design recommendation on rabbeted capping plate of equipment cell in nuclear chemical facility

    International Nuclear Information System (INIS)

    Zhang Jingyu; Yin Xiaozhan

    2013-01-01

    Rabbeted capping plates are widely used in the roof of equipment cells in order to meet the requirements of nuclear radiation protection. The key considerations in the design include vertical load, seismic load and repair load. This article establishes T shaped and Z-shaped plate model via FEM software (ANSYS), analyzes the bearing capacity and displacement distribution in different load cases, and provides recommendations to the design and construction accordingly. (authors)

  12. Hot cell works and related irradiation tests in fission reactor for development of new materials for nuclear application

    International Nuclear Information System (INIS)

    Shikama, Tatsuo

    1999-01-01

    Present status of research works in Oarai Branch, Institute for Materials Research, Tohoku University, utilizing Japan Materials Testing Reactor and related hot cells will be described.Topics are mainly related with nuclear materials studies, excluding fissile materials, which is mainly aiming for development of materials for advanced nuclear systems such as a nuclear fusion reactor. Conflict between traditional and routined procedures and new demands will be described and future perspective is discussed. (author)

  13. Gold-nanoparticle-assisted laser perturbation of chromatin assembly reveals unusual aspects of nuclear architecture within living cells.

    Science.gov (United States)

    Mazumder, Aprotim; Shivashankar, G V

    2007-09-15

    Chromatin organization within the nucleus is a vital regulator of genome function, yet its mechanical coupling to the nuclear architecture has remained elusive. To directly investigate this coupling, we locally modulated chromatin structure in living cells using nanoparticle-based laser perturbation. Unusual differences in the response of the cell nucleus were observed depending on the nuclear region that was perturbed--the heterochromatin, the euchromatin, and the nuclear envelope. This response varied under different conditions of cellular perturbations such as ATP depletion, apoptosis, and inhibition of histone deacetylases. Our studies implicate heterochromatin organization in imparting mechanical stability to the cell nucleus and suggest that nuclear size and shape are the result of interplay between nuclear and cytoplasmic anchors.

  14. Stereological estimation of nuclear volume and other quantitative histopathological parameters in the prognostic evaluation of supraglottic laryngeal squamous cell carcinoma

    DEFF Research Database (Denmark)

    Sørensen, Flemming Brandt; Bennedbaek, O; Pilgaard, J

    1989-01-01

    observers of the latter was poor in the material which consisted of 35 biopsy specimens. Unbiased estimates of nuclear Vv were on the average 385 microns3 (CV = 0.44), with more than 90% of the associated variance attributable to differences in nuclear Vv among individual lesions. Nuclear Vv was positively......The aim of this study was to investigate various approaches to the grading of malignancy in pre-treatment biopsies from patients with supraglottic laryngeal squamous cell carcinoma. The prospects of objective malignancy grading based on stereological estimation of the volume-weighted mean nuclear...... volume, nuclear Vv, and nuclear volume fraction, Vv(nuc/tis), along with morphometrical 2-dimensional estimation of nuclear density index, NI, and mitotic activity index, MI, were investigated and compared with the current morphological, multifactorial grading system. The reproducibility among two...

  15. Stereological estimation of nuclear volume and other quantitative histopathological parameters in the prognostic evaluation of supraglottic laryngeal squamous cell carcinoma

    DEFF Research Database (Denmark)

    Sørensen, Flemming Brandt; Bennedbaek, O; Pilgaard, J

    1989-01-01

    The aim of this study was to investigate various approaches to the grading of malignancy in pre-treatment biopsies from patients with supraglottic laryngeal squamous cell carcinoma. The prospects of objective malignancy grading based on stereological estimation of the volume-weighted mean nuclear...... volume, nuclear Vv, and nuclear volume fraction, Vv(nuc/tis), along with morphometrical 2-dimensional estimation of nuclear density index, NI, and mitotic activity index, MI, were investigated and compared with the current morphological, multifactorial grading system. The reproducibility among two...... observers of the latter was poor in the material which consisted of 35 biopsy specimens. Unbiased estimates of nuclear Vv were on the average 385 microns3 (CV = 0.44), with more than 90% of the associated variance attributable to differences in nuclear Vv among individual lesions. Nuclear Vv was positively...

  16. Inhibitory effect of benzene metabolites on nuclear DNA synthesis in bone marrow cells

    International Nuclear Information System (INIS)

    Lee, E.W.; Johnson, J.T.; Garner, C.D.

    1989-01-01

    Effects of endogenously produced and exogenously added benzene metabolites on the nuclear DNA synthetic activity were investigated using a culture system of mouse bone marrow cells. Effects of the metabolites were evaluated by a 30-min incorporation of [ 3 H]thymidine into DNA following a 30-min interaction with the cells in McCoy's 5a medium with 10% fetal calf serum. Phenol and muconic acid did not inhibit nuclear DNA synthesis. However, catechol, 1,2,4-benzenetriol, hydroquinone, and p-benzoquinone were able to inhibit 52, 64, 79, and 98% of the nuclear DNA synthetic activity, respectively, at 24 μM. In a cell-free DNA synthetic system, catechol and hydroquinone did not inhibit the incorporation of [ 3 H]thymidine triphosphate into DNA up to 24 μM but 1,2,4-benzenetriol and p-benzoquinone did. The effect of the latter two benzene metabolites was completely blocked in the presence of 1,4-dithiothreitol (1 mM) in the cell-free assay system. Furthermore, when DNA polymerase α, which requires a sulfhydryl (SH) group as an active site, was replaced by DNA polymerase 1, which does not require an SH group for its catalytic activity, p-benzoquinone and 1,2,4-benzenetriol were unable to inhibit DNA synthesis. Thus, the data imply the p-benzoquinone and 1,2,4-benzenetriol inhibited DNA polymerase α, consequently resulting in inhibition of DNA synthesis in both cellular and cell-free DNA synthetic systems. The present study identifies catechol, hydroquinone, p-benzoquinone, and 1,2,4-benzenetriol as toxic benzene metabolites in bone marrow cells and also suggests that their inhibitory action on DNA synthesis is mediated by mechanism(s) other than that involving DNA damage as a primary cause

  17. Serpent: an alternative for the nuclear fuel cells analysis of a BWR

    International Nuclear Information System (INIS)

    Silva A, L.; Del Valle G, E.; Gomez T, A. M.

    2013-10-01

    In the last ten years the diverse research groups in nuclear engineering of the Universidad Nacional Autonoma de Mexico and Instituto Politecnico Nacional (UNAM, IPN), as of research (Instituto Nacional de Investigaciones Nucleares, ININ) as well as the personnel of the Nuclear Plant Management of the Comision Federal de Electricidad have been using the codes Helios and /or CASMO-4 in the generation of cross sections (X S) of nuclear fuel cells of the cores corresponding to the Units 1 and 2 of the nuclear power plant of Laguna Verde. Both codes belong to the Studsvik-Scandpower Company who receives the payment for the use and their respective maintenance. In recent years, the code Serpent appears among the nuclear community distributed by the OECD/Nea which does not has cost neither in its use neither in its maintenance. The code is based on the Monte Carlo method and makes use of the processing in parallel. In the Escuela Superior de Fisica y Matematicas of the IPN, the personnel has accumulated certain experience in the use of Serpent under the direction of personal of the ININ; of this experience have been obtained for diverse fuel burned, the infinite multiplication factor for three cells of nuclear fuel, without control bar and with control bar for a known thermodynamic state fixed by: a) the fuel temperature (T f ), b) the moderator temperature (T m ) and c) the vacuums fraction (α). Although was not realized any comparison with the X S that the codes Helios and CASMO-4 generate, the results obtained for the infinite multiplication factor show the prospective tendencies with regard to the fuel burned so much in the case in that is not present the control bar like when it is. The results are encouraging and motivate to the study group to continue with the X S generation of a core in order to build the respective library of nuclear data as a following step and this can be used for the codes PARCS, of USA NRC, DYN3D of HZDR, or others developed locally in the

  18. Immunohistochemical detection for nuclear beta-catenin in sporadic basal cell carcinoma.

    Science.gov (United States)

    Yamazaki, F; Aragane, Y; Kawada, A; Tezuka, T

    2001-11-01

    Despite the increasing incidence of basal cell carcinoma (BCC), its pathogenesis has remained largely unknown. Recently, it was reported that genes involved in tissue morphogenesis, such as sonic hedgehog or patched, were found to be mutated in BCC, suggesting the involvement of those molecules in the pathogenesis of this tumour. Furthermore, there is evidence that the Wnt-mediated signalling pathway may be one of the downstream targets of sonic hedgehog-mediated signalling, which has led us to focus on molecular events on the Wnt pathway in BCC. Among the signal transducers involved in the Wnt pathway, it is clear that beta-catenin plays a pivotal role in the promotion of morphogenesis and cell growth. In respect to this, it has been reported that, in particular circumstances, as in colorectal cancers, beta-catenin migrates to the nuclei, where it exerts an ability to activate the transcription of various genes. To investigate the cellular distribution of beta-catenin in skin tumours, in particular, in BCC. Twenty skin biopsy specimens derived from BCC, 10 from inflammatory skin diseases and five from squamous cell carcinomas were immunostained with an antibody directed against beta-catenin. Fourteen of the 20 BCC samples tested showed nuclear localization of beta-catenin, while none of the other samples gave rise to positive nuclear staining. Nuclear localization of beta-catenin is a characteristic feature of BCC; this suggests its tumorigenic role in this tumour. This gives us a further insight into the molecular pathogenesis of BCC.

  19. Peripheral blood cells among community residents living near nuclear power plants

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yuan-Teh; Hsu, Hsiu-Ching; Chien, Kuo-Liong; Yang, Chi-Yu; Chen, Wen Jone [Department of Internal Medicine, National Taiwan University College of Medicine, No. 7 Chungshan South Road, 10020 Taipei (Taiwan, Province of China); Sung, Fung C. [Institute of Environmental Health, National Taiwan University College of Public Health, Taipei (Taiwan, Province of China); Lin, Ruey S. [Institute of Epidemiology, National Taiwan University College of Public Health, Taipei (Taiwan, Province of China)

    2001-12-03

    Information about hematopoieses as a result of exposure to very low levels of radiation is scarce. To investigate the human hematopoietic effect of very low level radiation exposure, measurements of peripheral blood components were performed among 3602 men and women, aged 35 and above, living in a community near two nuclear power installations in Chinshan, Taiwan. The radiation level that each individual was exposed to was represented by a surrogate level, '+', a transformed distance from each individual's residence to the two power plants D{sub 1} and D{sub 2}. In addition to comparing average hematology measurements, multiple regression analyses were done to include age, gender, smoking, drinking status and the surrogate radiation exposure level as independent variables. Univariate and bivariate analyses showed that the hematology measurements had significant associations with age, gender, smoking or drinking. The multiple regression analyses revealed that significant positive associations with '+' were found for hemoglobin, hematocrit, platelet, white blood cell and red blood cell. The platelet count might increase for 208.7x10{sup 3}/{mu}l if the exposure from the nuclear plants increased by one exposure unit. This type of association implies that those who lived closer to the nuclear power installation had a higher blood cell count; we suspect that this could be a type of radiation hormesis.

  20. Production of transgenic canine embryos using interspecies somatic cell nuclear transfer.

    Science.gov (United States)

    Hong, So Gun; Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Kim, Geon A; Koo, Ok Jae; Jang, Goo; Lee, Byeong Chun

    2012-02-01

    Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that

  1. Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

    Directory of Open Access Journals (Sweden)

    C. Soldani

    2010-05-01

    Full Text Available Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP-containing components (PANA, hnRNP-core proteins, fibrillarin or RNP-associated nuclear proteins (SC-35 splicing factor. Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures.

  2. The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

    DEFF Research Database (Denmark)

    Morovic, Martin; Murin, Matej; Strejcek, Frantisek

    2016-01-01

    One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription....... In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly...

  3. The nuclear receptor NR4A1 induces a form of cell death dependent on autophagy in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Jimena Bouzas-Rodríguez

    Full Text Available The control of cell death is a biological process essential for proper development, and for preventing devastating pathologies like cancer and neurodegeneration. On the other hand, autophagy regulation is essential for protein and organelle degradation, and its dysfunction is associated with overlapping pathologies like cancer and neurodegeneration, but also for microbial infection and aging. In the present report we show that two evolutionarily unrelated receptors--Neurokinin 1 Receptor (NK(1R, a G-protein coupled receptor, and Insulin-like Growth Factor 1 Receptor (IGF1R, a tyrosine kinase receptor--both induce non-apoptotic cell death with autophagic features and requiring the activity of the autophagic core machinery proteins PI3K-III, Beclin-1 and Atg7. Remarkably, this form of cell death occurs in apoptosis-competent cells. The signal transduction pathways engaged by these receptors both converged on the activation of the nuclear receptor NR4A1, which has previously been shown to play a critical role in some paradigms of apoptosis and in NK(1R-induced cell death. The activity of NR4A1 was necessary for IGF1R-induced cell death, as well as for a canonical model of cell death by autophagy induced by the presence of a pan-caspase inhibitor, suggesting that NR4A1 is a general modulator of this kind of cell death. During cell death by autophagy, NR4A1 was transcriptionally competent, even though a fraction of it was present in the cytoplasm. Interestingly, NR4A1 interacts with the tumor suppressor p53 but not with Beclin-1 complex. Therefore the mechanism to promote cell death by autophagy might involve regulation of gene expression, as well as protein interactions. Understanding the molecular basis of autophagy and cell death mediation by NR4A1, should provide novel insights and targets for therapeutic intervention.

  4. Nuclear Nox4 Role in Stemness Power of Human Amniotic Fluid Stem Cells

    Directory of Open Access Journals (Sweden)

    Tullia Maraldi

    2015-01-01

    Full Text Available Human amniotic fluid stem cells (AFSC are an attractive source for cell therapy due to their multilineage differentiation potential and accessibility advantages. However the clinical application of human stem cells largely depends on their capacity to expand in vitro, since there is an extensive donor-to-donor heterogeneity. Reactive oxygen species (ROS and cellular oxidative stress are involved in many physiological and pathophysiological processes of stem cells, including pluripotency, proliferation, differentiation, and stress resistance. The mode of action of ROS is also dependent on the localization of their target molecules. Thus, the modifications induced by ROS can be separated depending on the cellular compartments they affect. NAD(PH oxidase family, particularly Nox4, has been known to produce ROS in the nucleus. In the present study we show that Nox4 nuclear expression (nNox4 depends on the donor and it correlates with the expression of transcription factors involved in stemness regulation, such as Oct4, SSEA-4, and Sox2. Moreover nNox4 is linked with the nuclear localization of redox sensitive transcription factors, as Nrf2 and NF-κB, and with the differentiation potential. Taken together, these results suggest that nNox4 regulation may have important effects in stem cell capability through modulation of transcription factors and DNA damage.

  5. Nuclear Materials Characterization in the Materials and Fuels Complex Analytical Hot Cells

    International Nuclear Information System (INIS)

    Rodriquez, Michael

    2009-01-01

    As energy prices skyrocket and interest in alternative, clean energy sources builds, interest in nuclear energy has increased. This increased interest in nuclear energy has been termed the 'Nuclear Renaissance'. The performance of nuclear fuels, fuels and reactor materials and waste products are becoming a more important issue as the potential for designing new nuclear reactors is more immediate. The Idaho National Laboratory (INL) Materials and Fuels Complex (MFC) Analytical Laboratory Hot Cells (ALHC) are rising to the challenge of characterizing new reactor materials, byproducts and performance. The ALHC is a facility located near Idaho Falls, Idaho at the INL Site. It was built in 1958 as part of the former Argonne National Laboratory West Complex to support the operation of the second Experimental Breeder Reactor (EBR-II). It is part of a larger analytical laboratory structure that includes wet chemistry, instrumentation and radiochemistry laboratories. The purpose of the ALHC is to perform analytical chemistry work on highly radioactive materials. The primary work in the ALHC has traditionally been dissolution of nuclear materials so that less radioactive subsamples (aliquots) could be transferred to other sections of the laboratory for analysis. Over the last 50 years though, the capabilities within the ALHC have also become independent of other laboratory sections in a number of ways. While dissolution, digestion and subdividing samples are still a vitally important role, the ALHC has stand alone capabilities in the area of immersion density, gamma scanning and combustion gas analysis. Recent use of the ALHC for immersion density shows that extremely fine and delicate operations can be performed with the master-slave manipulators by qualified operators. Twenty milligram samples were tested for immersion density to determine the expansion of uranium dioxide after irradiation in a nuclear reactor. The data collected confirmed modeling analysis with very tight

  6. Moissanite anvil cell design for giga-pascal nuclear magnetic resonance

    Science.gov (United States)

    Meier, Thomas; Herzig, Tobias; Haase, Jürgen

    2014-04-01

    A new design of a non-magnetic high-pressure anvil cell for nuclear magnetic resonance (NMR) experiments at Giga-Pascal pressures is presented, which uses a micro-coil inside the pressurized region for high-sensitivity NMR. The comparably small cell has a length of 22 mm and a diameter of 18 mm, so it can be used with most NMR magnets. The performance of the cell is demonstrated with external-force vs. internal-pressure experiments, and the cell is shown to perform well at pressures up to 23.5 GPa using 800 μm 6H-SiC large cone Boehler-type anvils. 1H, 23Na, 27Al, 69Ga, and 71Ga NMR test measurements are presented, which show a resolution of better than 4.5 ppm, and an almost maximum possible signal-to-noise ratio.

  7. Nuclear EGFR-PKM2 axis induces cancer stem cell-like characteristics in irradiation-resistant cells.

    Science.gov (United States)

    Shi, Ying; Liu, Na; Lai, Weiwei; Yan, Bin; Chen, Ling; Liu, Shouping; Liu, Shuang; Wang, Xiang; Xiao, Desheng; Liu, Xiaoli; Mao, Chao; Jiang, Yiqun; Jia, Jiantao; Liu, Yating; Yang, Rui; Cao, Ya; Tao, Yongguang

    2018-05-28

    Radiation therapy has become an important tool in the treatment of cancer patients, but most patients relapse within 5 years. Relapse is due to the presence of cancer stem cells (CSCs), but the molecular mechanism of radioresistance in CSCs remains largely elusive. Here, we found that irradiation-resistant (IR) cells exhibited increased stem cell-like properties together with elevated anchorage-independent growth and metastasis ability. EGFR not only leads to increased acquisition of endometrial cancer stem cell markers in radioresistant sublines but is critical for the cancer stem-cell phenotype and tumorigenicity. Moreover, PKM2 functions as an interacting partner of EGFR, which induces the EMT phenotype and stem cell-like properties in IR cells. Finally, we found that the regulatory function of the EGFR-PKM2 axis is dependent on nuclear EGFR. In sum, our study indicated that EGFR and PKM2 directly interact and bind with each other to regulate the transcription of stemness-related genes and promote the stem-like phenotype, thus promoting invasion and metastasis. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Depletion of nuclear import protein karyopherin alpha 7 (KPNA7) induces mitotic defects and deformation of nuclei in cancer cells.

    Science.gov (United States)

    Vuorinen, Elisa M; Rajala, Nina K; Ihalainen, Teemu O; Kallioniemi, Anne

    2018-03-27

    Nucleocytoplasmic transport is a tightly regulated process carried out by specific transport machinery, the defects of which may lead to a number of diseases including cancer. Karyopherin alpha 7 (KPNA7), the newest member of the karyopherin alpha nuclear importer family, is expressed at a high level during embryogenesis, reduced to very low or absent levels in most adult tissues but re-expressed in cancer cells. We used siRNA-based knock-down of KPNA7 in cancer cell lines, followed by functional assays (proliferation and cell cycle) and immunofluorescent stainings to determine the role of KPNA7 in regulation of cancer cell growth, proper mitosis and nuclear morphology. In the present study, we show that the silencing of KPNA7 results in a dramatic reduction in pancreatic and breast cancer cell growth, irrespective of the endogenous KPNA7 expression level. This growth inhibition is accompanied by a decrease in the fraction of S-phase cells as well as aberrant number of centrosomes and severe distortion of the mitotic spindles. In addition, KPNA7 depletion leads to reorganization of lamin A/C and B1, the main nuclear lamina proteins, and drastic alterations in nuclear morphology with lobulated and elongated nuclei. Taken together, our data provide new important evidence on the contribution of KPNA7 to the regulation of cancer cell growth and the maintenance of nuclear envelope environment, and thus deepens our understanding on the impact of nuclear transfer proteins in cancer pathogenesis.

  9. Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer.

    Science.gov (United States)

    Jin, Jun-Xue; Li, Suo; Gao, Qing-Shan; Hong, Yu; Jin, Long; Zhu, Hai-Ying; Yan, Chang-Guo; Kang, Jin-Dan; Yin, Xi-Jun

    2013-10-01

    The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  10. Nuclear and cellular expression data from the whole 16-cell stage Arabidopsis thaliana embryo and a cell type-specific expression atlas of the early Arabidopsis embryo

    NARCIS (Netherlands)

    Palovaara, J.P.J.

    2017-01-01

    SuperSeries contain expression data from the nuclei of cell types involved in patterning events, with focus on root apical stem cell formation, at 16-cell stage, early globular stage and late globular stage in the early Arabidopsis embryo (atlas). Expression data comparing nuclear and cellular RNA

  11. Functions of Heterogeneous Nuclear Ribonucleoproteins in Stem Cell Potency and Differentiation

    Directory of Open Access Journals (Sweden)

    Qishan Chen

    2013-01-01

    Full Text Available Stem cells possess huge importance in developmental biology, disease modelling, cell replacement therapy, and tissue engineering in regenerative medicine because they have the remarkable potential for self-renewal and to differentiate into almost all the cell types in the human body. Elucidation of molecular mechanisms regulating stem cell potency and differentiation is essential and critical for extensive application. Heterogeneous nuclear ribonucleoproteins (hnRNPs are modular proteins consisting of RNA-binding motifs and auxiliary domains characterized by extensive and divergent functions in nucleic acid metabolism. Multiple roles of hnRNPs in transcriptional and posttranscriptional regulation enable them to be effective gene expression regulators. More recent findings show that hnRNP proteins are crucial factors implicated in maintenance of stem cell self-renewal and pluripotency and cell differentiation. The hnRNPs interact with certain sequences in target gene promoter regions to initiate transcription. In addition, they recognize 3′UTR or 5′UTR of specific gene mRNA forming mRNP complex to regulate mRNA stability and translation. Both of these regulatory pathways lead to modulation of gene expression that is associated with stem cell proliferation, cell cycle control, pluripotency, and committed differentiation.

  12. Growth Factor Midkine Promotes T-Cell Activation through Nuclear Factor of Activated T Cells Signaling and Th1 Cell Differentiation in Lupus Nephritis.

    Science.gov (United States)

    Masuda, Tomohiro; Maeda, Kayaho; Sato, Waichi; Kosugi, Tomoki; Sato, Yuka; Kojima, Hiroshi; Kato, Noritoshi; Ishimoto, Takuji; Tsuboi, Naotake; Uchimura, Kenji; Yuzawa, Yukio; Maruyama, Shoichi; Kadomatsu, Kenji

    2017-04-01

    Activated T cells play crucial roles in the pathogenesis of autoimmune diseases, including lupus nephritis (LN). The activation of calcineurin/nuclear factor of activated T cells (NFAT) and STAT4 signaling is essential for T cells to perform various effector functions. Here, we identified the growth factor midkine (MK; gene name, Mdk) as a novel regulator in the pathogenesis of 2,6,10,14-tetramethylpentadecane-induced LN via activation of NFAT and IL-12/STAT4 signaling. Wild-type (Mdk +/+ ) mice showed more severe glomerular injury than MK-deficient (Mdk -/- ) mice, as demonstrated by mesangial hypercellularity and matrix expansion, and glomerular capillary loops with immune-complex deposition. Compared with Mdk -/- mice, the frequency of splenic CD69 + T cells and T helper (Th) 1 cells, but not of regulatory T cells, was augmented in Mdk +/+ mice in proportion to LN disease activity, and was accompanied by skewed cytokine production. MK expression was also enhanced in activated CD4 + T cells in vivo and in vitro. MK induced activated CD4 + T cells expressing CD69 through nuclear activation of NFAT transcription and selectively increased in vitro differentiation of naive CD4 + T cells into Th1 cells by promoting IL-12/STAT4 signaling. These results suggest that MK serves an indispensable role in the NFAT-regulated activation of CD4 + T cells and Th1 cell differentiation, eventually leading to the exacerbation of LN. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  13. Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells

    Science.gov (United States)

    Lombardi, Maria; Castoria, Gabriella; Migliaccio, Antimo; Barone, Maria Vittoria; Di Stasio, Rosina; Ciociola, Alessandra; Bottero, Daniela; Yamaguchi, Hiroshi; Appella, Ettore; Auricchio, Ferdinando

    2008-01-01

    In breast cancer cells, cytoplasmic localization of the estradiol receptor α (ERα) regulates estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERα and show that its export is dependent on both estradiol-mediated phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome region maintenance 1 (CRM1). A Tat peptide containing the ERα NES disrupts ERα–CRM1 interaction and prevents nuclear export of ERα- and estradiol-induced DNA synthesis. NES-ERα mutants do not exit the nucleus and inhibit estradiol-induced S phase entry; ERα-dependent transcription is normal. ERα is associated with Forkhead proteins in the nucleus, and estradiol stimulates nuclear exit of both proteins. ERα knockdown or ERα NES mutations prevent ERα and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by estradiol-activated AKT, does not associate with ERα and is trapped in the nucleus, blocking S phase entry. In conclusion, estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERα, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry. PMID:18644889

  14. Telomere-surrounding regions are transcription-permissive 3D nuclear compartments in human cells

    International Nuclear Information System (INIS)

    Quina, Ana Sofia; Parreira, Leonor

    2005-01-01

    Positioning of genes relative to nuclear heterochromatic compartments is thought to help regulate their transcriptional activity. Given that human subtelomeric regions are rich in highly expressed genes, we asked whether human telomeres are related to transcription-permissive nuclear compartments. To address this question, we investigated in the nuclei of normal human lymphocytes the spatial relations of two constitutively expressed genes (ACTB and RARA) and three nuclear transcripts (ACTB, IL2RA and TCRB) to telomeres and centromeres, as a function of gene activity and transcription levels. We observed that genes and gene transcripts locate close to telomere clusters and away from chromocenters upon activation of transcription. These findings, together with the observation that SC35 domains, which are enriched in pre-mRNA processing factors, are in close proximity to telomeres, indicate that telomere-neighboring regions are permissive to gene expression in human cells. Therefore, the associations of telomeres observed in the interphase nucleus might contribute, as opposed to chromocenters, for the establishment of transcription-permissive 3D nuclear compartments

  15. Engineering membrane proteins for nuclear medicine. Applications for gene therapy and cell tracking

    International Nuclear Information System (INIS)

    Bogdanov Jr, A.A.; Simonova, M.; Weissleder, R.

    2000-01-01

    Nuclear imaging techniques such as PET and SPECT imaging are expected to play major roles in evaluating the efficacy of in vivo gene therapy. In particular, the quantification of vector delivery and imaging the efficacy of gene expression are of key interests in testing new treatment paradigms and in designing novel vectors. In this review article it has been illustrated how nuclear imaging can be used to image novel cell-surface expressed fusion proteins and how this strategy can be used to probe for phenotypic changes in genetically manipulated cells. Since the described approach uses new fusion proteins, typically not present on eukaryotic cells, such as artificial receptors can be designed to bind radioisotopes currently in clinical use. The described fusion proteins consists of 1) a binding domain such as a peptide based chelator that binds 99mT c oxotechnetate and 2) a membrane anchoring domain. A variety of fusion proteins have been tested so far and the most promising one to date consists of a metallothionein (MT)-derived C-terminal peptide fused a type II membrane protein markers containing the N-terminal membrane anchoring domain of neutral endopeptidase (PEP). Cell-surface expression of MT in transfected cells has been demonstrated using monoclonal antibodies in vitro. Both in vitro and in vivo transchelation experiments have confirmed expression of 99mT c-binding sites in eukaryotic cells. It was expected the described approach to evolve into a useful strategy to tag transfected cells with 99mT c and thus assessing efficiency of gene delivery and expression

  16. V79 cell survival after a single lithium ion nuclear traversal

    International Nuclear Information System (INIS)

    Pinto, M.; Buonanno, M.; Campajola, L.; Durante, M.; Grossi, G.; Publiese, G.; Scampoli, P.; Gialanella, G.; Manti, L.

    2003-01-01

    Full text: Biological studies on the effects of low doses of densely ionising radiation are highly influenced by the stochastic character of the energy deposition events. For several end-points, including clonogenic survival, to follow-up individual cells that have undergone an exactly determined number of charged particle traversals is highly desirable. While RBE-LET curves have been measured after conventional 'broad beam' irradiation with several ions of varying energies, the probability of cell survival after a single charged particle traversal has only been determined for accelerated protons and alpha particles, whereas the ability of single particle traversals at higher LET to cause clonogenic death is yet unknown. Recently, low dose studies have also shown phenomena of high interest, such as the hypersensitivity/induced radioresistance(HS/IRR) adaptive responses. However, for particles of high LET, even a single nuclear traversal may deliver an average dose to a single cell that may be beyond the dose region of the HS/IRR response. We ave set up an experimental apparatus for the determination of the inactivation cross section after an exactly known number of accelerated Lithium ions traversals (210 keV/micron when hitting the cell surface). Using a bio-stack approach (Pugliese et al, IJRB Oct;72(4):397-407 1997) LR115 thin nuclear track detectors have been employed for the direct visualisation of Lithium ion traversals in V79 cells nuclei that are labeled with Hoechst 33258. A computer software has been designed and implemented to control micro-meter movements of a motorised Marzhauser stage, mounted on a fluorescent microscope, for the acquisition of individual attached cell coordinates, type of traversal, as well as for re-visiting the registered coordinates for analysis of survivors. The V79 cell survival experiment after exactly known numbers of Lithium ions traversals is in progress, along with a classical 'broad beam' survival assay

  17. Successful reprogramming of epiblast stem cells by blocking nuclear localization of β-catenin.

    Science.gov (United States)

    Murayama, Hideyuki; Masaki, Hideki; Sato, Hideyuki; Hayama, Tomonari; Yamaguchi, Tomoyuki; Nakauchi, Hiromitsu

    2015-01-13

    Epiblast stem cells (EpiSCs) in mice and rats are primed pluripotent stem cells (PSCs). They barely contribute to chimeric embryos when injected into blastocysts. Reprogramming of EpiSCs to embryonic stem cell (ESC)-like cells (rESCs) may occur in response to LIF-STAT3 signaling; however, low reprogramming efficiency hampers potential use of rESCs in generating chimeras. Here, we describe dramatic improvement of conversion efficiency from primed to naive-like PSCs through upregulation of E-cadherin in the presence of the cytokine LIF. Analysis revealed that blocking nuclear localization of β-CATENIN with small-molecule inhibitors significantly enhances reprogramming efficiency of mouse EpiSCs. Although activation of Wnt/β-catenin signals has been thought desirable for maintenance of naive PSCs, this study provides the evidence that inhibition of nuclear translocation of β-CATENIN enhances conversion of mouse EpiSCs to naive-like PSCs (rESCs). This affords better understanding of gene regulatory circuits underlying pluripotency and reprogramming of PSCs. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Thermodynamic Paradigm for Solution Demixing Inspired by Nuclear Transport in Living Cells.

    Science.gov (United States)

    Wang, Ching-Hao; Mehta, Pankaj; Elbaum, Michael

    2017-04-14

    Living cells display a remarkable capacity to compartmentalize their functional biochemistry. A particularly fascinating example is the cell nucleus. Exchange of macromolecules between the nucleus and the surrounding cytoplasm does not involve traversing a lipid bilayer membrane. Instead, large protein channels known as nuclear pores cross the nuclear envelope and regulate the passage of other proteins and RNA molecules. Beyond simply gating diffusion, the system of nuclear pores and associated transport receptors is able to generate substantial concentration gradients, at the energetic expense of guanosine triphosphate hydrolysis. In contrast to conventional approaches to demixing such as reverse osmosis and dialysis, the biological system operates continuously, without application of cyclic changes in pressure or solvent exchange. Abstracting the biological paradigm, we examine this transport system as a thermodynamic machine of solution demixing. Building on the construct of free energy transduction and biochemical kinetics, we find conditions for the stable operation and optimization of the concentration gradients as a function of dissipation in the form of entropy production.

  19. Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

    International Nuclear Information System (INIS)

    Zhong Zhisheng; Zhang Gang; Meng Xiaoqian; Zhang Yanling; Chen Dayuan; Schatten, Heide; Sun Qingyuan

    2005-01-01

    Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, γ-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. γ-tubulin translocated into the two poles of the transient spindles, while no accumulated γ-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, γ-tubulin was translocated to the spindle poles. The distribution of γ-tubulin was similar in mouse intraspecies and rat-mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig-mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. γ-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the donor cell centrosome, defined as

  20. Decreased nuclear matrix DNA topoisomerase II in human leukemia cells resistant to VM-26 and m-AMSA

    International Nuclear Information System (INIS)

    Fernandes, D.J.; Danks, M.K.; Beck, W.T.

    1990-01-01

    CEM leukemia cells selected for resistance to VM-26 (CEM/VM-1) are cross-resistant to various other DNA topoisomerase II inhibitors but not to Vinca alkaloids. Since DNA topoisomerase II is a major protein of the nuclear matrix, the authors asked if alterations in nuclear matrix topoisomerase II might be important in this form of multidrug resistance. Pretreatment of drug-sensitive CEM cells for 2 h with either 5 μM VM-26 or 3 μM m-AMSA reduced the specific activity of newly replicated DNA on the nuclear matrix by 75 and 50%, respectively, relative to that of the bulk DNA. The decatenating and unknotting activities of DNA topoisomerase II were 6- and 7-fold lower, respectively, in the nuclear matrix preparations from the CEM/VM-1 cells compared to parental CEM cells. Western blot analysis revealed that the amount of immunoreactive topoisomerase II in the nuclear matrices of the CEM/VM-1 cells decreased 3.2-fold relative to that in CEM cells. Increasing the NaCl concentration used in the matrix isolation procedure from 0.2 to 1.8 M resulted in a progressive decrease in the specific activity of topoisomerase II in matrices of CEM/VM-1 but not CEM cells, which suggested that the association of the enzyme with the matrix is altered in the resistant cells. These data support the hypothesis that resistance to VM-26 and m-AMSA is directly related to the decreased activity of nuclear matrix topoisomerase II. In CEM/VM-1 cells the interaction of either VM-26 or m-AMSA with nuclear matrix topoisomerase II is specifically diminished

  1. Synaptic activity induces dramatic changes in the geometry of the cell nucleus: interplay between nuclear structure, histone H3 phosphorylation, and nuclear calcium signaling.

    Science.gov (United States)

    Wittmann, Malte; Queisser, Gillian; Eder, Anja; Wiegert, J Simon; Bengtson, C Peter; Hellwig, Andrea; Wittum, Gabriel; Bading, Hilmar

    2009-11-25

    Synaptic activity initiates many adaptive responses in neurons. Here we report a novel form of structural plasticity in dissociated hippocampal cultures and slice preparations. Using a recently developed algorithm for three-dimensional image reconstruction and quantitative measurements of cell organelles, we found that many nuclei from hippocampal neurons are highly infolded and form unequally sized nuclear compartments. Nuclear infoldings are dynamic structures, which can radically transform the geometry of the nucleus in response to neuronal activity. Action potential bursting causing synaptic NMDA receptor activation dramatically increases the number of infolded nuclei via a process that requires the ERK-MAP kinase pathway and new protein synthesis. In contrast, death-signaling pathways triggered by extrasynaptic NMDA receptors cause a rapid loss of nuclear infoldings. Compared with near-spherical nuclei, infolded nuclei have a larger surface and increased nuclear pore complex immunoreactivity. Nuclear calcium signals evoked by cytosolic calcium transients are larger in small nuclear compartments than in the large compartments of the same nucleus; moreover, small compartments are more efficient in temporally resolving calcium signals induced by trains of action potentials in the theta frequency range (5 Hz). Synaptic activity-induced phosphorylation of histone H3 on serine 10 was more robust in neurons with infolded nuclei compared with neurons with near-spherical nuclei, suggesting a functional link between nuclear geometry and transcriptional regulation. The translation of synaptic activity-induced signaling events into changes in nuclear geometry facilitates the relay of calcium signals to the nucleus, may lead to the formation of nuclear signaling microdomains, and could enhance signal-regulated transcription.

  2. Fluorine nuclear magnetic resonance-based assay in living mammalian cells.

    Science.gov (United States)

    Veronesi, Marina; Giacomina, Francesca; Romeo, Elisa; Castellani, Beatrice; Ottonello, Giuliana; Lambruschini, Chiara; Garau, Gianpiero; Scarpelli, Rita; Bandiera, Tiziano; Piomelli, Daniele; Dalvit, Claudio

    2016-02-15

    Nuclear magnetic resonance (NMR)-based screening has been recognized as a powerful approach for the identification and characterization of molecules interacting with pharmaceutical targets. Indeed, several NMR methods have been developed and successfully applied to many drug discovery projects. Whereas most of these approaches have targeted isolated biomolecular receptors, very few cases are reported with the screening performed in intact cells and cell extracts. Here we report the first successful application of the fluorine NMR-based assay n-FABS (n-fluorine atoms for biochemical screening) in living mammalian cells expressing the membrane protein fatty acid amide hydrolase (FAAH). This method allows the identification of both weak and potent inhibitors and the measurement of their potency in a physiological environment. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

    Science.gov (United States)

    Balbach, Sebastian T; Boiani, Michele

    2015-01-01

    Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.

  4. Nuclear trafficking and export of single, native mRNPs in Chironomus tentans salivary gland cells.

    Science.gov (United States)

    Kaminski, Tim P; Spille, Jan-Hendrik; Nietzel, Claudio; Siebrasse, Jan Peter; Kubitscheck, Ulrich

    2013-01-01

    Real-time observation of single molecules or biological nanoparticles with high spatial resolution in living cells provides detailed insights into the dynamics of cellular processes. The salivary gland cells of Chironomus tentans are a well-established model system to study the processing of RNA and the formation and fate of messenger ribonucleoprotein particles (mRNPs). For a long time, challenging imaging conditions limited the access to this system for in vivo fluorescence microscopy. Recent technical and methodical advantages now allow observing even single molecules in these cells. We describe here the experimental approach and the optical techniques required to analyze intranuclear trafficking and export of single native mRNPs across the nuclear envelope.

  5. Helicobacter pylori infection induces genetic instability of nuclear and mitochondrial DNA in gastric cells

    DEFF Research Database (Denmark)

    Machado, Ana Manuel Dantas; Figueiredo, Ceu; Touati, Eliette

    2009-01-01

    PURPOSE: Helicobacter pylori is a major cause of gastric carcinoma. To investigate a possible link between bacterial infection and genetic instability of the host genome, we examined the effect of H. pylori infection on known cellular repair pathways in vitro and in vivo. Moreover, various types...... of genetic instabilities in the nuclear and mitochondrial DNA (mtDNA) were examined. EXPERIMENTAL DESIGN: We observed the effects of H. pylori infection on a gastric cell line (AGS), on C57BL/6 mice, and on individuals with chronic gastritis. In AGS cells, the effect of H. pylori infection on base excision...... cells and chronic gastritis tissue were determined by PCR, single-stranded conformation polymorphism, and sequencing. H. pylori vacA and cagA genotyping was determined by multiplex PCR and reverse hybridization. RESULTS: Following H. pylori infection, the activity and expression of base excision repair...

  6. Monodisperse magnetite nanoparticles coupled with nuclear localization signal peptide for cell-nucleus targeting.

    Science.gov (United States)

    Xu, Chenjie; Xie, Jin; Kohler, Nathan; Walsh, Edward G; Chin, Y Eugene; Sun, Shouheng

    2008-03-07

    Functionalization of monodisperse superparamagnetic magnetite (Fe(3)O(4)) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe(3)O(4) nanoparticles functionalized with protein and nuclear localization signal (NLS) peptide. These NLS-coated nanoparticles were introduced into the HeLa cell cytoplasm and nucleus, where the particles were monodispersed and non-aggregated. The success of labeling was examined and identified by fluorescence microscopy and MRI. The work demonstrates that monodisperse magnetic nanoparticles can be readily functionalized and stabilized for potential diagnostic and therapeutic applications.

  7. Nuclear morphometry in male breast carcinoma: association with cell proliferative activity, oncogene expression, DNA content and prognosis.

    Science.gov (United States)

    Chiusa, L; Margaria, E; Pich, A

    2000-11-20

    To investigate the prognostic value of nuclear morphometry in male breast carcinoma (MBC), histological samples from 50 patients (mean age 62.2 years) were retrospectively analyzed by computerized nuclear morphometry. All patients received surgery; 35 had multiple combinations of adjuvant therapies. Mean follow-up was 67 months (range 1-230). In each case, 100 tumor cells were measured, and the mean nuclear area (MNA), standard deviation of the nuclear area (SDNA), mean nuclear perimeter (MNP), standard deviation of the nuclear perimeter (SDNP) and shape factor (SHF) were calculated. Morphometric features were compared with tumor histological grade, size, nodal status, DNA ploidy evaluated by flow-cytometry and cell proliferative activity assessed by the quantity of argyrophilic nucleolar organizer region-associated proteins (AgNORs), monoclonal antibody (MAb) PC10 against proliferating cell nuclear antigen and MAb MIB-1. Comparison was also made with the immunohistochemical detection of p53, bcl-2, c-erbB-2 and c-myc proteins. Significant association was found between nuclear morphometric parameters and tumor grade, DNA content and cell proliferation indices. SDNA was greater in p53-positive and bcl-2-negative cases; SDNP was greater in p53-positive cases; SHF was lower in p53- and c-myc-positive cases. Overall survival was shorter in carcinomas with high MNA, SDNA, MNP and SDNP and low SHF. In multivariate analysis, performed by testing nuclear morphometric parameters, histological grade, tumor size, nodal status and p53 immunostaining in the Cox model, p53 over-expression and histological grade retained independent prognostic significance. When p53 was excluded, only SDNP appeared as an independent prognostic variable. Our results indicate that nuclear morphometric parameters can identify an aggressive tumor phenotype and provide additional prognostic information for patients with MBC. Copyright 2000 Wiley-Liss, Inc.

  8. Selective nuclear morphometry as a prognostic factor of survival in renal cell carcinoma.

    Science.gov (United States)

    Monge, J M; Val-Bernal, J F; Buelta, L; García-Castrillo, L; Asensio, L

    1999-01-01

    In the present study, we sought to determine the predictive value of selective nuclear morphometry (SNM) for patient outcome in renal cell carcinoma (RCC). Tumor samples of 140 renal adenocarcinomas diagnosed and treated with radical nephrectomy and hilar lymphadenectomy between 1970 and 1988 with a minimum follow up of 5 years in all the cases were studied by SNM. The morphometric analysis was performed in the most malignant tumor selected zone. Selection was based on cytological criteria including nuclear grade. Nuclear morphometric features analyzed were: area, perimeter, major diameter, major and minor diameter of the equivalent ellipse, volume of the equivalent ellipse and sphere, circumference diameter, and shape factors. The results showed that in the selected zone tumor nuclei were larger than in the zones selected at random. There was an inverse correlation between morphometric parameters and survival and a direct one between tumoral grade and stage. Tumors of the long-term survival group of patients presented nuclei with smaller morphometric measurements than tumors of short term survival group, with significant differences between them (p < 0.05). In the survival analysis carried out by the Kaplan-Meier method significant differences existed between different groups formed from break point for: area, perimeter, major diameter, major and minor diameter of the ellipse, volume of the ellipse and sphere, circumference diameter and perimeter shape factor. In the multivariate analysis carried out by the Cox method, the feature with the most predictable value related to survival, was the tumor stage. Morphometric value with the highest punctuation in the test was major nuclear diameter. The rest of the morphometric values (except elliptic shape factor and elongation factor) were also significant but they did not improve prognostic information of the major nuclear diameter. SNM offers a useful aid in a more objective grading of RCC. Multivariate Cox analysis

  9. Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer.

    Science.gov (United States)

    Burgstaller, Jörg P; Schinogl, Pamela; Dinnyes, Andras; Müller, Mathias; Steinborn, Ralf

    2007-12-21

    The mitochondrial DNA (mtDNA) of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT) was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts. The three fetal fibroblast donor cells used for SCNT harboured low mtDNA copy numbers per cell (A: 753 +/- 54, B: 292 +/- 33 and C: 561 +/- 88). The ratio of donor to recipient oocyte mtDNAs was determined using a quantitative amplification refractory mutation system (ARMS) PCR (i.e. ARMS-qPCR). For quantification of SNP variants with frequencies below 0.1% we developed a restriction endonuclease-mediated selective quantitative PCR (REMS-qPCR). We report the first cases (n = 4 fetuses, n = 3 lambs) of recipient oocyte/nuclear donor mtDNA heteroplasmy in SCNT-derived ovine clones demonstrating that there is no species-effect hindering ovine nucleus-donor mtDNA from being transmitted to the somatic clonal offspring. Most of the heteroplasmic clones exhibited low-level heteroplasmy (0.1% to 0.9%, n = 6) indicating neutral transmission of parental mtDNAs. High-level heteroplasmy (6.8% to 46.5%) was observed in one case. This clone possessed a divergent recipient oocyte-derived mtDNA genotype with three rare amino acid changes compared to the donor including one substitution at an evolutionary conserved site. Our study using state-of-the-art techniques for mtDNA quantification, like ARMS-qPCR and the novel REMS-qPCR, documents for the first time the transmission of donor mtDNA into somatic sheep clones. MtDNA heteroplasmy was detected in seven of 12 clones tested, whereby all but one case revealed less

  10. Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Müller Mathias

    2007-12-01

    Full Text Available Abstract Background The mitochondrial DNA (mtDNA of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts. Results The three fetal fibroblast donor cells used for SCNT harboured low mtDNA copy numbers per cell (A: 753 ± 54, B: 292 ± 33 and C: 561 ± 88. The ratio of donor to recipient oocyte mtDNAs was determined using a quantitative amplification refractory mutation system (ARMS PCR (i.e. ARMS-qPCR. For quantification of SNP variants with frequencies below 0.1% we developed a restriction endonuclease-mediated selective quantitative PCR (REMS-qPCR. We report the first cases (n = 4 fetuses, n = 3 lambs of recipient oocyte/nuclear donor mtDNA heteroplasmy in SCNT-derived ovine clones demonstrating that there is no species-effect hindering ovine nucleus-donor mtDNA from being transmitted to the somatic clonal offspring. Most of the heteroplasmic clones exhibited low-level heteroplasmy (0.1% to 0.9%, n = 6 indicating neutral transmission of parental mtDNAs. High-level heteroplasmy (6.8% to 46.5% was observed in one case. This clone possessed a divergent recipient oocyte-derived mtDNA genotype with three rare amino acid changes compared to the donor including one substitution at an evolutionary conserved site. Conclusion Our study using state-of-the-art techniques for mtDNA quantification, like ARMS-qPCR and the novel REMS-qPCR, documents for the first time the transmission of donor mtDNA into somatic sheep clones. MtDNA heteroplasmy was detected in seven of 12 clones

  11. Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Betts Dean H

    2006-08-01

    Full Text Available Abstract Background Somatic cell nuclear transfer (SCNT provides an appealing alternative for the preservation of genetic material in non-domestic and endangered species. An important prerequisite for successful SCNT is the availability of good quality donor cells, as normal embryo development is dependent upon proper reprogramming of the donor genome so that embryonic genes can be appropriately expressed. The characteristics of donor cell lines and their ability to produce embryos by SCNT were evaluated by testing the effects of tissue sample collection (DART biopsy, PUNCH biopsy, post-mortem EAR sample and culture initiation (explant, collagenase digestion techniques. Results Differences in initial sample size based on sample collection technique had an effect on the amount of time necessary for achieving primary confluence and the number of population doublings (PDL produced. Thus, DART and PUNCH biopsies resulted in cultures with decreased lifespans (50 PDL and chromosomally stable (>70% normal cells at 20 PDL cultures produced by post-mortem EAR samples. Chromosome stability was influenced by sample collection technique and was dependent upon the culture's initial telomere length and its rate of shortening over cell passages. Following SCNT, short-lived cultures resulted in significantly lower blastocyst development (≤ 0.9% compared to highly proliferative cultures (11.8%. Chromosome stability and sample collection technique were significant factors in determining blastocyst development outcome. Conclusion These data demonstrate the influence of culture establishment techniques on cell culture characteristics, including the viability, longevity and normality of cells. The identification of a quantifiable marker associated with SCNT embryo developmental potential, chromosome stability, provides a means by which cell culture conditions can be monitored and improved.

  12. Micropillar displacements by cell traction forces are mechanically correlated with nuclear dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Li, Qingsen; Makhija, Ekta; Hameed, F.M. [Mechanobiology Institute, National University of Singapore (Singapore); Shivashankar, G.V., E-mail: shiva.gvs@gmail.com [Mechanobiology Institute, National University of Singapore (Singapore); Department of Biological Sciences, National University of Singapore (Singapore)

    2015-05-29

    Cells sense physical cues at the level of focal adhesions and transduce them to the nucleus by biochemical and mechanical pathways. While the molecular intermediates in the mechanical links have been well studied, their dynamic coupling is poorly understood. In this study, fibroblast cells were adhered to micropillar arrays to probe correlations in the physical coupling between focal adhesions and nucleus. For this, we used novel imaging setup to simultaneously visualize micropillar deflections and EGFP labeled chromatin structure at high spatial and temporal resolution. We observed that micropillar deflections, depending on their relative positions, were positively or negatively correlated to nuclear and heterochromatin movements. Our results measuring the time scales between micropillar deflections and nucleus centroid displacement are suggestive of a strong elastic coupling that mediates differential force transmission to the nucleus. - Highlights: • Correlation between focal adhesions and nucleus studied using novel imaging setup. • Micropillar and nuclear displacements were measured at high resolution. • Correlation timescales show strong elastic coupling between cell edge and nucleus.

  13. Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2011-12-01

    Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

  14. Micronuclei and other nuclear anomalies in buccal epithelial cells of children with chronic kidney disease.

    Science.gov (United States)

    Aykanat, Banu; Demircigil, Gonca Cakmak; Buyan, Necla; Baskin, Esra; Gulleroglu, Kaan; Fidan, Kibriya; Bayrakci, Umut Selda; Dalgic, Aydin; Karakayali, Hamdi; Haberal, Mehmet; Burgaz, Sema

    2016-12-01

    The objective of this study was to reveal the likely genomic instability in children with chronic kidney disease (CKD) using micronucleus (MN) assay on buccal epithelial cells (BEC). We investigated the frequencies of micronuclei and other nuclear anomalies, such as nuclear buds, binucleated cells, condensed chromatin, and karyorrhectic and pyknotic cells in BEC. Children with CKD were grouped as follows: children in the pre-dialysis (PreD) stage (N=17), children on regular haemodialysis (HD) (N=14), and children who have undergone transplantation (Tx) (N=17). As a control group, twenty age- and gender-matched healthy children were selected. The MN frequency in BEC of all groups of children with CKD was significantly elevated (5- to 7-fold) as compared to the control group (pchildren with CKD relative to the control group (p<0.001). Our results show that the BEC of pediatric PreD, HD, and Tx patients with CKD display increased cytogenetic, cytokinetic, and cytotoxic effects. They also point to the sensitivity and usefulness of the BEC MN assay in the assessment of genetic susceptibility of patients with CKD.

  15. Spongiatriol Inhibits Nuclear Factor Kappa B Activation and Induces Apoptosis in Pancreatic Cancer Cells

    Science.gov (United States)

    Guzmán, Esther; Maher, Michael; Temkin, Alexis; Pitts, Tara; Wright, Amy

    2013-01-01

    Pancreatic cancer, the fourth leading cause of cancer death in the US, is highly resistant to all current chemotherapies, and its growth is facilitated by chronic inflammation. The majority of pro-inflammatory cytokines initiate signaling cascades that converge at the activation of the Nuclear Factor Kappa B (NFκB), a signal transduction molecule that promotes cell survival, proliferation and angiogenesis. In an effort to identify novel inhibitors of NFκB, the HBOI library of pure compounds was screened using a reporter cell line that produces luciferin under the transcriptional control of NFκB. Seven compounds were identified through this screen, but in the case of five of them, their reported mechanism of action made them unlikely to be specific NFκB inhibitors. Spongiatriol, a marine furanoditerpenoid that was first isolated in the 1970s, is shown here to inhibit NFκB transcriptional activity in a reporter cell line, to reduce levels of phosphorylated (active) NFκB in the AsPC-1 cell line, to have an IC50 for cytotoxicity in the low micromolar range against the AsPC-1, BxPC-3, MiaPaCa-2 and Panc-1 pancreatic cancer cell lines, and to induce moderate but significant apoptosis in both the AsPC-1 and the Panc-1 cell lines. PMID:23549285

  16. Chromosome conformation maps in fission yeast reveal cell cycle dependent sub nuclear structure.

    Science.gov (United States)

    Grand, Ralph S; Pichugina, Tatyana; Gehlen, Lutz R; Jones, M Beatrix; Tsai, Peter; Allison, Jane R; Martienssen, Robert; O'Sullivan, Justin M

    2014-11-10

    Successful progression through the cell cycle requires spatial and temporal regulation of gene transcript levels and the number, positions and condensation levels of chromosomes. Here we present a high resolution survey of genome interactions in Schizosaccharomyces pombe using synchronized cells to investigate cell cycle dependent changes in genome organization and transcription. Cell cycle dependent interactions were captured between and within S. pombe chromosomes. Known features of genome organization (e.g. the clustering of telomeres and retrotransposon long terminal repeats (LTRs)) were observed throughout the cell cycle. There were clear correlations between transcript levels and chromosomal interactions between genes, consistent with a role for interactions in transcriptional regulation at specific stages of the cell cycle. In silico reconstructions of the chromosome organization within the S. pombe nuclei were made by polymer modeling. These models suggest that groups of genes with high and low, or differentially regulated transcript levels have preferred positions within the S. pombe nucleus. We conclude that the S. pombe nucleus is spatially divided into functional sub-nuclear domains that correlate with gene activity. The observation that chromosomal interactions are maintained even when chromosomes are fully condensed in M phase implicates genome organization in epigenetic inheritance and bookmarking. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Nesprin-1α-Dependent Microtubule Nucleation from the Nuclear Envelope via Akap450 Is Necessary for Nuclear Positioning in Muscle Cells.

    Science.gov (United States)

    Gimpel, Petra; Lee, Yin Loon; Sobota, Radoslaw M; Calvi, Alessandra; Koullourou, Victoria; Patel, Rutti; Mamchaoui, Kamel; Nédélec, François; Shackleton, Sue; Schmoranzer, Jan; Burke, Brian; Cadot, Bruno; Gomes, Edgar R

    2017-10-09

    The nucleus is the main microtubule-organizing center (MTOC) in muscle cells due to the accumulation of centrosomal proteins and microtubule (MT) nucleation activity at the nuclear envelope (NE) [1-4]. The relocalization of centrosomal proteins, including Pericentrin, Pcm1, and γ-tubulin, depends on Nesprin-1, an outer nuclear membrane (ONM) protein that connects the nucleus to the cytoskeleton via its N-terminal region [5-7]. Nesprins are also involved in the recruitment of kinesin to the NE and play a role in nuclear positioning in skeletal muscle cells [8-12]. However, a function for MT nucleation from the NE in nuclear positioning has not been established. Using the proximity-dependent biotin identification (BioID) method [13, 14], we found several centrosomal proteins, including Akap450, Pcm1, and Pericentrin, whose association with Nesprin-1α is increased in differentiated myotubes. We show that Nesprin-1α recruits Akap450 to the NE independently of kinesin and that Akap450, but not other centrosomal proteins, is required for MT nucleation from the NE. Furthermore, we demonstrate that this mechanism is disrupted in congenital muscular dystrophy patient myotubes carrying a nonsense mutation within the SYNE1 gene (23560 G>T) encoding Nesprin-1 [15, 16]. Finally, using computer simulation and cell culture systems, we provide evidence for a role of MT nucleation from the NE on nuclear spreading in myotubes. Our data thus reveal a novel function for Nesprin-1α/Nesprin-1 in nuclear positioning through recruitment of Akap450-mediated MT nucleation activity to the NE. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  18. Diesel exhaust particulate extracts inhibit transcription of nuclear respiratory factor-1 and cell viability in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mattingly, Kathleen A.; Klinge, Carolyn M. [University of Louisville School of Medicine, Department of Biochemistry and Molecular Biology, Center for Genetics and Molecular Medicine, Louisville, KY (United States)

    2012-04-15

    Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1-regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17{beta}-estradiol (E{sub 2}), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription, and this suppression was not ablated by concomitant treatment with E{sub 2}, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E{sub 2} increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. (orig.)

  19. High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation.

    Science.gov (United States)

    Vega, Sebastián L; Liu, Er; Arvind, Varun; Bushman, Jared; Sung, Hak-Joon; Becker, Matthew L; Lelièvre, Sophie; Kohn, Joachim; Vidi, Pierre-Alexandre; Moghe, Prabhas V

    2017-02-01

    Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative "imaging-derived" parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. A hybrid approach to solving the problem of design of nuclear fuel cells; Un enfoque hibrido para la solucion del problema del diseno de celdas de combustible nuclear

    Energy Technology Data Exchange (ETDEWEB)

    Montes T, J. L.; Perusquia del C, R.; Ortiz S, J. J.; Castillo, A., E-mail: joseluis.montes@inin.gob.mx [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2015-09-15

    An approach to solving the problem of fuel cell design for BWR power reactor is presented. For this purpose the hybridization of a method based in heuristic knowledge rules called S15 and the advantages of a meta-heuristic method is proposed. The synergy of potentialities of both techniques allows finding solutions of more quality. The quality of each solution is obtained through a multi-objective function formed from the main cell parameters that are provided or obtained during the simulation with the CASMO-4 code. To evaluate this alternative of solution nuclear fuel cells of reference of nuclear power plant of Laguna Verde were used. The results show that in a systematic way the results improve when both methods are coupled. As a result of the hybridization process of the mentioned techniques an improvement is achieved in a range of 2% with regard to the achieved results in an independent way by the S15 method. (Author)

  1. High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation

    Energy Technology Data Exchange (ETDEWEB)

    Vega, Sebastián L. [Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ (United States); Liu, Er; Arvind, Varun [Department of Biomedical Engineering, Rutgers University, Piscataway, NJ (United States); Bushman, Jared [Department of Chemistry and Chemical Biology, New Jersey Center for Biomaterials, Piscataway, NJ (United States); School of Pharmacy, University of Wyoming, Laramie, WY (United States); Sung, Hak-Joon [Department of Chemistry and Chemical Biology, New Jersey Center for Biomaterials, Piscataway, NJ (United States); Department of Biomedical Engineering, Vanderbilt University, Nashville, TN (United States); Becker, Matthew L. [Department of Polymer Science and Engineering, University of Akron, Akron, OH (United States); Lelièvre, Sophie [Department of Basic Medical Sciences, Purdue University, West Lafayette, IN (United States); Kohn, Joachim [Department of Chemistry and Chemical Biology, New Jersey Center for Biomaterials, Piscataway, NJ (United States); Vidi, Pierre-Alexandre, E-mail: pvidi@wakehealth.edu [Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC (United States); Moghe, Prabhas V., E-mail: moghe@rutgers.edu [Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ (United States); Department of Biomedical Engineering, Rutgers University, Piscataway, NJ (United States)

    2017-02-01

    Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative “imaging-derived” parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions. - Highlights: • High-content analysis of nuclear shape and organization classify stem and progenitor cells poised for distinct lineages. • Early oncogenic changes in mesenchymal stem cells (MSCs) are also detected with nuclear descriptors. • A new class of cancer-mitigating biomaterials was identified based on image

  2. Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer.

    Science.gov (United States)

    Feng, Xiujing; Cao, Shaoxian; Wang, Huili; Meng, Chunhua; Li, Jingxin; Jiang, Jin; Qian, Yong; Su, Lei; He, Qiang; Zhang, Qingxiao

    2015-02-01

    Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat β-lactoglobulin (βLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

  3. Reprogrammed transcriptome in rhesus-bovine interspecies somatic cell nuclear transfer embryos.

    Directory of Open Access Journals (Sweden)

    Kai Wang

    Full Text Available Global activation of the embryonic genome (EGA, one of the most critical steps in early mammalian embryo development, is recognized as the time when interspecies somatic cell nuclear transfer (iSCNT embryos fail to thrive.In this study, we analyzed the EGA-related transcriptome of rhesus-bovine iSCNT 8- to 16-cell embryos and dissected the reprogramming process in terms of embryonic gene activation, somatic gene silencing, and maternal RNA degradation. Compared with fibroblast donor cells, two thousand and seven genes were activated in iSCNT embryos, one quarter of them reaching expression levels comparable to those found in in vitro fertilized (IVF rhesus embryos. This suggested that EGA in iSCNT embryos had partially recapitulated rhesus embryonic development. Eight hundred and sixty somatic genes were not silenced properly and continued to be expressed in iSCNT embryos, which indicated incomplete nuclear reprogramming. We compared maternal RNA degradation in bovine oocytes between bovine-bovine SCNT and iSCNT embryos. While maternal RNA degradation occurred in both SCNT and iSCNT embryos, we saw more limited overall degradation of maternal RNA in iSCNT embryos than in SCNT embryos. Several important maternal RNAs, like GPF9, were not properly processed in SCNT embryos.Our data suggested that iSCNT embryos are capable of triggering EGA, while a portion of somatic cell-associated genes maintain their expression. Maternal RNA degradation seems to be impaired in iSCNT embryos. Further understanding of the biological roles of these genes, networks, and pathways revealed by iSCNT may expand our knowledge about cell reprogramming, pluripotency, and differentiation.

  4. Insulin-induced translocation of IR to the nucleus in insulin responsive cells requires a nuclear translocation sequence.

    Science.gov (United States)

    Kesten, Dov; Horovitz-Fried, Miriam; Brutman-Barazani, Tamar; Sampson, Sanford R

    2018-04-01

    Insulin binding to its cell surface receptor (IR) activates a cascade of events leading to its biological effects. The Insulin-IR complex is rapidly internalized and then is either recycled back to the plasma membrane or sent to lysosomes for degradation. Although most of the receptor is recycled or degraded, a small amount may escape this pathway and migrate to the nucleus of the cell where it might be important in promulgation of receptor signals. In this study we explored the mechanism by which insulin induces IR translocation to the cell nucleus. Experiments were performed cultured L6 myoblasts, AML liver cells and 3T3-L1 adipocytes. Insulin treatment induced a rapid increase in nuclear IR protein levels within 2 to 5 min. Treatment with WGA, an inhibitor of nuclear import, reduced insulin-induced increases nuclear IR protein; IR was, however, translocated to a perinuclear location. Bioinformatics tools predicted a potential nuclear localization sequence (NLS) on IR. Immunofluorescence staining showed that a point mutation on the predicted NLS blocked insulin-induced IR nuclear translocation. In addition, blockade of nuclear IR activation in isolated nuclei by an IR blocking antibody abrogated insulin-induced increases in IR tyrosine phosphorylation and nuclear PKCδ levels. Furthermore, over expression of mutated IR reduced insulin-induced glucose uptake and PKB phosphorylation. When added to isolated nuclei, insulin induced IR phosphorylation but had no effect on nuclear IR protein levels. These results raise questions regarding the possible role of nuclear IR in IR signaling and insulin resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Differences in Nuclear DNA Organization Between Lymphocytes, Hodgkin and Reed–Sternberg Cells Revealed by Structured Illumination Microscopy

    NARCIS (Netherlands)

    Righolt, C.H.; Guffei, A.; Knecht, H.; Young, I.T.; Stallinga, S.; Van Vliet, L.J.; Mai, S.

    2014-01-01

    Advances in light microscopy have enabled the visualization of DNA in the interphase nucleus with more detail than is visible with conventional light microscopy. The nuclear architecture is assumed to be different in cancer cells compared to normal cells. In this paper we have studied, for the first

  6. Differentiation alters stem cell nuclear architecture, mechanics, and mechano-sensitivity

    Science.gov (United States)

    Heo, Su-Jin; Driscoll, Tristan P; Thorpe, Stephen D; Nerurkar, Nandan L; Baker, Brendon M; Yang, Michael T; Chen, Christopher S; Lee, David A; Mauck, Robert L

    2016-01-01

    Mesenchymal stem cell (MSC) differentiation is mediated by soluble and physical cues. In this study, we investigated differentiation-induced transformations in MSC cellular and nuclear biophysical properties and queried their role in mechanosensation. Our data show that nuclei in differentiated bovine and human MSCs stiffen and become resistant to deformation. This attenuated nuclear deformation was governed by restructuring of Lamin A/C and increased heterochromatin content. This change in nuclear stiffness sensitized MSCs to mechanical-loading-induced calcium signaling and differentiated marker expression. This sensitization was reversed when the ‘stiff’ differentiated nucleus was softened and was enhanced when the ‘soft’ undifferentiated nucleus was stiffened through pharmacologic treatment. Interestingly, dynamic loading of undifferentiated MSCs, in the absence of soluble differentiation factors, stiffened and condensed the nucleus, and increased mechanosensitivity more rapidly than soluble factors. These data suggest that the nucleus acts as a mechanostat to modulate cellular mechanosensation during differentiation. DOI: http://dx.doi.org/10.7554/eLife.18207.001 PMID:27901466

  7. Comparison of the Thermal Response of Two Calorimetric Cells Dedicated to Nuclear Heating Measurements during Calibration

    International Nuclear Information System (INIS)

    Brun, J.; Reynard, C.; De-Vita, C.; Carette, M.; Muraglia, M.; Lyoussi, A.; Fourmentel, D.; Guimbal, P.; Villard, J-F.

    2013-06-01

    Nuclear heating is a key parameter which contributes to the thermal design and the quality of in-pile experiments performed in Material Testing Reactors (MTRs) for the study of nuclear materials and fuels under irradiation. Nuclear heating is typically measured in MTRs by radiometric calorimeters. However this kind of sensor has to be suited and improved in perspective of the new experimental conditions inside the channels of Jules Horowitz Reactor (JHR). In this paper, we study the responses of two non adiabatic differential calorimeter cells having the same geometric design, but different dimensions. These experimental works are carried out during a preliminary out-of-pile calibration operating procedure of these sensors which consists in simulating the sample heating by Joule effect. The influence of the imposed electrical power and of the forced cooling flow is determined on the sensor calibration curves. A more sensitive sensor leads to a quadratic calibration curve. This behavior difference of the two calorimetric configurations is explained by means of temperature and heat flux measurements performed with a new instrumented jacket. (authors)

  8. Limit power of nuclear fuel cells with biconcave cross sections; Potencia limite de celulas de combustivel nuclear cilindricas de secao biconcava

    Energy Technology Data Exchange (ETDEWEB)

    Alves, Thiago Antonini; Pelegrini, Marcelo Ferreira; Woiski, Emanuel Rocha; Maia, Cassio Roberto Macedo [UNESP, Ilha Solteira, SP (Brazil). Dept. de Engenharia Mecanica]. E-mail: antonini@dem.feis.unesp.br; woiski@dem.feis.unesp.br; cassio@dem.feis.unesp.br

    2004-07-01

    Diffusive media with distributed sources, such as the case of nuclear fuel cells, represent a major role in engineering. Due to the nuclear fission of the chemical element, fuel cells are capable of releasing an enormous amount of thermal energy in spite of their reduced dimensions, in such a way that the maximum power of the reactor is closely related to the fusion temperature of the fuel, and consequently to the maximum temperature in the cell. The cell maximum temperature is, therefore, a chief parameter in nuclear reactor design. Limiting power, of course, depends not only of the fuel thermo physical properties, but also of the cell shape and dimensions. The present work purports the study of the effects of some parameters of cell geometry on the limiting power, especially for cell with biconcave cross sections. Given the large temperature gradients in the cell, the thermal conductivity must be assumed as a generic function of temperature. Therefore, the problem has been modeled as a nonlinear 2 D Poisson-like PDE, with a nontrivial geometry of the boundary. For the analytical solution, Kirchhoff transform has been employed to turn the equation into a linear Poisson equation, a conformal transform brought it to a rectangular domain and Generalized Integral Transform method applied in order to solve the resulting equation. For the numerical solution of the linearized equation, a program has been developed in Python, reusing classes of Ellipt2d, an open-source elliptic solver. The domain has been divided into linear triangular finite elements, and the system of equations resulting of Galerkin method application has been solved, for each parameter set. The trend in critical power has been discussed, as well as the numerical results compared to the analytical solutions and to the literature. (author)

  9. Construction of synthetic nucleoli and what it tells us about propagation of sub-nuclear domains through cell division.

    Science.gov (United States)

    Grob, Alice; McStay, Brian

    2014-01-01

    The cell nucleus is functionally compartmentalized into numerous membraneless and dynamic, yet defined, bodies. The cell cycle inheritance of these nuclear bodies (NBs) is poorly understood at the molecular level. In higher eukaryotes, their propagation is challenged by cell division through an "open" mitosis, where the nuclear envelope disassembles along with most NBs. A deeper understanding of the mechanisms involved can be achieved using the engineering principles of synthetic biology to construct artificial NBs. Successful biogenesis of such synthetic NBs demonstrates knowledge of the basic mechanisms involved. Application of this approach to the nucleolus, a paradigm of nuclear organization, has highlighted a key role for mitotic bookmarking in the cell cycle propagation of NBs.

  10. Effects of ultraviolet irradiation and postirradiation incubation on heterogeneous nuclear RNA size in murine cells

    International Nuclear Information System (INIS)

    Ali, R.; Sauerbier, W.

    1978-01-01

    We have analyzed the decrease in synthesis of individual size classes of heterogeneous nuclear RNA (hnRNA) in ultraviolet (uv)-irradiated Merwin plasmacytoma (MPC-11) cells at various times of postirradiation incubation. HnRNA from nonirradiated control cells is distributed over a wide range from approximately 60S to 5S, with 42S RNA carrying more label than any other size class. HnRNA from uv-irradiated cells shows a dose-dependent shift in size distribution toward lower molecular weight. The size distribution of hnRNA synthesized after prolonged times of postirradiation incubation is restored toward normal, i.e., synthesis of long RNA molecules increases relative to the synthesis of short ones. Analysis of the total number of hnRNA chains synthesized during a 20-min [ 3 H]uridine pulse shows a considerable eduction in their number with increasing uv dose. Murine cell lines are excision-repair-deficient but capable of post replication repair inhibited by caffeine. HnRNA transcripts of cells incubated in its presence were studied. The caffeine, which has no effect on hnRNA size in control cells, inhibits to a considerable extent the restoration of full-length transcripts during postirradiation incubation. The lack of excision repair in MPC-11 was confirmed by the analysis of pyrimidine dimers in trichloracetic acid-insoluble and soluble fractions within 8 h of postirradiation incubation. The size of parental and daughter strand DNA in uv-irradiated cells was correlated with RNA transcript size. The parental DNA in these experiments does not change its size as a consequence of uv exposure and postirradiation incubation. In contrast, daughter DNA strands are short in uv-irradiated cells and they increase in size during postirradiation incubation to reach the size of parental strands after 8 h

  11. Nuclear magnetic resonance spectroscopic investigation of anode exhaust of direct methanol fuel cells without isotope enrichment

    Energy Technology Data Exchange (ETDEWEB)

    Byun, Young Seok; Hwang, Reo Yun; Han, Ochee [Western Seoul Center, Korea Basic Science Institute, Seoul (Korea, Republic of)

    2016-12-15

    Fuel cells are devices that electrochemically convert the chemical energy of fuels such as natural gas, gasoline, and methanol, into electricity. Fuel cells more efficiently use energy than internal combustion engines and do not produce undesirable pollutants, such as NO{sub x} ,SO{sub x} and particulates. Fuel cells can be distinguished from one another by their electrolytes. Among the various direct alcohol fuel cells, direct methanol fuel cells (DMFCs) have been developed most. However, DMFCs have several practical problems such as methanol crossove r from an anode to a cathode and slow methanol oxidation reaction rates. Therefore, understanding the electrochemical reaction mechanisms of DMFCs may provide clues to solve these problems, and various analytical methods have been employed to examine these mechanisms. We demonstrated that {sup 1}H and {sup 13}C NMR spectroscopy can be used for analyzing anode exhausts of DMFCs operated with methanol without any isotope enrichment. However, the low sensitivity of NMR spectroscopy hindered our efforts to detect minor reaction intermediates. Therefore, sensitivity enhancement techniques such as dynamic nuclear polarization (DNP) NMR methods and/or presaturation methods to increase the dynamic range of the proton spectra by pre-saturating large water signals, are expected to be useful to detect low-concentration species.

  12. Nuclear fallout provides a new link between aPKC and polarized cell trafficking.

    Science.gov (United States)

    Calero-Cuenca, Francisco J; Espinosa-Vázquez, José Manuel; Reina-Campos, Miguel; Díaz-Meco, María T; Moscat, Jorge; Sotillos, Sol

    2016-04-18

    Cell polarity, essential for cell physiology and tissue coherence, emerges as a consequence of asymmetric localization of protein complexes and directional trafficking of cellular components. Although molecules required in both processes are well known their relationship is still poorly understood. Here we show a molecular link between Nuclear Fallout (Nuf), an adaptor of Rab11-GTPase to the microtubule motor proteins during Recycling Endosome (RE) trafficking, and aPKC, a pivotal kinase in the regulation of cell polarity. We demonstrate that aPKC phosphorylates Nuf modifying its subcellular distribution. Accordingly, in aPKC mutants Nuf and Rab11 accumulate apically indicating altered RE delivery. We show that aPKC localization in the apico-lateral cortex is dynamic. When we block exocytosis, by means of exocyst-sec mutants, aPKC accumulates inside the cells. Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling. We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution. We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached. Thus, we provide a novel link between cell polarity regulation and traffic control in epithelia.

  13. MEK inhibitor, PD98059, promotes breast cancer cell migration by inducing β-catenin nuclear accumulation.

    Science.gov (United States)

    Zhao, Ying; Ge, Chao-Chao; Wang, Jun; Wu, Xiao-Xiao; Li, Xiao-Min; Li, Wei; Wang, Sha-Sha; Liu, Tong; Hou, Jiu-Zhou; Sun, Hua; Fang, Dong; Xie, Song-Qiang

    2017-11-01

    Abnormal activation of the RAF/MEK/ERK signaling pathway has been observed in breast cancer. Thus, a number of MEK inhibitors have been designed as one treatment option for breast cancer. Although some studies have found that these MEK inhibitors inhibit the growth of a variety of human cancer cells, some trials have shown that the use of MEK inhibitors as a treatment for breast cancer does not adequately improve survival for unknown reasons. In the present study, MEK inhibitor PD98059 was used to evaluate its anticancer effects on human breast cancer MCF-7 and MDA-MB-231 cells and to explore the possible mechanism of action. Our results revealed that MEK inhibitor PD98059 exhibited antiproliferative effects in a dose- and time-dependent manner in MCF-7 and MDA-MB-231 breast cancer cells. Conversely, incubation of MCF-7 and MDA-MB-231 cells with PD98059 promoted their migration. Further investigation disclosed that the enhanced ability of migration promoted by PD98059 was dependent on β-catenin nuclear translocation in the MCF-7 and MDA-MB‑231 cells. Subsequent experiments documented that activation of EGFR signaling induced by PD98059 increased the amount of β-catenin in the nucleus. Taken together, our findings may elucidate a possible mechanism explaining the ineffectiveness of MEK inhibitors in breast cancer treatment and improve our understanding of the role of MEK in cancer.

  14. Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer.

    Science.gov (United States)

    Li, Honghui; Wang, Gui; Hao, Zhiqiang; Zhang, Guozhong; Qing, Yubo; Liu, Shuanghui; Qing, Lili; Pan, Weirong; Chen, Lei; Liu, Guichun; Zhao, Ruoping; Jia, Baoyu; Zeng, Luyao; Guo, Jianxiong; Zhao, Lixiao; Zhao, Heng; Lv, Chaoxiang; Xu, Kaixiang; Cheng, Wenmin; Li, Hushan; Zhao, Hong-Ye; Wang, Wen; Wei, Hong-Jiang

    2016-09-22

    Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth.

  15. Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer

    Science.gov (United States)

    Li, Honghui; Wang, Gui; Hao, Zhiqiang; Zhang, Guozhong; Qing, Yubo; Liu, Shuanghui; Qing, Lili; Pan, Weirong; Chen, Lei; Liu, Guichun; Zhao, Ruoping; Jia, Baoyu; Zeng, Luyao; Guo, Jianxiong; Zhao, Lixiao; Zhao, Heng; Lv, Chaoxiang; Xu, Kaixiang; Cheng, Wenmin; Li, Hushan; Zhao, Hong-Ye; Wang, Wen; Wei, Hong-Jiang

    2016-01-01

    Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth. PMID:27654750

  16. Development of maintenance equipment for nuclear material fabrication equipment in a highly active hot cell

    Energy Technology Data Exchange (ETDEWEB)

    Park, J. J.; Yang, M. S.; Kim, K. H. and others

    2000-09-01

    This report presents the development of a maintenance system for a highly contaminated nuclear material handling equipment at a hot-cell. This maintenance system has mainly three subsystems - a gamma-radiation measurement module for detecting a gamma-radiation level and identifying its distribution in-situ, a dry-type decontamination device for cleaning up contaminated particles, and a maintenance chamber for isolating contaminated equipment. The mechanical design considerations, controller, capabilities and remote operation and manipulation of the maintenance system are described. Such subsystems developed were installed and tested in the IMEF (Irradiated Material Examination Facility) M6 hot-cell after mock-up tests and performed their specific tasks successfully.

  17. Development of maintenance equipment for nuclear material fabrication equipment in a highly active hot cell

    International Nuclear Information System (INIS)

    Park, J. J.; Yang, M. S.; Kim, K. H. and others

    2000-09-01

    This report presents the development of a maintenance system for a highly contaminated nuclear material handling equipment at a hot-cell. This maintenance system has mainly three subsystems - a gamma-radiation measurement module for detecting a gamma-radiation level and identifying its distribution in-situ, a dry-type decontamination device for cleaning up contaminated particles, and a maintenance chamber for isolating contaminated equipment. The mechanical design considerations, controller, capabilities and remote operation and manipulation of the maintenance system are described. Such subsystems developed were installed and tested in the IMEF (Irradiated Material Examination Facility) M6 hot-cell after mock-up tests and performed their specific tasks successfully

  18. Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells.

    Science.gov (United States)

    Li, LiQi; Jothi, Raja; Cui, Kairong; Lee, Jan Y; Cohen, Tsadok; Gorivodsky, Marat; Tzchori, Itai; Zhao, Yangu; Hayes, Sandra M; Bresnick, Emery H; Zhao, Keji; Westphal, Heiner; Love, Paul E

    2011-02-01

    The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.

  19. Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies

    International Nuclear Information System (INIS)

    Loucaides, Eva M.; Kirchbach, Johann C. von; Foeglein, Agnes; Sharps, Jane; Fodor, Ervin; Digard, Paul

    2009-01-01

    The negative sense RNA genome of influenza A virus is transcribed and replicated in the nuclei of infected cells by the viral RNA polymerase. Only four viral polypeptides are required but multiple cellular components are potentially involved. We used fluorescence recovery after photobleaching (FRAP) to characterise the dynamics of GFP-tagged viral ribonucleoprotein (RNP) components in living cells. The nucleoprotein (NP) displayed very slow mobility that significantly increased on formation of transcriptionally active RNPs. Conversely, single or dimeric polymerase subunits showed fast nuclear dynamics that decreased upon formation of heterotrimers, suggesting increased interaction of the full polymerase complex with a relatively immobile cellular component(s). Treatment with inhibitors of cellular transcription indicated that in part, this reflected an interaction with cellular RNA polymerase II. Analysis of mutated influenza virus polymerase complexes further suggested that this was through an interaction between PB2 and RNA Pol II separate from PB2 cap-binding activity.

  20. A sup 1 H nuclear magnetic resonance study of structural and organisational changes in the cell

    CERN Document Server

    Tunnah, S K

    2000-01-01

    Increasing importance is being placed on understanding the role of membrane lipids in many different areas of biochemistry. It is of interest to determine what interactions may occur between membrane lipids and drug species. Furthermore, an increasing body of evidence suggests that membrane lipids are involved in the pathology of numerous diseases such as rheumatoid arthritis, cancer and HIV. Clearly, the more information available on the mechanisms involved in diseases, the greater the potential for identifying a cure or even a prevention. sup 1 H nuclear magnetic resonance (NMR) spectroscopy was used to study the alterations in membrane lipid organisation and structure in intact, viable cultured cells. Changes in the sup 1 H NMR spectra and the spin-lattice relaxation measurements of the human K562 and the rat FRTL-5 cell lines were observed on the addition of the fatty acid species: triolein, evening primrose oil, arachidonic acid and ITF 1779. Results indicate that the membrane lipids are reorganised to a...

  1. Silibinin induces apoptosis via calpain-dependent AIF nuclear translocation in U87MG human glioma cell death

    Directory of Open Access Journals (Sweden)

    Kim Yong K

    2011-04-01

    Full Text Available Abstract Background Silibinin, a natural polyphenolic flavonoid, has been reported to induce cell death in various cancer cell types. However, the molecular mechanism is not clearly defined. Our previous study showed that silibinin induces glioma cell death and its effect was effectively prevented by calpain inhibitor. The present study was therefore undertaken to examine the role of calpain in the silibinin-induced glioma cell death. Methods U87MG cells were grown on well tissue culture plates and cell viability was measured by MTT assay. ROS generation and △ψm were estimated using the fluorescence dyes. PKC activation and Bax expression were measured by Western blot analysis. AIF nuclear translocation was determined by Western blot and immunocytochemistry. Results Silibinin induced activation of calpain, which was blocked by EGTA and the calpain inhibitor Z-Leu-Leu-CHO. Silibinin caused ROS generation and its effect was inhibited by calpain inhibitor, the general PKC inhibitor GF 109203X, the specific PKCδ inhibitor rottlerin, and catalase. Silibinin-induce cell death was blocked by calpain inhibitor and PKC inhibitors. Silibinin-induced PKCδ activation and disruption of △ψm were prevented by the calpain inhibitor. Silibinin induced AIF nuclear translocation and its effect was prevented by calpain inhibitor. Transfection of vector expressing microRNA of AIF prevented the silibinin-induced cell death. Conclusions Silibinin induces apoptotic cell death through a calpain-dependent mechanism involving PKC, ROS, and AIF nuclear translocation in U87MG human glioma cells.

  2. Nuclear abnormalities in cells from nasal epithelium: a promising assay to evaluate DNA damage related to air pollution in infants.

    Science.gov (United States)

    Mergener, Michelle; Rhoden, Cláudia R; Amantéa, Sérgio L

    2014-01-01

    This study intends to provide a quick, easy, and inexpensive way to assess nuclear abnormalities such as micronuclei and bud frequencies; binucleated, karyorrhectic, karyolytic, pycnotic, and condensed chromatin cells in nasal scrapings of infants, which are particularly important for conducting genotoxic studies related to the inhaled atmosphere in pediatric populations. Nasal swab samples were collected from 40 infants under 12 months of age using a small cytobrush. 2,000 cells from each infant sample were analyzed and classified according to the frequency of nuclear abnormalities. Rates of nuclear abnormalities found agree with values reported in other studies of neonates and children. This study found 0.13% of cells with micronuclei; 1.20% karyorrhexis; 0.03% pyknosis; 10.85% karyolysis; 1.11% condensed chromatin; 0.54 binucleated cells; and 0.02% nuclear bud. Differences were not observed between genders or environmental passive smoking, nor was any age correlation found. The assay proposed here is suitable for assessing the frequency of nuclear abnormalities from nasal cells in infants. Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  3. Nuclear abnormalities in cells from nasal epithelium: a promising assay to evaluate DNA damage related to air pollution in infants

    Directory of Open Access Journals (Sweden)

    Michelle Mergener

    2014-12-01

    Full Text Available OBJECTIVES: This study intends to provide a quick, easy, and inexpensive way to assess nuclear abnormalities such as micronuclei and bud frequencies; binucleated, karyorrhectic, karyolytic, pycnotic, and condensed chromatin cells in nasal scrapings of infants, which are particularly important for conducting genotoxic studies related to the inhaled atmosphere in pediatric populations. METHODS: Nasal swab samples were collected from 40 infants under 12 months of age using a small cytobrush. 2,000 cells from each infant sample were analyzed and classified according to the frequency of nuclear abnormalities. RESULTS: Rates of nuclear abnormalities found agree with values reported in other studies of neonates and children. This study found 0.13% of cells with micronuclei; 1.20% karyorrhexis; 0.03% pyknosis; 10.85% karyolysis; 1.11% condensed chromatin; 0.54 binucleated cells; and 0.02% nuclear bud. Differences were not observed between genders or environmental passive smoking, nor was any age correlation found. CONCLUSION: The assay proposed here is suitable for assessing the frequency of nuclear abnormalities from nasal cells in infants.

  4. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Choi, Seong-Jun [Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of)

    2014-10-03

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

  5. The role of nuclear receptors in controlling the fine balance between proliferation and differentiation of neural stem cells.

    Science.gov (United States)

    Stergiopoulos, Athanasios; Politis, Panagiotis K

    2013-06-01

    In the central nervous system (CNS) of vertebrates a large variety of cell types are specified from a pool of highly plastic neural stem/progenitor cells (NSCs) via a combined action of extrinsic morphogenetic cues and intrinsic transcriptional regulatory networks. Nuclear receptors and their ligands are key regulators of fate decisions in NSCs during development and adulthood, through their ability to control transcription of downstream genes. In the last few years considerable progress has been made towards the understanding of the actions of nuclear receptors in NSCs as well as elucidating the mechanistic basis for these actions. Here we summarize recent progress in the role of nuclear receptors in the biology of NSCs. These studies highlight the importance of this family of transcriptional regulators in CNS development and function in health and disease. Furthermore, they raise the intriguing possibility of using nuclear receptors as therapeutic targets for nervous system related diseases and traumas. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

    Science.gov (United States)

    Waraky, Ahmed; Lin, Yingbo; Warsito, Dudi; Haglund, Felix; Aleem, Eiman; Larsson, Olle

    2017-11-03

    We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases ( e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G 1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Comparison of reprogramming genes in induced pluripotent stem cells and nuclear transfer cloned embryos.

    Science.gov (United States)

    Duan, Lian; Wang, Zhendong; Shen, Jingling; Shan, Zhiyan; Shen, Xinghui; Wu, Yanshuang; Sun, Ruizhen; Li, Tong; Yuan, Rui; Zhao, Qiaoshi; Bai, Guangyu; Gu, Yanli; Jin, Lianhong; Lei, Lei

    2014-08-01

    The most effective reprogramming methods, somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs), are widely used in biological research and regenerative medicine, yet the mechanism that reprograms somatic cells to totipotency remains unclear and thus reprogramming efficiency is still low. Microarray technology has been employed in analyzing the transcriptomes changes during iPS reprogramming. Unfortunately, it is difficult to obtain enough DNA from SCNT reconstructed embryos to take advantage of this technology. In this study, we aimed to identify critical genes from the transcriptional profile for iPS reprogramming and compared expression levels of these genes in SCNT reprogramming. By integrating gene expression information from microarray databases and published studies comparing somatic cells with either miPSCs or mouse embryonic stem cells (ESCs), we obtained two lists of co-upregulated genes. The gene ontology (GO) enriched analysis of these two lists demonstrated that the reprogramming process is associated with numerous biological processes. Specifically, we selected 32 genes related to heterochromatin, embryonic development, and cell cycle from our co-upregulated gene datasets and examined the gene expression level in iPSCs and SCNT embryos by qPCR. The results revealed that some reprogramming related genes in iPSCs were also expressed in SCNT reprogramming. We established the network of gene interactions that occur with genes differentially expressed in iPS and SCNT reprogramming and then performed GO analysis on the genes in the network. The network genes function in chromatin organization, heterochromatin, transcriptional regulation, and cell cycle. Further researches to improve reprogramming efficiency, especially in SCNT, will focus on functional studies of these selected genes.

  8. Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus

    OpenAIRE

    Zanta, Maria Antonietta; Belguise-Valladier, Pascale; Behr, Jean-Paul

    1999-01-01

    Translocation of exogenous DNA through the nuclear membrane is a major concern of gene delivery technologies. To take advantage of the cellular import machinery, we have synthesized a capped 3.3-kbp CMVLuciferase-NLS gene containing a single nuclear localization signal peptide (PKKKRKVEDPYC). Transfection of cells with the tagged gene remained effective down to nanogram amounts of DNA. Transfection enhancement (10- to 1,000-fold) as a result of the signal peptide was observed irrespective of ...

  9. Nuclear Translocation of b-Catenin during Mesenchymal Stem Cells Differentiation into Hepatocytes Is Associated with a Tumoral Phenotype

    OpenAIRE

    Herencia, Carmen; Martínez-Moreno, Julio M.; Herrera, Concepción; Corrales, Fernando J.; Santiago-Mora, Raquel; Espejo, Isabel; Barcos, Montserrat; Almadén Peña, Yolanda; Mata, Manuel de la; Rodríguez-Ariza, Antonio; Muñoz-Castañeda, Juan R.

    2012-01-01

    Wnt/b-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/b-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, b-catenin nuclear t...

  10. Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: Members of the nuclear DAPC associate with the nuclear matrix

    International Nuclear Information System (INIS)

    Fuentes-Mera, Lizeth; Rodriguez-Munoz, Rafael; Gonzalez-Ramirez, Ricardo; Garcia-Sierra, Francisco; Gonzalez, Everardo; Mornet, Dominique; Cisneros, Bulmaro

    2006-01-01

    Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. In this work, we report the identification and characterization of a novel DAPC present in the nuclei of HeLa cells, which contains dystrophin Dp71 as a key component. Using confocal microscopy and cell fractionation analyses, we found the presence of Dp71, β-sarcoglycan, β-dystroglycan, α- and β-syntrophin, α1- and β-dystrobrevin and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Next, we analyze the possible association of the nuclear DAPC with the nuclear matrix. We found the presence of Dp71, β-dystroglycan, nNOS, β-sarcoglycan, α/β syntrophin, α1-dystrobrevin and β-dystrobrevin in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells. Moreover, we found that Dp71, β-dystroglycan and β-dystrobrevin co-immunoprecipitated with the nuclear matrix proteins lamin B1 and actin. The association of members of the nuclear DAPC with the nuclear matrix indicates that they may work as scaffolding proteins involved in nuclear architecture

  11. MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Teng, E-mail: tengyu33@yahoo.com [Department of Dermatology, Shandong Ji-ning No. 1 People’s Hospital, Shandong Province 272011 (China); Ji, Jiang [Department of Dermatology, The Second Hospital Affiliated of Soochow University, SuZhou, Jiangsu Province 215000 (China); Guo, Yong-li [Department of Oncology, Shandong Ji-ning No. 1 People’s Hospital, Shandong Province 272011 (China)

    2013-11-08

    Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen species (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells.

  12. MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

    International Nuclear Information System (INIS)

    Yu, Teng; Ji, Jiang; Guo, Yong-li

    2013-01-01

    Highlights: •Curcumin activates MST1 in melanoma cells. •MST1 mediates curcumin-induced apoptosis of melanoma cells. •ROS production is involved in curcumin-induced MST1 activation. •MST1 mediates curcumin-induced JNK activation in melanoma cells. •MST1 mediates curcumin-induced Foxo3a nuclear translocation and Bim expression. -- Abstract: Different groups including ours have shown that curcumin induces melanoma cell apoptosis, here we focused the role of mammalian Sterile 20-like kinase 1 (MST1) in it. We observed that curcumin activated MST1-dependent apoptosis in cultured melanoma cells. MST1 silencing by RNA interference (RNAi) suppressed curcumin-induced cell apoptosis, while MST1 over-expressing increased curcumin sensitivity. Meanwhile, curcumin induced reactive oxygen species (ROS) production in melanoma cells, and the ROS scavenger, N-acetyl-cysteine (NAC), almost blocked MST1 activation to suggest that ROS might be required for MST1 activation by curcumin. c-Jun N-terminal protein kinase (JNK) activation by curcumin was dependent on MST1, since MST1 inhibition by RNAi or NAC largely inhibited curcumin-induced JNK activation. Further, curcumin induced Foxo3 nuclear translocation and Bim-1 (Foxo3 target gene) expression in melanoma cells, such an effect by curcumin was inhibited by MST1 RNAi. In conclusion, we suggested that MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells

  13. In vitro and in vivo genotoxic effects of somatic cell nuclear transfer cloned cattle meat.

    Science.gov (United States)

    Lee, Nam-Jin; Yang, Byoung-Chul; Jung, Yu-Ri; Lee, Jung-Won; Im, Gi-Sun; Seong, Hwan-Hoo; Park, Jin-Ki; Kang, Jong-Koo; Hwang, Seongsoo

    2011-09-01

    Although the nutritional composition and health status after consumption of the meat and milk derived from both conventionally bred (normal) and somatic cell nuclear transferred (cloned) animals and their progeny are not different, little is known about their food safeties like genetic toxicity. This study is performed to examine both in vitro (bacterial mutation and chromosome aberration) and in vivo (micronucleus) genotoxicity studies of cloned cattle meat. The concentrations of both normal and cloned cattle meat extracts (0-10×) were tested to five strains of bacteria (Salmonella typhimurium: TA98, TA100, TA1535, and TA1537; Escherichia coli: WP2uvrA) for bacterial mutation and to Chinese hamster lung (CHL/IU) cells for chromosome aberration, respectively. For micronucleus test, ICR mice were divided into five dietary groups: commercial pellets (control), pellets containing 5% (N-5) and 10% (N-10) normal cattle meat, and pellets containing 5% (C-5) and 10% (C-10) cloned cattle meat. No test substance-related genotoxicity was noted in the five bacterial strains, CHL/IU cells, or mouse bone marrow cells, suggesting that the cloned cattle meat potentially may be safe in terms of mutagenic hazards. Thus, it can be postulated that the cloned cattle meat do not induce any harmful genotoxic effects in vitro and in vivo. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Investigation of stroke in sickle cell disease by [sup 1]H nuclear magnetic resonance spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Z. (Dept. of Radiology, Pennsylvania Univ., Philadelphia, PA (United States)); Bogdan, A.R. (Siemens Medical Systems, Aston, PA (United States)); Zimmermann, R.A. (Dept. of Radiology, Pennsylvania Univ., Philadelphia, PA (United States)); Gusnard, D.A. (Dept. of Radiology, Pennsylvania Univ., Philadelphia, PA (United States)); Leigh, J.S. (Dept. of Radiology, Pennsylvania Univ., Philadelphia, PA (United States)); Ohene-Frempong, K. (Div. of Hematology, Children' s Hospital, Pennsylvania Univ., Philadelphia, PA (United States))

    1992-12-01

    Localized proton nuclear magnetic resonance spectroscopy (MRS), obtained with stimulated echo and spin echo sequences, MR imaging (MRI) and MR angiography (MRA) were used to study the brain in 13 children and adolescents with sickle cell disease. Regions of interest (ROI) studied by MRS included regions appearing normal on MRI as well as regions showing complications of sickle cell disease, including focal deep white matter areas of high signal intensity (deep white matter ischemia, DWMI) seen on long TR images, focal atropic brain areas, and infarcts. The findings in these studies are summarized as follows: Normal-appearing regions on MRI have normal MRS. In ROI including small areas of DWMI, lactate elevation was not detected, but the levels of N-acetyl-aspartate (NAA) appeared slightly elevated. In areas of DWMI 1-2 cm in size, reduced blood flow could be seen on MRA and lactate elevation could be detected with MRS. When blood flow to a DWMI region was normal, NAA was reduced and there was little lactate elevation, as cell death had already occurred. ROI consisting of atrophic tissue had reduced NAA levels but total creatine levels were not changed. Sometimes lipids, presumably from broken cell membrane, could be detected. In regions of past massive stroke, all metabolites were absent except for small amounts of lactate or lipids. (orig.)

  15. Investigation of stroke in sickle cell disease by 1H nuclear magnetic resonance spectroscopy

    International Nuclear Information System (INIS)

    Wang, Z.; Bogdan, A.R.; Zimmermann, R.A.; Gusnard, D.A.; Leigh, J.S.; Ohene-Frempong, K.

    1992-01-01

    Localized proton nuclear magnetic resonance spectroscopy (MRS), obtained with stimulated echo and spin echo sequences, MR imaging (MRI) and MR angiography (MRA) were used to study the brain in 13 children and adolescents with sickle cell disease. Regions of interest (ROI) studied by MRS included regions appearing normal on MRI as well as regions showing complications of sickle cell disease, including focal deep white matter areas of high signal intensity (deep white matter ischemia, DWMI) seen on long TR images, focal atropic brain areas, and infarcts. The findings in these studies are summarized as follows: Normal-appearing regions on MRI have normal MRS. In ROI including small areas of DWMI, lactate elevation was not detected, but the levels of N-acetyl-aspartate (NAA) appeared slightly elevated. In areas of DWMI 1-2 cm in size, reduced blood flow could be seen on MRA and lactate elevation could be detected with MRS. When blood flow to a DWMI region was normal, NAA was reduced and there was little lactate elevation, as cell death had already occurred. ROI consisting of atrophic tissue had reduced NAA levels but total creatine levels were not changed. Sometimes lipids, presumably from broken cell membrane, could be detected. In regions of past massive stroke, all metabolites were absent except for small amounts of lactate or lipids. (orig.)

  16. Serpent: an alternative for the nuclear fuel cells analysis of a BWR; SERPENT: una alternativa para el analisis de celdas de combustible nuclear de un BWR

    Energy Technology Data Exchange (ETDEWEB)

    Silva A, L.; Del Valle G, E. [IPN, Escuela Superior de Fisica y Matematicas, Av. Instituto Politecnico Nacional s/n, U.P. Adolfo Lopez Mateos, Edificio 9, Col. San Pedro Zacatenco, 07738 Mexico D. F. (Mexico); Gomez T, A. M., E-mail: lidi.s.albarran@gmail.com [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2013-10-15

    In the last ten years the diverse research groups in nuclear engineering of the Universidad Nacional Autonoma de Mexico and Instituto Politecnico Nacional (UNAM, IPN), as of research (Instituto Nacional de Investigaciones Nucleares, ININ) as well as the personnel of the Nuclear Plant Management of the Comision Federal de Electricidad have been using the codes Helios and /or CASMO-4 in the generation of cross sections (X S) of nuclear fuel cells of the cores corresponding to the Units 1 and 2 of the nuclear power plant of Laguna Verde. Both codes belong to the Studsvik-Scandpower Company who receives the payment for the use and their respective maintenance. In recent years, the code Serpent appears among the nuclear community distributed by the OECD/Nea which does not has cost neither in its use neither in its maintenance. The code is based on the Monte Carlo method and makes use of the processing in parallel. In the Escuela Superior de Fisica y Matematicas of the IPN, the personnel has accumulated certain experience in the use of Serpent under the direction of personal of the ININ; of this experience have been obtained for diverse fuel burned, the infinite multiplication factor for three cells of nuclear fuel, without control bar and with control bar for a known thermodynamic state fixed by: a) the fuel temperature (T{sub f}), b) the moderator temperature (T{sub m}) and c) the vacuums fraction (α). Although was not realized any comparison with the X S that the codes Helios and CASMO-4 generate, the results obtained for the infinite multiplication factor show the prospective tendencies with regard to the fuel burned so much in the case in that is not present the control bar like when it is. The results are encouraging and motivate to the study group to continue with the X S generation of a core in order to build the respective library of nuclear data as a following step and this can be used for the codes PARCS, of USA NRC, DYN3D of HZDR, or others developed locally

  17. Nuclear accumulation of beta-catenin occurs commonly in the epithelial cells of juvenile polyps.

    Science.gov (United States)

    Iwamoto, Michiko; Hoffenberg, Edward J; Carethers, John M; Doctolero, Ryan; Tajima, Akihiro; Sugano, Kentaro; Franklin, Wilbur A; Ahnen, Dennis J

    2005-01-01

    In the two conditions juvenile polyps (JPs) and juvenile polyposis coli (JPC), colonic polyps may have overlapping histologic and phenotypic appearance, but JPC confers a significant risk for colon adenocarcinoma. Although not thought to contain adenomatous polyposis coli (APC) mutations, the status of beta-catenin and full-length APC protein expression in JPs is not known. We evaluated beta-catenin and full-length APC protein expression in JPs from children with JPs and JPC. Cases were identified through endoscopic procedure records. Immunohistochemistry was performed for beta-catenin and full-length APC protein. Loss of heterozygosity at the APC gene locus on chromosome 5 was assessed using two APC-linked microsatellite markers. Polyp and normal colonic tissue were analyzed from 36 children with JPs and 9 with JPC. Both APC and beta-catenin immunoreactivity were present in epithelial cells from all samples but in different patterns. In all normal colon and polyp samples, APC expression was cytoplasmic with maximal immunoreactivity in the goblet cells. In contrast, beta-catenin immunoreactivity in epithelial cells was limited to the plasma membrane in normal colon but was both cytoplasmic and nuclear in all 45 JPs. No evidence of APC gene loss of heterozygosity was found. In polyps from children with JPs and JPC, nuclear beta-catenin accumulation is a consistent feature, and it is not due to APC gene mutation or loss of full-length APC protein expression. Thus, beta-catenin accumulation may be intrinsic to the formation of juvenile-type polyps through an as-yet-undefined mechanism.

  18. TR4 nuclear receptor suppresses HCC cell invasion via downregulating the EphA2 expression.

    Science.gov (United States)

    Jin, Ren'an; Lin, Hui; Li, Gonghui; Xu, Junjie; Shi, Liang; Chang, Chawnshang; Cai, Xiujun

    2018-02-15

    Early studies indicated that testicular nuclear receptor 4 (TR 4 ) could function as a suppressor in the transcriptional regulation of the HBV core gene expression, which might then influence the development of hepatocellular carcinoma (HCC). The direct linkage between TR 4 and HCC progression, however, remained unclear. Here, via a human clinical sample survey, we found that 13 of the 18 HCC patients studied had lower TR 4 expression in metastatic lesions than in matched primary HCC lesions, suggesting that TR 4 may play a negative role in HCC metastasis. Results from in vitro cell migration/invasion studied confirmed that TR 4 could suppress HCC cell migration/invasion. Mechanism dissection revealed that TR 4 might function through downregulating ephrin type-A receptor 2 (EphA2) expression at the transcriptional level via direct binding to the TR 4 REs located on the 5' promoter of EphA2 to suppress HCC cell migration/invasion. Targeting the EphA2 via EphA2-siRNA partially reversed the enhanced HCC cell migration/invasion with confirmed TR 4 knockdown. Notably, results from preclinical studies using in vivo mouse model with orthotopic xenograft of HCC LM3 cells also confirmed the in vitro findings. Taking these findings together, preclinical studies using multiple in vitro HCC cell lines and an in vivo mouse model all led to the conclusion that TR 4 may function as a suppressor of HCC metastasis and that targeting this newly identified TR 4 -EphA2 signaling may improve our ability to suppress HCC metastasis.

  19. The Nuclear Factor of Activated T Cells (Nfat) Transcription Factor Nfatp (Nfatc2) Is a Repressor of Chondrogenesis

    Science.gov (United States)

    Ranger, Ann M.; Gerstenfeld, Louis C.; Wang, Jinxi; Kon, Tamiyo; Bae, Hyunsu; Gravallese, Ellen M.; Glimcher, Melvin J.; Glimcher, Laurie H.

    2000-01-01

    Nuclear factor of activated T cells (NFAT) transcription factors regulate gene expression in lymphocytes and control cardiac valve formation. Here, we report that NFATp regulates chondrogenesis in the adult animal. In mice lacking NFATp, resident cells in the extraarticular connective tissues spontaneously differentiate to cartilage. These cartilage cells progressively differentiate and the tissue undergoes endochondral ossification, recapitulating the development of endochondral bone. Proliferation of already existing articular cartilage cells also occurs in some older animals. At both sites, neoplastic changes in the cartilage cells occur. Consistent with these data, NFATp expression is regulated in mesenchymal stem cells induced to differentiate along a chondrogenic pathway. Lack of NFATp in articular cartilage cells results in increased expression of cartilage markers, whereas overexpression of NFATp in cartilage cell lines extinguishes the cartilage phenotype. Thus, NFATp is a repressor of cartilage cell growth and differentiation and also has the properties of a tumor suppressor. PMID:10620601

  20. FTS is responsible for radiation-induced nuclear phosphorylation of EGFR and repair of DNA damage in cervical cancer cells.

    Science.gov (United States)

    Muthusami, Sridhar; Prabakaran, D S; Yu, Jae-Ran; Park, Woo-Yoon

    2015-02-01

    Radiation-induced nuclear stabilization and phosphorylation of epidermal growth factor receptor (EGFR) confers radioresistance. Understanding of the factor(s) regulating the nuclear stabilization and phosphorylation of EGFR is important for the modulation of radioresistance. Present study was designed to delineate the regulation of EGFR nuclear stabilization and phosphorylation by fused toes homolog (FTS), an oncoprotein, which is responsible for the radioresistance in cervical cancer cells. A cervical cancer cell line, ME180 was used. Radiation-induced change in the levels of EGFR, p-EGFR and FTS were evaluated in the cytoplasm and nucleus using Western blot analyses. FTS was silenced using siRNA-based approach. Interaction between EGFR and FTS was assessed using immunofluorescence and immunoprecipitation analyses. Double-strand breaks (DSB) of DNA were assessed using γ H2AX. Radiation increased the levels of EGFR and FTS in the cytoplasm and nucleus. EGFR and FTS are in physical association with each other and are co-localized in the cells. FTS silencing largely reduced the nuclear stabilization and phosphorylation of EGFR and DNA-protein kinase along with increased initial and residual DSBs. EGFR and FTS physically associate with each other and FTS silencing radiosensitizes ME180 cells through impaired nuclear EGFR signaling.

  1. UNcleProt (Universal Nuclear Protein database of barley): The first nuclear protein database that distinguishes proteins from different phases of the cell cycle

    Czech Academy of Sciences Publication Activity Database

    Blavet, Nicolas; Uřinovská, J.; Jeřábková, Hana; Chamrád, I.; Vrána, Jan; Lenobel, R.; Beinhauer, D.; Šebela, M.; Doležel, Jaroslav; Petrovská, Beáta

    2017-01-01

    Roč. 8, č. 1 (2017), s. 70-80 ISSN 1949-1034 R&D Projects: GA ČR(CZ) GA14-28443S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : cicer-arietinum l. * rice oryza-sativa * chromatin-associated proteins * proteomic analysis * mitotic chromosomes * dehydration * localization * chickpea * network * phosphoproteome * barley * cell cycle * database * flow-cytometry * localization * mass spectrometry * nuclear proteome * nucleus Subject RIV: CE - Biochemistry OBOR OECD: Cell biology Impact factor: 2.387, year: 2016

  2. Knock-down of methyl CpG-binding protein 2 (MeCP2 causes alterations in cell proliferation and nuclear lamins expression in mammalian cells

    Directory of Open Access Journals (Sweden)

    Babbio Federica

    2012-07-01

    Full Text Available Abstract Background MeCP2 (CpG-binding protein 2 is a nuclear multifunctional protein involved in several cellular processes, like large-scale chromatin reorganization and architecture, and transcriptional regulation. In recent years, a non-neuronal role for MeCP2 has emerged in cell growth and proliferation. Mutations in the MeCP2 gene have been reported to determine growth disadvantages in cultured lymphocyte cells, and its functional ablation suppresses cell growth in glial cells and proliferation in mesenchymal stem cells and prostate cancer cells. MeCP2 interacts with lamin B receptor (LBR and with Heterochromatin Protein 1 (HP1 at the nuclear envelope (NE, suggesting that it could be part of complexes involved in attracting heterochromatin at the nuclear periphery and in mediating gene silencing. The nuclear lamins, major components of the lamina, have a role in maintaining NE integrity, in orchestrating mitosis, in DNA replication and transcription, in regulation of mitosis and apoptosis and in providing anchoring sites for chromatin domains. In this work, we inferred that MeCP2 might have a role in nuclear envelope stability, thereby affecting the proliferation pattern of highly proliferating systems. Results By performing knock-down (KD of MeCP2 in normal murine (NIH-3 T3 and in human prostate transformed cells (PC-3 and LNCaP, we observed a strong proliferation decrease and a defect in the cell cycle progression, with accumulation of cells in S/G2M, without triggering a strong apoptotic and senescent phenotype. In these cells, KD of MeCP2 evidenced a considerable decrease of the levels of lamin A, lamin C, lamin B1 and LBR proteins. Moreover, by confocal analysis we confirmed the reduction of lamin A levels, but we also observed an alteration in the shape of the nuclear lamina and an irregular nuclear rim. Conclusions Our results that indicate reduced levels of NE components, are consistent with a hypothesis that the deficiency of Me

  3. Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fate.

    Science.gov (United States)

    Lin, Yin C; Benner, Christopher; Mansson, Robert; Heinz, Sven; Miyazaki, Kazuko; Miyazaki, Masaki; Chandra, Vivek; Bossen, Claudia; Glass, Christopher K; Murre, Cornelis

    2012-12-01

    The genome is folded into domains located in compartments that are either transcriptionally inert or transcriptionally permissive. Here we used genome-wide strategies to characterize domains during B cell development. Structured interaction matrix analysis showed that occupancy by the architectural protein CTCF was associated mainly with intradomain interactions, whereas sites bound by the histone acetyltransferase p300 or the transcription factors E2A or PU.1 were associated with intra- and interdomain interactions that are developmentally regulated. We identified a spectrum of genes that switched nuclear location during early B cell development. In progenitor cells, the transcriptionally inactive locus encoding early B cell factor (Ebf1) was sequestered at the nuclear lamina, which thereby preserved their multipotency. After development into the pro-B cell stage, Ebf1 and other genes switched compartments to establish new intra- and interdomain interactions associated with a B lineage-specific transcription signature.

  4. A role for aberrantly expressed nuclear localized decorin in migration and invasion of dysplastic and malignant oral epithelial cells.

    Science.gov (United States)

    Dil, Nyla; Banerjee, Abhijit G

    2011-09-29

    Oral cancer is the sixth most common malignancy worldwide with a mortality rate that is higher than many other cancers. Death usually occurs as a result of local invasion and regional lymph node metastases. Decorin is a multifunctional proteoglycan of the extracellular matrix that affects the biology of various types of cancer. Previously; we have shown that decorin is aberrantly expressed in the nucleus in human dysplastic oral keratinocytes (DOK) and malignant squamous cells carcinoma (SCC-25) and human biopsy tissues. In this study, we examined the role of nuclear decorin in oral cancer progression. We have used a post-transcriptional gene silencing (RNA interference) approach to stably knockdown nuclear decorin gene expression in DOK and SCC-25 cells using a specific shRNA plasmid and a combination of immunological and molecular techniques to study nuclear decorin function in these oral epithelial cell lines. More than 80% decorin silencing/knockdown was achieved as confirmed by real time PCR and western blot analysis in both DOK and SCC-25 cells. This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively. Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines. Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype. Furthermore, we found that nuclear localized decorin interacts with EGFR in the nuclear fractions of both DOK and SCC-25 cells. Interestingly, EGFR (trans) activation has previously been shown to be involved in IL-8 production in various epithelia. Taken together, our results indicate that nuclear localized decorin plays an

  5. The Cellular Distribution of RanGAP1 Is Regulated by CRM1-Mediated Nuclear Export in Mammalian Cells.

    Directory of Open Access Journals (Sweden)

    Keith Cha

    Full Text Available The Ran GTPase activating protein RanGAP1 plays an essential role in nuclear transport by stimulating RanGTP hydrolysis in the cytoplasmic compartment. In mammalian cells, unmodified RanGAP1 is predominantly cytoplasmic, whereas modification by small ubiquitin-related modifier protein (SUMO targets RanGAP1 to the cytoplasmic filaments of nuclear pore complex (NPC. Although RanGAP1 contains nine putative nuclear export signals and a nuclear localization signal, little is known if RanGAP1 shuttles between the nuclear and cytoplasmic compartments and how its primary localization in the cytoplasm and at the NPC is regulated. Here we show that inhibition of CRM1-mediated nuclear export using RNAi-knockdown of CRM1 and inactivation of CRM1 by leptomycin B (LMB results in nuclear accumulation of RanGAP1. LMB treatment induced a more robust redistribution of RanGAP1 from the cytoplasm to the nucleoplasm compared to CRM1 RNAi and also uniquely triggered a decrease or loss of RanGAP1 localization at the NPC, suggesting that LMB treatment is more effective in inhibiting CRM1-mediated nuclear export of RanGAP1. Our time-course analysis of LMB treatment reveals that the NPC-associated RanGAP1 is much more slowly redistributed to the nucleoplasm than the cytoplasmic RanGAP1. Furthermore, LMB-induced nuclear accumulation of RanGAP1 is positively correlated with an increase in levels of SUMO-modified RanGAP1, suggesting that SUMOylation of RanGAP1 may mainly take place in the nucleoplasm. Lastly, we demonstrate that the nuclear localization signal at the C-terminus of RanGAP1 is required for its nuclear accumulation in cells treated with LMB. Taken together, our results elucidate that RanGAP1 is actively transported between the nuclear and cytoplasmic compartments, and that the cytoplasmic and NPC localization of RanGAP1 is dependent on CRM1-mediated nuclear export.

  6. The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

    Science.gov (United States)

    Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi

    2017-05-01

    Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.

  7. Internet accessible hot cell with gamma spectroscopy at the Missouri S and T nuclear reactor

    International Nuclear Information System (INIS)

    Grant, Edwin; Mueller, Gary; Castano, Carlos; Usman, Shoaib; Kumar, Arvind

    2011-01-01

    Highlights: → A dual-chambered internet-accessible heavily shielded facility has been built. → The facility allows distance users to analyze neutron irradiated samples remotely. → The Missouri S and T system uses computer automation with user feedback. → The system can analyze multiple samples and assist several researchers concurrently. - Abstract: A dual-chambered internet-accessible heavily shielded facility with pneumatic access to the University of Missouri Science and Technology (Missouri S and T) 200 kW Research Nuclear Reactor (MSTR) core has been built and is currently available for irradiation and analysis of samples. The facility allows authorized distance users engaged in collaborative activities with Missouri S and T to remotely manipulate and analyze neutron irradiated samples. The system consists of two shielded compartments, one for multiple sample storage, and the other dedicated exclusively for radiation measurements and spectroscopy. The second chamber has multiple detector ports, with graded shielding, and has the capability to support gamma spectroscopy using radiation detectors such as an HPGe detector. Both these chambers are connected though a rapid pneumatic system with access to the MSTR nuclear reactor core. This new internet-based system complements the MSTR's current bare pneumatic tube (BPT) and cadmium lined pneumatic tube (CPT) facilities. The total transportation time between the core and the hot cell, for samples weighing 10 g, irradiated in the MSTR core, is roughly 3.0 s. This work was funded by the DOE grant number DE-FG07-07ID14852 and expands the capabilities of teaching and research at the MSTR. It allows individuals who do not have on-site access to a nuclear reactor facility to remotely participate in research and educational activities.

  8. High-pressure nuclear magnetic resonance studies of fuel cell membranes

    Science.gov (United States)

    Mananga, Eugene Stephane

    This thesis focuses on the use of high pressure NMR to study transport properties in electrolyte membranes used for fuel cells. The main concern is in studying the self-diffusion coefficients of ions and molecules in membranes and solutions, which can be used to characterize electrolytes in fuel cells. For this purpose, a high-pressure fringe field NMR method to study transport properties in material systems useful for fuel cell and battery electrolytes, was designed, developed, and implemented. In this investigation, pressure is the thermodynamic variable to obtain additional information about the ionic transport process, which could yield the crucial parameter, activation volume. Most of the work involves proton NMR, with additional investigations of others nuclei, such as fluorine, phosphorus and lithium. Using the FFG method, two fuel cell membrane types (NAFION-117, SPTES), and different dilutions of phosphoric acid were investigated, as was LiTf salt in Diglyme solution, which is used as a lithium battery electrolyte. In addition to high-pressure NMR diffusion measurements carried out in the fringe field gradient for the investigation of SPTES, pulse field gradient spin echo NMR was also used to characterize the water diffusion, in addition to measuring diffusion rates as a function of temperature. This second method allows us to measure distinct diffusion coefficients in cases where the different nuclear (proton) environments can be resolved in the NMR spectrum. Polymer electrolyte systems, in which the mobility of both cations and anions is probed by NMR self-diffusion measurements using standard pulsed field gradient methods and static gradient measurements as a function of applied hydrostatic pressure, were also investigated. The material investigated is the low molecular weight liquid diglyme/LiCF3SO3 (LiTf) complexes which can be used as electrolytes in lithium batteries. Finally, high-pressure diffusion coefficient measurements of phosphoric acid in

  9. Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

    Science.gov (United States)

    Zhang, Rong; Jack, Gregory S; Rao, Nagesh; Zuk, Patricia; Ignarro, Louis J; Wu, Benjamin; Rodríguez, Larissa V

    2012-03-01

    Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion. Copyright © 2011 AlphaMed Press.

  10. Quantification of the spatial organization of the nuclear lamina as a tool for cell classification

    NARCIS (Netherlands)

    Righolt, C.H.; Zatreanu, D.A.; Raz, V.

    2013-01-01

    The nuclear lamina is the structural scaffold of the nuclear envelope that plays multiple regulatory roles in chromatin organization and gene expression as well as a structural role in nuclear stability. The lamina proteins, also referred to as lamins, determine nuclear lamina organization and

  11. Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)

    Energy Technology Data Exchange (ETDEWEB)

    Do, Minhwa; Jang, Won-Gu; Hwang, Jeong Hee; Jang, Hoon; Kim, Eun-Jung; Jeong, Eun-Jeong [Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806 (Korea, Republic of); Shim, Hosup [Department of Physiology, Dankook University School of Medicine, Cheonan 330 714 (Korea, Republic of); Hwang, Sung Soo; Oh, Keon Bong; Byun, Sung June [Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Suwon (Korea, Republic of); Kim, Jin-Hoi [Department of Animal Biotechnology, Konkuk University, Seoul 143 701 (Korea, Republic of); Lee, Jeong Woong, E-mail: jwlee@kribb.re.kr [Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806 (Korea, Republic of)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer We success serial SCNT through the third generation using pig fibroblasts. Black-Right-Pointing-Pointer Donor-specific mtDNA in the recloned pigs was detected. Black-Right-Pointing-Pointer SCNT affect mtDNA mounts. -- Abstract: Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A{yields}T), 16062 (T{yields}C), and 16135 (G{yields}A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor's mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.

  12. Differential nuclear envelope assembly at the end of mitosis in suspension-cultured Apium graveolens cells.

    Science.gov (United States)

    Kimura, Yuta; Kuroda, Chie; Masuda, Kiyoshi

    2010-04-01

    NMCP1 is a plant protein that has a long coiled-coil domain within the molecule. Newly identified NMCP2 of Daucus carota and Apium graveolens showed similar peripheral localization in the interphase nucleus, and the sequence spanning the coiled-coil domain exhibited significant similarity with the corresponding region of NMCP1. To better understand disassembly and assembly of the nuclear envelope (NE) during mitosis, subcellular distribution of NMCP1 and NMCP2 was examined using A. graveolens cells. AgNMCP1 (NMCP1 in Apium) disassembled at prometaphase, dispersed mainly within the spindle, and accumulated on segregating chromosomes, while AgNMCP2 (NMCP2 in Apium), following disassembly at prometaphase with timing similar to that of AgNMCP1, dispersed throughout the mitotic cytoplasm at metaphase and anaphase. The protein accumulated at the periphery of reforming nuclei at telophase. A probe for the endomembrane indicated that the nuclear membrane (NM) disappears at prometaphase and begins to reappear at early telophase. Growth of the NM continued after mitosis was completed. NMCP2 in the mitotic cytoplasm localized in vesicular structures that could be distinguished from the bulk endomembrane system. These results suggest that NMCP1 and NMCP2 are recruited for NE assembly in different pathways in mitosis and that NMCP2 associates with NM-derived vesicles in the mitotic cytoplasm.

  13. Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.

    Science.gov (United States)

    Kieffer-Kwon, Kyong-Rim; Nimura, Keisuke; Rao, Suhas S P; Xu, Jianliang; Jung, Seolkyoung; Pekowska, Aleksandra; Dose, Marei; Stevens, Evan; Mathe, Ewy; Dong, Peng; Huang, Su-Chen; Ricci, Maria Aurelia; Baranello, Laura; Zheng, Ying; Tomassoni Ardori, Francesco; Resch, Wolfgang; Stavreva, Diana; Nelson, Steevenson; McAndrew, Michael; Casellas, Adriel; Finn, Elizabeth; Gregory, Charles; St Hilaire, Brian Glenn; Johnson, Steven M; Dubois, Wendy; Cosma, Maria Pia; Batchelor, Eric; Levens, David; Phair, Robert D; Misteli, Tom; Tessarollo, Lino; Hager, Gordon; Lakadamyali, Melike; Liu, Zhe; Floer, Monique; Shroff, Hari; Aiden, Erez Lieberman; Casellas, Rafael

    2017-08-17

    50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells. Published by Elsevier Inc.

  14. Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes.

    Science.gov (United States)

    Hwang, Insung; Jeong, Yeon Woo; Kim, Joung Joo; Lee, Hyo Jeong; Kang, Mina; Park, Kang Bae; Park, Jung Hwan; Kim, Yeun Wook; Kim, Woo Tae; Shin, Taeyoung; Hyun, Sang Hwan; Jeung, Eui-Bae; Hwang, Woo Suk

    2013-01-01

    Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature's diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (Pcloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones' inheritance of maternal domestic dog mitochondrial DNA.

  15. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue; Pang, Daxin; Ouyang, Hongsheng

    2011-07-29

    The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50μg/mL vitamin C 15h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. The design of in-cell crane handling systems for nuclear plants

    International Nuclear Information System (INIS)

    Hansford, S.M.; Scott, R.

    1992-01-01

    The reprocessing and waste management facilities at (BNFL's) British Nuclear Fuels Limited's Sellafield site make extensive use of crane handling systems. These range from conventional mechanical handling operations as used generally in industry to high integrity applications through to remote robotic handling operations in radiation environments. This paper describes the design methodologies developed for the design of crane systems for remote handling operations - in-cell crane systems. In most applications the in-cell crane systems are an integral part of the plant process equipment and reliable and safe operations are a key design parameter. Outlined are the techniques developed to achieve high levels of crane system availability for operations in hazardous radiation environments. These techniques are now well established and proven through many years of successful plant operation. A recent application of in-cell crane handling systems design for process duty application is described. The benefits of a systematic design approach and a functionally-based engineering organization are also highlighted. (author)

  17. Nuclear morphometric analysis of osteoblast precursor cells in periodontal ligament, SL-3 rats

    Science.gov (United States)

    Roberts, W. E.; Fielder, P. J.; Rosenoer, L. M.; Maese, A. C.; Gonsalves, M. R.; Morey, E. R.; Morey-Holton, E. R. (Principal Investigator)

    1987-01-01

    Five small (55 days old, 196 +/- 5 g) (mean +/- SE) and five large (83 days old, 382 +/- 4 g) Sprague-Dawley strain, specific pathogen-free rats were exposed to a 7-day spaceflight and 12-h postflight recovery period. As measured in 3-micron sections, periodontal ligament (PDL) fibroblastlike cells were classified according to nuclear size: A + A' (40-79), B (80-119), C (120-169), and D (greater than or equal to 170 microns 3). Since the histogenesis sequence is A----A'----C----D----osteoblast, the relative incidence of A + A' to C + D is an osteogenic index. No difference in A + A' or C + D cells in small rats may reflect partial recovery of preosteoblast formation (A----C) during the 12-h postflight period. Large flight rats demonstrated increased numbers of A + A', indicating an inhibition of preosteoblast formation (A----C). At least in the older group, a 7-day flight is adequate to reduce PDL osteogenic potential (inhibition in PDL osteoblast differentiation and/or specific attrition of C + D cells) that does not recover by 12-h postflight.

  18. Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

    Energy Technology Data Exchange (ETDEWEB)

    Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong; Chung, Hyun Joo [Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Kim, Myoung Hee, E-mail: mhkim1@yuhs.ac [Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

    2010-02-19

    Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna was similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.

  19. Characterization of the radiation field and evaluation of the nuclear responses in the ITER cryopump port cell

    Energy Technology Data Exchange (ETDEWEB)

    Moro, Fabio, E-mail: fabio.moro@enea.it [ENEA, Fusion Technical Unit, Nuclear Technologies Laboratory, 45th Enrico Fermi St., 00044 Frascati, Rome (Italy); Villari, Rosaria; Flammini, Davide; Podda, Salvatore [ENEA, Fusion Technical Unit, Nuclear Technologies Laboratory, 45th Enrico Fermi St., 00044 Frascati, Rome (Italy); Antipenkov, Alexander; Dremel, Matthias; Levesy, Bruno; Loughlin, Michael [ITER Organization, Route de Vinon sur Verdon, 13115 Saint Paul-lez-Durance (France); Cambi, Gilio; Cepraga, Dan Gabriel [Bologna University, Physics Department, 46th Irnerio St., I-401226 Bologna (Italy); Petrizzi, Luigino [European Commission, DG Research & Innovation G5, CDMA 00/030, B-1049 Brussels (Belgium)

    2016-11-01

    The ITER main vacuum system consists of six torus exhaust pumps integrated in the Cryostat through dedicated housings at the building B1 level. The Port Cell area outside the cryopump plug is affected by neutrons streaming through the housing structure and diagnostics penetrations. The aim of the study presented in this paper is to perform a complete assessment of the nuclear responses in the Cryopump Port Cell #12 by means of the MCNP-5 Monte Carlo code in a full 3-D geometry. The results of the neutronic analyses provide guidelines for the design and maintenance of the embedded components, ensuring their structural integrity and proper operation. Radiation transport calculations have been carried out to determine the radiation field inside the Port Cell through 3-D neutrons and gamma maps. Nuclear heating induced by neutrons and photons and the absorbed dose during the ITER lifetime on steel and silicon have been estimated in the Port Cell area, in order to assess the nuclear loads that the diagnostics and related electronic components have to withstand. Furthermore, the impact of the gamma-rays emitted by neutron-activated water circulating in the Primary Heat Transfer System have been evaluated on the Port Cell environment: 3-D maps of the gamma flux, nuclear loads on steel/silicon during plasma operation are provided.

  20. Distinct nuclear arrangement of active and inactive c-myc genes in control and differentiated colon carcinoma cells

    International Nuclear Information System (INIS)

    Harnicarova, Andrea; Kozubek, Stanislav; Pachernik, Jiri; Krejci, Jana; Bartova, Eva

    2006-01-01

    Using sequential RNA-DNA fluorescence in situ hybridization, the nuclear arrangement of both the active and inactive c-myc gene as well as its transcription was investigated in colon cancer HT-29 cells induced to differentiate into enterocytes. Cytogenetic studies revealed the presence of two chromosomes 8 in HT-29 cells, of which the one containing c-myc gene amplicons was substantially larger and easily distinguished from the normal chromosome. This observation enabled detection of both activity and nuclear localization of c-myc genes in single cells and in individual chromosome territories. Similar transcriptional activity of the c-myc gene was observed in both the normal and derivative chromosome 8 territories showing no influence of the amplification on the c-myc gene expression. Our experiments demonstrate strikingly specific nuclear and territorial arrangements of active genes as compared with inactive ones: on the periphery of their territories facing to the very central region of the cell nucleus. Nuclear arrangement of c-myc genes and transcripts was conserved during cell differentiation and, therefore, independent of the level of differentiation-specific c-myc gene expression. However, after the induction of differentiation, a more internal territorial location was found for the single copy c-myc gene of normal chromosome 8, while amplicons conserved their territorial topography

  1. In silico analyses reveal nuclear asymmetry of spongiocytes and compact cells of adrenocorticotrophic hormone-independent macronodular adrenocortical hyperplasia.

    Science.gov (United States)

    Zhang, Qian; Dou, Jingtao; Gu, Weijun; Yang, Guoqing; Mu, Yiming; Lu, Juming

    2014-05-01

    Little information is available about the risk of progression of seemingly benign adrenocortical hyperplasias to carcinomatous conditions. Using in silico approaches of digitally archived tissue sections, the nuclear morphometric parameters were compared to assess nuclear asymmetry as an index for nuclear atypia. Four groups of nuclei were used for the current study: spongiocytes and compact cells obtained from adrenocorticotropic hormone (ACTH)-independent macronodular hyperplasia, which were hypothesized to be high risk for nuclear asymmetry, and primary pigmented nodular adrenocortical disease and micronodular adrenocortical hyperplasia samples were used as internal controls. Analyses reveal high nuclear irregularity index of spongiocytes and shape factor abnormalities of both spongiocytes and compact cells of ACTH-independent macronodular adrenal hyperplasia compared with the other 2 groups (high F values and very low P values after analyses of variances), thus confirming the hypothesis that ACTH-independent macronodular adrenal hyperplasia present with subtle morphometric features of nuclear atypia. This probably puts this class of adrenocortical tumors at risk of dysplastic progression, and more studies are needed to test the hypothesis.

  2. In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

    Science.gov (United States)

    Kim, Dong-Hoon; No, Jin-Gu; Choi, Mi-Kyung; Yeom, Dong-Hyeon; Kim, Dong-Kyo; Yang, Byoung-Chul; Yoo, Jae Gyu; Kim, Min Kyu; Kim, Hong-Tea

    2015-01-01

    The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.

  3. The phosphorylation-dependent regulation of nuclear SREBP1 during mitosis links lipid metabolism and cell growth

    Science.gov (United States)

    Bengoechea-Alonso, Maria Teresa; Ericsson, Johan

    2016-01-01

    ABSTRACT The SREBP transcription factors are major regulators of lipid metabolism. Disturbances in lipid metabolism are at the core of several health issues facing modern society, including cardiovascular disease, obesity and diabetes. In addition, the role of lipid metabolism in cancer cell growth is receiving increased attention. Transcriptionally active SREBP molecules are unstable and rapidly degraded in a phosphorylation-dependent manner by Fbw7, a ubiquitin ligase that targets several cell cycle regulatory proteins for degradation. We have previously demonstrated that active SREBP1 is stabilized during mitosis. We have now delineated the mechanisms involved in the stabilization of SREBP1 in mitotic cells. This process is initiated by the phosphorylation of a specific serine residue in nuclear SREBP1 by the mitotic kinase Cdk1. The phosphorylation of this residue creates a docking site for a separate mitotic kinase, Plk1. Plk1 interacts with nuclear SREBP1 in mitotic cells and phosphorylates a number of residues in the C-terminal domain of the protein, including a threonine residue in close proximity of the Fbw7 docking site in SREBP1. The phosphorylation of these residues by Plk1 blocks the interaction between SREBP1 and Fbw7 and attenuates the Fbw7-dependent degradation of nuclear SREBP1 during cell division. Inactivation of SREBP1 results in a mitotic defect, suggesting that SREBP1 could regulate cell division. We propose that the mitotic phosphorylation and stabilization of nuclear SREBP1 during cell division provides a link between lipid metabolism and cell proliferation. Thus, the current study provides additional support for the emerging hypothesis that SREBP-dependent lipid metabolism may be important for cell growth. PMID:27579997

  4. Nuclear transfer of synchronized African wild cat somatic cells into enucleated domestic cat oocytes

    Science.gov (United States)

    Gomez, M.C.; Jenkins, J.A.; Giraldo, A.; Harris, R.F.; King, A.; Dresser, B.L.; Pope, C.E.

    2003-01-01

    The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei

  5. Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer.

    Science.gov (United States)

    Park, JungEun; Oh, HyunJu; Hong, SoGun; Kim, MinJung; Kim, GeonA; Koo, OkJae; Kang, SungKeun; Jang, Goo; Lee, ByeongChun

    2011-03-01

    As shown by the birth of the first cloned dog 'Snuppy', a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Review: Placental perturbations induce the developmental abnormalities often observed in bovine somatic cell nuclear transfer.

    Science.gov (United States)

    Chavatte-Palmer, P; Camous, S; Jammes, H; Le Cleac'h, N; Guillomot, M; Lee, R S F

    2012-02-01

    Since the first success in cloning sheep, the production of viable animals by somatic cell nuclear transfer (SCNT) has developed significantly. Cattle are by far the most successfully cloned species but, despite this, the technique is still associated with a high incidence of pregnancy failure and accompanying placental and fetal pathologies. Pre- and early post-implantation losses can affect up to 70% of the pregnancies. In the surviving pregnancies, placentomegaly and fetal overgrowth are commonly observed, but the incidence varies widely, depending on the genotype of the nuclear donor cell and differences in SCNT procedures. In all cases, the placenta is central to the onset of the pathologies. Although cellular organisation of the SCNT placenta appears normal, placental vascularisation is modified and fetal-to-maternal tissue ratios are slightly increased in the SCNT placentomes. In terms of functionality, steroidogenesis is perturbed and abnormal estrogen production and metabolism probably play an important part in the increased gestation length and lack of preparation for parturition observed in SCNT recipients. Maternal plasma concentrations of pregnancy-associated glycoproteins are increased, mostly due to a reduction in turnover rate rather than increased placental production. Placental glucose transport and fructose synthesis appear to be modified and hyperfructosemia has been observed in neonatal SCNT calves. Gene expression analyses of the bovine SCNT placenta show that multiple pathways and functions are affected. Abnormal epigenetic re-programming appears to be a key component of the observed pathologies, as shown by studies on the expression of imprinted genes in SCNT placenta. Copyright © 2012. Published by Elsevier Ltd.

  7. Nuclear localization of the transcriptional regulator MIER1α requires interaction with HDAC1/2 in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Shengnan Li

    Full Text Available MIER1α is a transcriptional regulator that functions in gene repression through its ability to interact with various chromatin modifiers and transcription factors. We have also shown that MIER1α interacts with ERα and inhibits estrogen-stimulated growth. While MIER1α is localized in the nucleus of MCF7 cells, previous studies have shown that it does not contain a nuclear localization signal. In this report, we investigate the mechanism involved in transporting MIER1α into the nucleus. We explored the possibility that MIER1α is transported into the nucleus through a 'piggyback' mechanism. One obvious choice is via interaction with ERα, however we demonstrate that nuclear targeting of MIER1α does not require ERα. Knockdown of ERα reduced protein expression to 22% of control, but did not alter the percentage of cells with nuclear MIER1α (98% nuclear with scrambled shRNA vs. 95% with ERα shRNA. Further evidence was obtained using two stable transfectants derived from the ER-negative MDA231 cell line: MC2 (ERα+ and VC5 (ERα-. Confocal analysis showed no difference in MIER1α localization (86% nuclear in MC2 vs. 89% in VC5. These data demonstrate that ERα is not involved in nuclear localization of MIER1α. To identify the critical MIER1α sequence, we performed a deletion analysis and determined that the ELM2 domain was necessary and sufficient for nuclear localization. This domain binds HDAC1 & 2, therefore we investigated their role. Confocal analysis of an MIER1α containing an ELM2 point mutation previously shown to abolish HDAC binding revealed that this mutation results in almost complete loss of nuclear targeting: 10% nuclear vs. 97% with WT-MIER1α. Moreover, double knockdown of HDAC1 and 2 caused a reduction in percent nuclear from 86% to 44%. The results of this study demonstrate that nuclear targeting of MIER1α requires an intact ELM2 domain and is dependent on interaction with HDAC1/2.

  8. v-Src-induced nuclear localization of YAP is involved in multipolar spindle formation in tetraploid cells.

    Science.gov (United States)

    Kakae, Keiko; Ikeuchi, Masayoshi; Kuga, Takahisa; Saito, Youhei; Yamaguchi, Naoto; Nakayama, Yuji

    2017-01-01

    The protein-tyrosine kinase, c-Src, is involved in a variety of signaling events, including cell division. We have reported that v-Src, which is a mutant variant of the cellular proto-oncogene, c-Src, causes delocalization of Aurora B kinase, resulting in a furrow regression in cytokinesis and the generation of multinucleated cells. However, the effect of v-Src on mitotic spindle formation is unknown. Here we show that v-Src-expressing HCT116 and NIH3T3 cells undergo abnormal cell division, in which cells separate into more than two cells. Upon v-Src expression, the proportion of multinucleated cells is increased in a time-dependent manner. Flow cytometry analysis revealed that v-Src increases the number of cells having a ≥4N DNA content. Microscopic analysis showed that v-Src induces the formation of multipolar spindles with excess centrosomes. These results suggest that v-Src induces multipolar spindle formation by generating multinucleated cells. Tetraploidy activates the tetraploidy checkpoint, leading to a cell cycle arrest of tetraploid cells at the G1 phase, in which the nuclear exclusion of the transcription co-activator YAP plays a critical role. In multinucleated cells that are induced by cytochalasin B and the Plk1 inhibitor, YAP is excluded from the nucleus. However, v-Src prevents this nuclear exclusion of YAP through a decrease in the phosphorylation of YAP at Ser127 in multinucleated cells. Furthermore, v-Src decreases the expression level of p53, which also plays a critical role in the cell cycle arrest of tetraploid cells. These results suggest that v-Src promotes abnormal spindle formation in at least two ways: generation of multinucleated cells and a weakening of the tetraploidy checkpoint. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Deguelin regulates nuclear pore complex proteins Nup98 and Nup88 in U937 cells in vitro.

    Science.gov (United States)

    Liu, Hong-li; Chen, Yan; Cui, Guo-hui; Wu, Qiu-ling; He, Jing; Chen, Wei-hua; Zhou, Jian-feng

    2005-10-01

    To investigate the anticancer effects and the molecular mechanisms of deguelin on human U937 leukemia cells, and to explore the underlying mechanism regulating nucleoporin 98 (Nup98) and nucleoporin 88 (Nup88) in vitro. The effects of deguelin on the growth of U937 cells were studied by MTT assay. The effect of deguelin on the cell cycle of U937 cells was studied by using a propidium iodide method. The localization of the nuclear pore complex proteins Nup98 and Nup88 was investigated by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 and Nup88 in U937 cells was investigated by using flow cytometry and Western blot. The proliferation of U937 cells was inhibited in the deguelin-treated group, with a 24-h IC(50) value of 21.61 nmol/L and a 36-h IC(50) value of 17.07 nmol/L. U937 cells treated with deguelin had reduced percentages of cells in the G(0)/G(1) phase, whereas cells accumulated in the S and G(2)/M phases. Nup88 and Nup98 were found on both the nuclear and cytoplasmic sides of the U937 cells by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 was upregulated and that of the Nup88 protein was downregulated in U937 cells treated with deguelin. Deguelin is able to inhibit the proliferation of U937 cells by regulating the cell cycle such that cells are arrested at the S and G(2)/M phases, so that the proportion of cells in the G(0)/G(1) phase decreases. The antitumor effects of deguelin are related to upregulating the expression of Nup98 and downregulating the expression of Nup88 protein in U937 cells.

  10. [Effect of Nm23-H1 Nuclear Localization on Proliferation of 
Human Lung Adenocarcinoma Cell Line A549].

    Science.gov (United States)

    Sheng, Ya; Xiong, Yanli; Xu, Mingfang; Kuang, Xunjie; Wang, Dong; Yang, Xueqin

    2017-04-20

    Recent studies have indicated that Nm23-H1 is found in the nucleus, but previous studies have been based on the overexpression or suppression of Nm23-H1 in the cytoplasm. Due to the lacking nuclear localization signal of Nm23-H1, these results cannot reflect or repeat cells in which Nm23-H1 mainly positioned in nuclei and whether they cause clinical biological effects. Therefore, to explore the effects of transposing Nm23-H1 from the cytoplasm to the nucleus during lung cancer cell proliferation, a vector with a nuclear localization signal of Nm23-H1 was constructed and A549 cells were transfected. Gene recombination technology was used to construct pLentis-CMV-NME1-IRES2-PURO lentiviral vectors using a nuclear localization signal sequence, and the recombinant plasmid was verified using restriction enzyme analysis and sequencing. Nm23-H1 positioning and expression were performed after the stably transfected A549 cells were assessed by Western blot and confocal laser scanning microscope. The A549 cell proliferation was assessed using a cell counting kit-8. Flow cytometry was performed to assess the cell cycle distribution of A549 cells. The directional Nm23-H1 lentiviral vector was successfully constructed within the nucleus. Compared with that of the empty vector group, the proliferation rates of the transfection groups at 72 h, 96 h, and 120 h were remarkably increased (PA549 cells in the G0/G1 phase proportion was 35.69%, which was higher than the 28.28% of the transfection group (t=1.461, P=0.217); furthermore, the transfection group of A549 cells in the G2/M phase proportion was 58.7% and that of the empty vector group was 31.30% (t=4.560, P=0.010). Human lung adenocarcinoma cell line A549 cells of Nm23-H1 nuclear localized mainly in the G2/M phase and the nuclear Nm23-H1 promoted A549 cell proliferation in vitro.

  11. Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

    International Nuclear Information System (INIS)

    Sun, Hengwen; Yang, Shana; Li, Jianhua; Zhang, Yajie; Gao, Dongsheng; Zhao, Shenting

    2016-01-01

    Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.

  12. Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Hengwen [Department of Radiation, Cancer Center of Guangdong General Hospital (Guangdong Academy of Medical Science), Guangzhou, 510080, Guangdong (China); Yang, Shana; Li, Jianhua [Department of Physiology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China); Zhang, Yajie [Department of Pathology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China); Gao, Dongsheng [Department of Oncology, Guangdong Medical College Affiliated Pengpai Memorial Hospital, Hai Feng, 516400, Gungdong (China); Zhao, Shenting, E-mail: zhaoshenting@126.com [Department of Physiology, Guangzhou Medical University, Guangzhou, 510182, Guangdong (China)

    2016-03-25

    Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.

  13. Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate.

    Science.gov (United States)

    Castore, Reneau; Hughes, Cameron; Debeaux, Austin; Sun, Jingxin; Zeng, Cailing; Wang, Shih-Ya; Tatchell, Kelly; Shi, Runhua; Lee, Kyung-Jong; Chen, David J; Harrison, Lynn

    2011-11-01

    Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments.

  14. Quantitative and subcellular localization analysis of the nuclear isoform dUTP pyrophosphatase in alkylating agent-induced cell responses

    International Nuclear Information System (INIS)

    Hu, Xiaolan; Yu, Yingnian; Li, Qian; Wu, Danxiao; Tan, Zhengning; Wang, Cheng; Wang, Jvping; Wu, Meiping

    2011-01-01

    Highlights: → MNNG-induced appearance of DUT-N in the extracellular fluid has cellular specificity. → MNNG alters the subcellular distribution of DUT-N in human cells in different ways. → DUT-N may be a potential biomarker to assess the risk of alkylating agents exposure. -- Abstract: Our previous proteome analysis showed that the nuclear isoform of dUTP pyrophosphatase (DUT-N) was identified in the culture medium of human amnion FL cells after exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). These results suggest that DUT-N may be a potential early biomarker to assess the risk of alkylating agents exposure. DUT-N is one of the two isoforms of deoxyuridine triphosphate nucleotidohydrolase (dUTPase). Our current knowledge of DUT-N expression in human cells is very limited. In the current study, we first investigated the appearance of DUT-N in the culture medium of different human cell lines in response to a low concentration of MNNG exposure. We verified that the MNNG-induced appearance of DUT-N in the extracellular environment is cell-specific. Western blot analysis confirmed that the intracellular DUT-N changes responded to MNNG in a concentration-dependent and cell-specific manner. Furthermore, subcellular fraction experiments showed that 0.25 μM MNNG treatment dramatically increased the DUT-N expression levels in the cytoplasmic extracts prepared from both FL and HepG2 cells, increased DUT-N levels in nuclear extracts prepared from HepG2 cells, and decreased DUT-N levels in nuclear extracts from FL cells. Morphological studies using immunofluorescence showed that a low concentration of MNNG could alter the distribution of DUT-N in FL and HepG2 cells in different ways. Taken together, these studies indicate a role of DUT-N in alkylating agent-induced cell responses.

  15. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer.

    Science.gov (United States)

    Zhou, Xiaoqing; Xin, Jige; Fan, Nana; Zou, Qingjian; Huang, Jiao; Ouyang, Zhen; Zhao, Yu; Zhao, Bentian; Liu, Zhaoming; Lai, Sisi; Yi, Xiaoling; Guo, Lin; Esteban, Miguel A; Zeng, Yangzhi; Yang, Huaqiang; Lai, Liangxue

    2015-03-01

    The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals. However, these animals have been generated by co-injection of Cas9 mRNA and single-guide RNA (sgRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. One or two rounds of further breeding should be performed to obtain homozygotes with identical genotype and phenotype. To address this issue, gene-targeted somatic cells can be used as donor for somatic cell nuclear transfer (SCNT) to produce gene-targeted animals with single and identical mutations. In this study, we applied Cas9/sgRNAs to effectively direct gene editing in porcine fetal fibroblasts and then mutant cell colonies were used as donor to generate homozygous gene-targeted pigs through single round of SCNT. As a result, we successfully obtained 15 tyrosinase (TYR) biallelic mutant pigs and 20 PARK2 and PINK1 double-gene knockout (KO) pigs. They were all homozygous and no off-target mutagenesis was detected by comprehensive analysis. TYR (-/-) pigs showed typical albinism and the expression of parkin and PINK1 were depleted in PARK2 (-/-)/PINK1 (-/-) pigs. The results demonstrated that single- or double-gene targeted pigs can be effectively achieved by using the CRISPR/Cas9 system combined with SCNT without mosaic mutation and detectable off-target effects. This gene-editing system provides an efficient, rapid, and less costly manner to generate genetically modified pigs or other large animals.

  16. Distinct functional domains of PNMA5 mediate protein-protein interaction, nuclear localization, and apoptosis signaling in human cancer cells.

    Science.gov (United States)

    Lee, Yong Hoi; Pang, Siew Wai; Poh, Chit Laa; Tan, Kuan Onn

    2016-09-01

    Members of paraneoplastic Ma (PNMA) family have been identified as onconeuronal antigens, which aberrant expressions in cancer cells of patients with paraneoplastic disorder (PND) are closely linked to manifestation of auto-immunity, neuro-degeneration, and cancer. The purpose of present study was to determine the role of PNMA5 and its functional relationship to MOAP-1 (PNMA4) in human cancer cells. PNMA5 mutants were generated through deletion or site-directed mutagenesis and transiently expressed in human cancer cell lines to investigate their role in apoptosis, subcellular localization, and potential interaction with MOAP-1 through apoptosis assays, fluorescence microscopy, and co-immunoprecipitation studies, respectively. Over-expressed human PNMA5 exhibited nuclear localization pattern in both MCF-7 and HeLa cells. Deletion mapping and mutagenesis studies showed that C-terminus of PNMA5 is responsible for nuclear localization, while the amino acid residues (391KRRR) within the C-terminus of PNMA5 are required for nuclear targeting. Deletion mapping and co-immunoprecipitation studies showed that PNMA5 interacts with MOAP-1 and N-terminal domain of PNMA5 is required for interaction with MOAP-1. Furthermore, co-expression of PNMA5 and MOAP-1 in MCF-7 cells significantly enhanced chemo-sensitivity of MCF-7 to Etoposide treatment, indicating that PNMA5 and MOAP-1 interact synergistically to promote apoptotic signaling in MCF-7 cells. Our results show that PNMA5 promotes apoptosis signaling in HeLa and MCF-7 cells and interacts synergistically with MOAP-1 through its N-terminal domain to promote apoptosis and chemo-sensitivity in human cancer cells. The C-terminal domain of PNMA5 is required for nuclear localization; however, both N-and C-terminal domains of PNMA5 appear to be required for pro-apoptotic function.

  17. Simulation of the burnup in cell calculation using the WIMSD-5B Code considering different nuclear data libraries

    Energy Technology Data Exchange (ETDEWEB)

    Tavares, Desirée Yael de Sena; Silva, Adilson Costa da; Lima, Zelmo Rodrigues de, E-mail: zelmolima@yahoo.com.br [Instituto de Engenharia Nuclear (IEN/CNEN-RJ), Rio de Janeiro, RJ (Brazil)

    2017-07-01

    This work proposes to implement the cell calculation considering the fuel burning using the WIMSD-5B code. The cell calculation procedure allows to determine the nuclear parameters present in the multi-group neutron diffusion equation and for this purpose the neutron transport theory is used in a problem with dimensional reduction, but in contrast is considered a large number of groups associated with the neutron spectrum. There are a variety of reactor physics codes that determine the nuclear parameters by solving the neutron transport equation applied to an equivalent cell representing a fuel element. The WIMSD-5B code is a deterministic code that solves the transport equation using collision probability method. The simulation of fuel burning in the cell calculation took into account different nuclear data libraries. The WIMSD-5B code supports several nuclear data libraries and in the present work the following libraries were used: IAEA, ENDFB-VII.1, JENDL3.2, JEFF3.1 and JEF2.2, all formatted for 69 energy groups. (author)

  18. High frequency of tumor cells with nuclear Egr-1 protein expression in human bladder cancer is associated with disease progression

    International Nuclear Information System (INIS)

    Egerod, Frederikke Lihme; Bartels, Annette; Fristrup, Niels; Borre, Michael; Ørntoft, Torben F; Oleksiewicz, Martin B; Brünner, Nils; Dyrskjøt, Lars

    2009-01-01

    Egr-1 (early growth response-1 transcription factor) has been proposed to be involved in invasion and metastasis processes of human bladder cancer, but Egr-1 protein expression levels in human bladder cancer have not been investigated. In the present study we investigated the expression levels of Egr-1 protein in early stages of human bladder cancer and correlated it to later progression. Expression of Egr-1 protein in human bladder cancer was examined by immunohistochemistry, on a tissue microarray constructed from tumors from 289 patients with non-muscle invasive urothelial bladder cancer. The frequency of tumor cells with nuclear Egr-1 immunolabelling correlated to bladder cancer stage, grade and to later progression to muscle-invasive bladder cancer (T2-4). Stage T1 tumors exhibited significantly higher frequencies of tumor cells with nuclear Egr-1 immunolabelling than Ta tumors (P = 0.001). Furthermore, Kaplan-Meier survival analysis showed that a high frequency of tumor cells with nuclear Egr-1 immunolabelling was significantly associated with a higher risk of progression to stage T2-4 (log-rank test, P = 0.035). Tumor cells with nuclear Egr-1 immunolabelling were found to localize at the tumor front in some of the tumor biopsies. The results from this study support a potential involvement of Egr-1 in the progression from non-muscle invasive bladder cancers to muscle invasive bladder cancer

  19. Stereological estimates of nuclear volume in squamous cell carcinoma of the uterine cervix and its precursors

    DEFF Research Database (Denmark)

    Sørensen, Flemming Brandt; Bichel, P; Jakobsen, A

    1991-01-01

    Using modern stereology, this study was carried out to obtain base-line data concerning three-dimensional, mean nuclear size in precancerous and invasive lesions of the uterine cervix. Unbiased estimates of the volume-weighted mean nuclear volume (nuclear vv) were obtained by point-sampling of nu......Using modern stereology, this study was carried out to obtain base-line data concerning three-dimensional, mean nuclear size in precancerous and invasive lesions of the uterine cervix. Unbiased estimates of the volume-weighted mean nuclear volume (nuclear vv) were obtained by point...

  20. The effect of the lamin A and its mutants on nuclear structure, cell proliferation, protein stability, and mobility in embryonic cells.

    Science.gov (United States)

    Piekarowicz, Katarzyna; Machowska, Magdalena; Dratkiewicz, Ewelina; Lorek, Daria; Madej-Pilarczyk, Agnieszka; Rzepecki, Ryszard

    2017-08-01

    LMNA gene encodes for nuclear intermediate filament proteins lamin A/C. Mutations in this gene lead to a spectrum of genetic disorders, collectively referred to as laminopathies. Lamin A/C are widely expressed in most differentiated somatic cells but not in early embryos and some undifferentiated cells. To investigate the role of lamin A/C in cell phenotype maintenance and differentiation, which could be a determinant of the pathogenesis of laminopathies, we examined the role played by exogenous lamin A and its mutants in differentiated cell lines (HeLa, NHDF) and less-differentiated HEK 293 cells. We introduced exogenous wild-type and mutated (H222P, L263P, E358K D446V, and ∆50) lamin A into different cell types and analyzed proteins' impact on proliferation, protein mobility, and endogenous nuclear envelope protein distribution. The mutants give rise to a broad spectrum of nuclear phenotypes and relocate lamin C. The mutations ∆50 and D446V enhance proliferation in comparison to wild-type lamin A and control cells, but no changes in exogenous protein mobility measured by FRAP were observed. Interestingly, although transcripts for lamins A and C are at similar level in HEK 293 cells, only lamin C protein is detected in western blots. Also, exogenous lamin A and its mutants, when expressed in HEK 293 cells underwent posttranscriptional processing. Overall, our results provide new insight into the maintenance of lamin A in less-differentiated cells. Embryonic cells are very sensitive to lamin A imbalance, and its upregulation disturbs lamin C, which may influence gene expression and many regulatory pathways.

  1. Ultraviolet B Radiation Stimulates the Interaction between Nuclear Factor of Activated T Cells 5 (NFAT5) and Nuclear Factor-Kappa B (NF-κB) in Human Lens Epithelial Cells.

    Science.gov (United States)

    Chung, Inyoung; Hah, Young-Sool; Ju, SunMi; Kim, Ji-Hye; Yoo, Woong-Sun; Cho, Hee-Young; Yoo, Ji-Myong; Seo, Seong-Wook; Choi, Wan-Sung; Kim, Seong-Jae

    2017-07-01

    Nuclear factor-kappa B (NF-κB) has been proposed as a therapeutic target for the treatment of cataracts. The authors investigated the relationship between nuclear factor of activated T cells 5 (NFAT5) and NF-κB in ultraviolet B (UVB)-irradiated human lens epithelial (HLE) cells. Human lens epithelial B-3 (HLE-B3) cells were exposed to UVB light at a dose of 10 mJ/cm 2 and then incubated for 24 h. Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) assay. Gene expression level of NFAT5 was determined using real-time quantitative polymerase chain reaction (qPCR). Protein expression levels of NFAT5, NF-κB p65, and α-smooth muscle actin (α-SMA) and the association of NFAT5 with the NF-κB p65 subunit were measured by Western blot analysis and a co-immunoprecipitation assay, respectively. The cellular distribution of NFAT5 and NF-κB p65 was examined by triple immunofluorescence staining. At 24 h after UVB exposure, cell viability significantly decreased in a dose-dependent manner, and UVB light (15 and 20 mJ/cm 2 ) significantly increased the ROS generation. UVB irradiation increased NFAT5 mRNA and protein levels and increased phosphorylation of NF-κB in HLE-B3 cells. α-SMA protein levels were increased in the irradiated cells. In addition, NFAT5 and NF-κB translocated from the cytoplasm to the nucleus, and binding between the p65 subunit and NFAT5 was increased. Exposure to UVB radiation induces nuclear translocation and stimulates binding between NFAT5 and NF-κB proteins in HLE-B3 cells. These interactions may form part of the biochemical mechanism of cataractogenesis in UVB-irradiated HLECs.

  2. Current status and applications of somatic cell nuclear transfer in dogs.

    Science.gov (United States)

    Jang, Goo; Kim, Min Kyu; Lee, Byeong Chun

    2010-11-01

    Although somatic cell nuclear transfer (SCNT) technology and applications are well developed in most domesticated and laboratory animals, their use in dogs has advanced only slowly. Many technical difficulties had to be overcome before preliminary experiments could be conducted. First, due to the very low efficiency of dog oocyte maturation in vitro, in vivo matured oocytes were generally used. The nucleus of an in vivo matured oocyte was removed and a donor cell (from fetal or adult fibroblasts) was injected into the oocyte. Secondly, fusion of the reconstructed oocytes was problematic, and it was found that a higher electrical voltage was necessary, in comparison to other mammalian species. By transferring the resulting fused oocytes into surrogate females, several cloned offspring were born. SCNT was also used for producing cloned wolves, validating reproductive technologies for aiding conservation of endangered or extinct breeds. Although examples of transgenesis in canine species are very sparse, SCNT studies are increasing, and together with the new field of gene targeting technology, they have been applied in many fields of veterinary or bio-medical science. This review summarizes the current status of SCNT in dogs and evaluates its potential future applications. Copyright © 2010 Elsevier Inc. All rights reserved.

  3. MutLα and Proliferating Cell Nuclear Antigen Share Binding Sites on MutSβ*

    Science.gov (United States)

    Iyer, Ravi R.; Pluciennik, Anna; Genschel, Jochen; Tsai, Miaw-Sheue; Beese, Lorena S.; Modrich, Paul

    2010-01-01

    MutSβ (MSH2-MSH3) mediates repair of insertion-deletion heterologies but also triggers triplet repeat expansions that cause neurological diseases. Like other DNA metabolic activities, MutSβ interacts with proliferating cell nuclear antigen (PCNA) via a conserved motif (QXX(L/I)XXFF). We demonstrate that MutSβ-PCNA complex formation occurs with an affinity of ∼0.1 μm and a preferred stoichiometry of 1:1. However, up to 20% of complexes are multivalent under conditions where MutSβ is in molar excess over PCNA. Conformational studies indicate that the two proteins associate in an end-to-end fashion in solution. Surprisingly, mutation of the PCNA-binding motif of MutSβ not only abolishes PCNA binding, but unlike MutSα, also dramatically attenuates MutSβ-MutLα interaction, MutLα endonuclease activation, and bidirectional mismatch repair. As predicted by these findings, PCNA competes with MutLα for binding to MutSβ, an effect that is blocked by the cell cycle regulator p21CIP1. We propose that MutSβ-MutLα interaction is mediated in part by residues ((L/I)SRFF) embedded within the MSH3 PCNA-binding motif. To our knowledge this is the first case where residues important for PCNA binding also mediate interaction with a second protein. These findings also indicate that MutSβ- and MutSα-initiated repair events differ in fundamental ways. PMID:20154325

  4. Detection of molecules and cells using nuclear magnetic resonance with magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Rümenapp, Christine, E-mail: ruemenapp@tum.de [Zentralinstitut für Medizintechnik (IMETUM), Technische Universität München, Garching (Germany); Gleich, Bernhard [Zentralinstitut für Medizintechnik (IMETUM), Technische Universität München, Garching (Germany); Mannherz, Hans Georg [Abteilung für Anatomie und Molekulare Embryologie, Ruhr Universität Bochum, Bochum (Germany); Haase, Axel [Zentralinstitut für Medizintechnik (IMETUM), Technische Universität München, Garching (Germany)

    2015-04-15

    For the detection of small molecules, proteins or even cells in vitro, functionalised magnetic nanoparticles and nuclear magnetic resonance measurements can be applied. In this work, magnetic nanoparticles with the size of 5–7 nm were functionalised with antibodies to detect two model systems of different sizes, the protein avidin and Saccharomyces cerevisiae as the model organism. The synthesised magnetic nanoparticles showed a narrow size distribution, which was determined using transmission electron microscopy and dynamic light scattering. The magnetic nanoparticles were functionalised with the according antibodies via EDC/NHS chemistry. The binding of the antigen to magnetic nanoparticles was detected through the change in the NMR T{sub 2} relaxation time at 0.5 T (≈21.7 MHz). In case of a specific binding the particles cluster and the T{sub 2} relaxation time of the sample changes. The detection limit in buffer for FITC-avidin was determined to be 1.35 nM and 10{sup 7} cells/ml for S. cerevisiae. For fluorescent microscopy the avidin molecules were labelled with FITC and for the detection of S. cerevisiae the magnetic nanoparticles were additionally functionalised with rhodamine. The binding of the particles to S. cerevisiae and the resulting clustering was also seen by transmission electron microscopy.

  5. Characterization of proton exchange membrane materials for fuel cells by solid state nuclear magnetic resonance

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Zueqian [Iowa State Univ., Ames, IA (United States)

    2010-01-01

    Solid-state nuclear magnetic resonance (NMR) has been used to explore the nanometer-scale structure of Nafion, the widely used fuel cell membrane, and its composites. We have shown that solid-state NMR can characterize chemical structure and composition, domain size and morphology, internuclear distances, molecular dynamics, etc. The newly-developed water channel model of Nafion has been confirmed, and important characteristic length-scales established. Nafion-based organic and inorganic composites with special properties have also been characterized and their structures elucidated. The morphology of Nafion varies with hydration level, and is reflected in the changes in surface-to-volume (S/V) ratio of the polymer obtained by small-angle X-ray scattering (SAXS). The S/V ratios of different Nafion models have been evaluated numerically. It has been found that only the water channel model gives the measured S/V ratios in the normal hydration range of a working fuel cell, while dispersed water molecules and polymer ribbons account for the structures at low and high hydration levels, respectively.

  6. Comparative analyses of the two proliferating cell nuclear antigens from the hyperthermophilic archaeon, Thermococcus kodakarensis.

    Science.gov (United States)

    Kuba, Yumani; Ishino, Sonoko; Yamagami, Takeshi; Tokuhara, Masahiro; Kanai, Tamotsu; Fujikane, Ryosuke; Daiyasu, Hiromi; Atomi, Haruyuki; Ishino, Yoshizumi

    2012-11-01

    The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen (PCNA) is the sliding clamp. The ring-shaped PCNA encircles double-stranded DNA within its central hole and tethers other proteins on DNA. The majority of Crenarchaeota, a subdomain of Archaea, have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. In contrast, most organisms in Euryarchaeota, the other major subdomain, have a single PCNA forming a homotrimeric ring structure. Among the Euryarchaeota whose genome is sequenced, Thermococcus kodakarensis is the only species with two genes encoding PCNA homologues on its genome. We cloned the two genes from the T. kodakarensis genome, and the gene products, PCNA1 and PCNA2, were characterized. PCNA1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T. kodakarensis in vitro. PCNA2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA2, whereas gene disruption for PCNA1 was not possible, suggesting that PCNA1 is essential for DNA replication. The sensitivities of the Δpcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate (MMS) and mitomycin C (MMC) were indistinguishable from those of the wild-type strain. © 2012 The Authors Genes to Cells © 2012 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

  7. Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

    International Nuclear Information System (INIS)

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa; Yoon, Hyun-Joo; Yoo, Hae Yong; Choi, Cheol Yong

    2014-01-01

    Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis

  8. Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Yoon, Hyun-Joo [TissueGene Inc. 9605 Medical Center Dr., Rockville, MD 20850 (United States); Yoo, Hae Yong [Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Samsung Medical Center, Sungkyunkwan University, Seoul 135-710 (Korea, Republic of); Choi, Cheol Yong, E-mail: choicy@skku.ac.kr [Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2014-01-03

    Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

  9. Human NTH1 physically interacts with p53 and proliferating cell nuclear antigen

    International Nuclear Information System (INIS)

    Oyama, Masaki; Wakasugi, Mitsuo; Hama, Takashi; Hashidume, Hatsuho; Iwakami, Yasutaka; Imai, Rika; Hoshino, Sanae; Morioka, Hiroshi; Ishigaki, Yasuhito; Nikaido, Osamu; Matsunaga, Tsukasa

    2004-01-01

    Thymine glycol (Tg) is one of predominant oxidative DNA lesions caused by ionizing radiation and other oxidative stresses. Human NTH1 is a bifunctional enzyme with DNA glycosylase and AP lyase activities and removes Tg as the first step of base excision repair (BER). We have searched for the factors interacting with NTH1 by using a pull-down assay and found that GST-NTH1 fusion protein precipitates proliferating cell nuclear antigen (PCNA) and p53 as well as XPG from human cell-free extracts. GST-NTH1 also bound to recombinant FLAG-tagged XPG, PCNA, and (His) 6 -tagged p53 proteins, indicating direct protein-protein interaction between those proteins. Furthermore, His-p53 and FLAG-XPG, but not PCNA, stimulated the Tg DNA glycosylase/AP lyase activity of GST-NTH1 or NTH1. These results provide an insight into the positive regulation of BER reaction and also suggest a possible linkage between BER of Tg and other cellular mechanisms

  10. Methylene blue photodynamic therapy in malignant melanoma decreases expression of proliferating cell nuclear antigen and heparanases.

    Science.gov (United States)

    Wagner, M; Suarez, E R; Theodoro, T R; Machado Filho, C D A S; Gama, M F M; Tardivo, J P; Paschoal, F M; Pinhal, M A S

    2012-07-01

    Malignant melanoma (MM) is a very aggressive tumour. Although surgical excision of MM in the early stages has a very good prognosis, it often fails to completely inhibit tumour progression. Methylene blue photodynamic therapy (MB-PDT) is a technique that induces tissue damage by reactive oxygen species (ROS). To investigate the efficacy of and potential use of MB-PDT in restraining the aggressiveness of MM by analysing levels of proliferating cell nuclear antigen (PCNA) and heparanase (HPSE, a molecular marker of cell invasion) in a mouse model. Expression of PCNA and two HPSE isoforms were analysed using immunohistochemistry (IHC) after MB-PDT in mice. Tumour volume and weight were also measured. Two treatments with MB-PDT promoted a decrease of 99% decrease in tumour volume and 75% in tumour weight compared with untreated mice (P < 0.05). Using IHC, a decrease in expression of 75% for PCNA and 95% for both HPSE isoforms (P < 0.05) was found. MB-PDT is a cheap and efficient method of decreasing MM volume and thus disease progression. This reduction is mediated by downregulation of PCNA and heparanases. © The Author(s). CED © 2012 British Association of Dermatologists.

  11. Cell cycle regulation of human immunodeficiency virus type 1 integration in T cells: antagonistic effects of nuclear envelope breakdown and chromatin condensation

    International Nuclear Information System (INIS)

    Mannioui, Abdelkrim; Schiffer, Cecile; Felix, Nathalie

    2004-01-01

    We examined the influence of mitosis on the kinetics of human immunodeficiency virus type 1 integration in T cells. Single-round infection of cells arrested in G1b or allowed to synchronously proceed through division showed that mitosis delays virus integration until 18-24 h postinfection, whereas integration reaches maximum levels by 15 h in G1b-arrested cells. Subcellular fractionation of metaphase-arrested cells indicated that, while nuclear envelope disassembly facilitates docking of viral DNA to chromatin, chromosome condensation directly antagonizes and therefore delays integration. As a result of the balance between the two effects, virus integration efficiency is eventually up to threefold greater in dividing cells. At the single-cell level, using a green fluorescent protein-expressing reporter virus, we found that passage through mitosis leads to prominent asymmetric segregation of the viral genome in daughter cells without interfering with provirus expression

  12. Nuclear IL-33 is a transcriptional regulator of NF-κB p65 and induces endothelial cell activation

    International Nuclear Information System (INIS)

    Choi, Yeon-Sook; Park, Jeong Ae; Kim, Jihye; Rho, Seung-Sik; Park, Hyojin; Kim, Young-Myeong; Kwon, Young-Guen

    2012-01-01

    Highlights: ► IL-33 as nuclear factor regulated expression of ICAM-1 and VCAM-1. ► Nuclear IL-33 increased the transcription of NF-κB p65 by binding to the p65 promoter. ► Nuclear IL-33 controls NF-κB-dependent inflammatory responses. -- Abstract: Interleukin (IL)-33, an IL-1 family member, acts as an extracellular cytokine by binding its cognate receptor, ST2. IL-33 is also a chromatin-binding transcriptional regulator highly expressed in the nuclei of endothelial cells. However, the function of IL-33 as a nuclear factor is poorly defined. Here, we show that IL-33 is a novel transcriptional regulator of the p65 subunit of the NF-κB complex and is involved in endothelial cell activation. Quantitative reverse transcriptase PCR and Western blot analyses indicated that IL-33 mediates the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in endothelial cells basally and in response to tumor necrosis factor-α-treatment. IL-33-induced ICAM-1/VCAM-1 expression was dependent on the regulatory effect of IL-33 on the nuclear factor (NF)-κB pathway; NF-κB p65 expression was enhanced by IL-33 overexpression and, conversely, reduced by IL-33 knockdown. Moreover, NF-κB p65 promoter activity and chromatin immunoprecipitation analysis revealed that IL-33 binds to the p65 promoter region in the nucleus. Our data provide the first evidence that IL-33 in the nucleus of endothelial cells participates in inflammatory reactions as a transcriptional regulator of NF-κB p65.

  13. MFTF-α+T end cell vacuum vessel and nuclear shield trade studies

    International Nuclear Information System (INIS)

    Kirchner, J.

    1984-01-01

    Three separate and distinct vacuum vessel and nuclear shield trade studies were performed in series. The studies are: vacuum topology, nuclear shield location and composition, and water bulk shield location and material selection

  14. A novel nuclear FGF Receptor-1 partnership with retinoid and Nur receptors during developmental gene programming of embryonic stem cells.

    Science.gov (United States)

    Lee, Yu-Wei; Terranova, Christopher; Birkaya, Barbara; Narla, Sridhar; Kehoe, Daniel; Parikh, Abhirath; Dong, Shuo; Ratzka, Andreas; Brinkmann, Hella; Aletta, John M; Tzanakakis, Emmanuel S; Stachowiak, Ewa K; Claus, Peter; Stachowiak, Michal K

    2012-09-01

    FGF Receptor-1 (FGFR1), a membrane-targeted protein, is also involved in independent direct nuclear signaling. We show that nuclear accumulation of FGFR1 is a common response to retinoic acid (RA) in pluripotent embryonic stem cells (ESC) and neural progenitors and is both necessary and sufficient for neuronal-like differentiation and accompanying neuritic outgrowth. Dominant negative nuclear FGFR1, which lacks the tyrosine kinase domain, prevents RA-induced differentiation while full-length nuclear FGFR1 elicits differentiation in the absence of RA. Immunoprecipitation and GST assays demonstrate that FGFR1 interacts with RXR, RAR and their Nur77 and Nurr1 partners. Conditions that promote these interactions decrease the mobility of nuclear FGFR1 and RXR in live cells. RXR and FGFR1 co-associate with 5'-Fluorouridine-labeled transcription sites and with RA Responsive Elements (RARE). RA activation of neuronal (tyrosine hydroxylase) and neurogenic (fgf-2 and fgfr1) genes is accompanied by increased FGFR1, Nur, and histone H3.3 binding to their regulatory sequences. Reporter-gene assays show synergistic activations of RARE, NBRE, and NurRE by FGFR1, RAR/RXR, and Nurs. As shown for mESC differentiation, FGFR1 mediates gene activation by RA and augments transcription in the absence of RA. Cooperation of FGFR1 with RXR/RAR and Nurs at targeted genomic sequences offers a new mechanism in developmental gene regulation. Copyright © 2012 Wiley Periodicals, Inc.

  15. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Pang, Daxin, E-mail: pdx@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Ouyang, Hongsheng, E-mail: ouyh@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China)

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  16. A {sup 1}H nuclear magnetic resonance study of structural and organisational changes in the cell

    Energy Technology Data Exchange (ETDEWEB)

    Tunnah, Susan K

    2000-07-01

    Increasing importance is being placed on understanding the role of membrane lipids in many different areas of biochemistry. It is of interest to determine what interactions may occur between membrane lipids and drug species. Furthermore, an increasing body of evidence suggests that membrane lipids are involved in the pathology of numerous diseases such as rheumatoid arthritis, cancer and HIV. Clearly, the more information available on the mechanisms involved in diseases, the greater the potential for identifying a cure or even a prevention. {sup 1}H nuclear magnetic resonance (NMR) spectroscopy was used to study the alterations in membrane lipid organisation and structure in intact, viable cultured cells. Changes in the {sup 1}H NMR spectra and the spin-lattice relaxation measurements of the human K562 and the rat FRTL-5 cell lines were observed on the addition of the fatty acid species: triolein, evening primrose oil, arachidonic acid and ITF 1779. Results indicate that the membrane lipids are reorganised to accommodate the interpolation of these molecules. The spatial arrangement adopted by each of these species appeared to dictate its effect on the lipids. Doxorubicin and menadione, both known to cause oxidative stress, were added to K562 cells. Although both agents are known to act by different mechanisms, the NMR data and scanning electron microscopy suggested that both caused similar alterations in the membrane organisation and lipid fluidity. Protrusions were formed indicating areas of weakness in the membrane. Spin-echo NMR was employed to investigate the action of the thiol-containing compounds, penicillamine, captopril and N-acetylcysteine in erythrocytes under conditions of oxidative stress. Results indicate that while captopril acts as a free radical scavenger, penicillamine may act as either oxidant or reductant. N-acetylcysteine was observed to act as a reducing agent. (author)

  17. Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Séverine A Degrelle

    Full Text Available Somatic cell nuclear transfer (SCNT is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each; one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling". Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538, we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity and subsequent pregnancy loss. Finally

  18. Bovine Lhx8, a Germ Cell-Specific Nuclear Factor, Interacts with Figla.

    Directory of Open Access Journals (Sweden)

    Liyuan Fu

    Full Text Available LIM homeobox 8 (Lhx8 is a germ cell-specific transcription factor essential for the development of oocytes during early oogenesis. In mice, Lhx8 deficiency causes postnatal oocyte loss and affects the expression of many oocyte-specific genes. The aims of this study were to characterize the bovine Lhx8 gene, determine its mRNA expression during oocyte development and early embryogenesis, and evaluate its interactions with other oocyte-specific transcription factors. The bovine Lhx8 gene encodes a protein of 377 amino acids. A splice variant of Lhx8 (Lhx8_v1 was also identified. The predicted bovine Lhx8 protein contains two LIM domains and one homeobox domain. However, one of the LIM domains in Lhx8_v1 is incomplete due to deletion of 83 amino acids near the N terminus. Both Lhx8 and Lhx8_v1 transcripts were only detected in the gonads but none of the somatic tissues examined. The expression of Lhx8 and Lhx8_v1 appears to be restricted to oocytes as none of the transcripts was detectable in granulosa or theca cells. The maternal Lhx8 transcript is abundant in GV and MII stage oocytes as well as in early embryos but disappear by morula stage. A nuclear localization signal that is required for the import of Lhx8 into nucleus was identified, and Lhx8 is predominantly localized in the nucleus when ectopically expressed in mammalian cells. Finally, a novel interaction between Lhx8 and Figla, another transcription factor essential for oogenesis, was detected. The results provide new information for studying the mechanisms of action for Lhx8 in oocyte development and early embryogenesis.

  19. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    International Nuclear Information System (INIS)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue; Pang, Daxin; Ouyang, Hongsheng

    2011-01-01

    Highlights: → Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. → The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. → A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 μg/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  20. A 1H nuclear magnetic resonance study of structural and organisational changes in the cell

    International Nuclear Information System (INIS)

    Tunnah, Susan K.

    2000-01-01

    Increasing importance is being placed on understanding the role of membrane lipids in many different areas of biochemistry. It is of interest to determine what interactions may occur between membrane lipids and drug species. Furthermore, an increasing body of evidence suggests that membrane lipids are involved in the pathology of numerous diseases such as rheumatoid arthritis, cancer and HIV. Clearly, the more information available on the mechanisms involved in diseases, the greater the potential for identifying a cure or even a prevention. 1 H nuclear magnetic resonance (NMR) spectroscopy was used to study the alterations in membrane lipid organisation and structure in intact, viable cultured cells. Changes in the 1 H NMR spectra and the spin-lattice relaxation measurements of the human K562 and the rat FRTL-5 cell lines were observed on the addition of the fatty acid species: triolein, evening primrose oil, arachidonic acid and ITF 1779. Results indicate that the membrane lipids are reorganised to accommodate the interpolation of these molecules. The spatial arrangement adopted by each of these species appeared to dictate its effect on the lipids. Doxorubicin and menadione, both known to cause oxidative stress, were added to K562 cells. Although both agents are known to act by different mechanisms, the NMR data and scanning electron microscopy suggested that both caused similar alterations in the membrane organisation and lipid fluidity. Protrusions were formed indicating areas of weakness in the membrane. Spin-echo NMR was employed to investigate the action of the thiol-containing compounds, penicillamine, captopril and N-acetylcysteine in erythrocytes under conditions of oxidative stress. Results indicate that while captopril acts as a free radical scavenger, penicillamine may act as either oxidant or reductant. N-acetylcysteine was observed to act as a reducing agent. (author)

  1. Stereological estimates of nuclear volume in squamous cell carcinoma of the uterine cervix and its precursors

    DEFF Research Database (Denmark)

    Sørensen, Flemming Brandt; Bichel, P; Jakobsen, A

    1991-01-01

    Using modern stereology, this study was carried out to obtain base-line data concerning three-dimensional, mean nuclear size in precancerous and invasive lesions of the uterine cervix. Unbiased estimates of the volume-weighted mean nuclear volume (nuclear vv) were obtained by point-sampling of nu...

  2. NGF-induced cell differentiation and gene activation is mediated by integrative nuclear FGFR1 signaling (INFS).

    Science.gov (United States)

    Lee, Yu-Wei; Stachowiak, Ewa K; Birkaya, Barbara; Terranova, Christopher; Capacchietti, Mariolina; Claus, Peter; Aletta, John M; Stachowiak, Michal K

    2013-01-01

    Nerve growth factor (NGF) is the founding member of the polypeptide neurotrophin family responsible for neuronal differentiation. To determine whether the effects of NGF rely upon novel Integrative Nuclear FGF Receptor-1 (FGFR1) Signaling (INFS) we utilized the PC12 clonal cell line, a long-standing benchmark model of sympathetic neuronal differentiation. We demonstrate that NGF increases expression of the fgfr1 gene and promotes trafficking of FGFR1 protein from cytoplasm to nucleus by inhibiting FGFR1 nuclear export. Nuclear-targeted dominant negative FGFR1 antagonizes NGF-induced neurite outgrowth, doublecortin (dcx) expression and activation of the tyrosine hydroxylase (th) gene promoter, while active constitutive nuclear FGFR1 mimics the effects of NGF. NGF increases the expression of dcx, th, βIII tubulin, nurr1 and nur77, fgfr1and fibroblast growth factor-2 (fgf-2) genes, while enhancing binding of FGFR1and Nur77/Nurr1 to those genes. NGF activates transcription from isolated NurRE and NBRE motifs. Nuclear FGFR1 transduces NGF activation of the Nur dimer and raises basal activity of the Nur monomer. Cooperation of nuclear FGFR1 with Nur77/Nurr1 in NGF signaling expands the integrative functions of INFS to include NGF, the first discovered pluripotent neurotrophic factor.

  3. NGF-induced cell differentiation and gene activation is mediated by integrative nuclear FGFR1 signaling (INFS.

    Directory of Open Access Journals (Sweden)

    Yu-Wei Lee

    Full Text Available Nerve growth factor (NGF is the founding member of the polypeptide neurotrophin family responsible for neuronal differentiation. To determine whether the effects of NGF rely upon novel Integrative Nuclear FGF Receptor-1 (FGFR1 Signaling (INFS we utilized the PC12 clonal cell line, a long-standing benchmark model of sympathetic neuronal differentiation. We demonstrate that NGF increases expression of the fgfr1 gene and promotes trafficking of FGFR1 protein from cytoplasm to nucleus by inhibiting FGFR1 nuclear export. Nuclear-targeted dominant negative FGFR1 antagonizes NGF-induced neurite outgrowth, doublecortin (dcx expression and activation of the tyrosine hydroxylase (th gene promoter, while active constitutive nuclear FGFR1 mimics the effects of NGF. NGF increases the expression of dcx, th, βIII tubulin, nurr1 and nur77, fgfr1and fibroblast growth factor-2 (fgf-2 genes, while enhancing binding of FGFR1and Nur77/Nurr1 to those genes. NGF activates transcription from isolated NurRE and NBRE motifs. Nuclear FGFR1 transduces NGF activation of the Nur dimer and raises basal activity of the Nur monomer. Cooperation of nuclear FGFR1 with Nur77/Nurr1 in NGF signaling expands the integrative functions of INFS to include NGF, the first discovered pluripotent neurotrophic factor.

  4. Pathogenic and Diagnostic Potential of BLCA-1 and BLCA-4 Nuclear Proteins in Urothelial Cell Carcinoma of Human Bladder

    Directory of Open Access Journals (Sweden)

    Matteo Santoni

    2012-01-01

    Full Text Available Transitional cell carcinoma (TCC of the bladder is one of the most common malignancies of genitourinary tract. Patients with bladder cancer need a life-long surveillance, directly due to the relatively high recurrence rate of this tumor. The use of cystoscopy represents the gold standard for the followup of previously treated patients. Nevertheless, several factors, including cost and invasiveness, render cystoscopy not ideal for routine controls. Advances in the identification of specific alterations in the nuclear structure of bladder cancer cells have opened novel diagnostic landscapes. The members of nuclear matrix protein family BLCA-1 and BLCA-4, are currently under evaluation as bladder cancer urinary markers. They are involved in tumour cell proliferation, survival, and angiogenesis. In this paper, we illustrate the role of BLCA-1 and BLCA-4 in bladder carcinogenesis and their potential exploitation as biomarkers in this cancer.

  5. Structural dynamics of the cell nucleus: basis for morphology modulation of nuclear calcium signaling and gene transcription.

    Science.gov (United States)

    Queisser, Gillian; Wiegert, Simon; Bading, Hilmar

    2011-01-01

    Neuronal morphology plays an essential role in signal processing in the brain. Individual neurons can undergo use-dependent changes in their shape and connectivity, which affects how intracellular processes are regulated and how signals are transferred from one cell to another in a neuronal network. Calcium is one of the most important intracellular second messengers regulating cellular morphologies and functions. In neurons, intracellular calcium levels are controlled by ion channels in the plasma membrane such as NMDA receptors (NMDARs), voltage-gated calcium channels (VGCCs) and certain α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as well as by calcium exchange pathways between the cytosol and internal calcium stores including the endoplasmic reticulum and mitochondria. Synaptic activity and the subsequent opening of ligand and/or voltage-gated calcium channels can initiate cytosolic calcium transients which propagate towards the cell soma and enter the nucleus via its nuclear pore complexes (NPCs) embedded in the nuclear envelope. We recently described the discovery that in hippocampal neurons the morphology of the nucleus affects the calcium dynamics within the nucleus. Here we propose that nuclear infoldings determine whether a nucleus functions as an integrator or detector of oscillating calcium signals. We outline possible ties between nuclear mophology and transcriptional activity and discuss the importance of extending the approach to whole cell calcium signal modeling in order to understand synapse-to-nucleus communication in healthy and dysfunctional neurons.

  6. Multiparameter breast cancer cell image analysis for objective estimation of nuclear grade: comparison with light microscopic observational data

    Science.gov (United States)

    Berzins, Juris; Sneiders, Uldis; Plegere, Daina; Freivalds, Talivaldis; Grigalinovica, Romalda

    2000-04-01

    We performed a multi parameter image analysis assessment of breast cancer cell population nuclear grade (NG), which is regarded as one of the main prognostic factors for treatment efficacy and survival of the patients and compared it with light microscopic estimation of NG. Cytological imprint slides from 20 ductal carcinomas were stained according to Leischmann-AzureII-eosine method, and NG was estimated by light microscopic observation according to Black in Fisher's modification. Simultaneously, using specially elaborated software, in each patient 100 cancer cells were analyzed for nuclear perimeter, diameter, area, nucleolar area, and average intensity of staining. The chromatin structure was assessed using mean diameter of chromatin grains and relatively chromatic are within the nucleus. Light microscopic estimation revealed 4/15 grade 2 and 7/15 grade 3 tumors out of 15 filtrating ductal carcinomas, with 4/15 classified as intermediate between grade 2-3. Multifactoral linear correlation coefficient r equals 0.39, p < 0.001 for ductal cancer, higher NG comes with increasing nucleolar area, nuclear roundness factor, nuclear are, and chromatin area within the cell nucleus. Image analysis may yield precise information on NG as a prognostic factor in breast cancer patients.

  7. Goat red blood cells as precursor for iron oxide nanocrystal synthesis to develop nuclear targeted lung cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Sreevani, Vellingiri; Shanthi, Krishnamurthy; Kannan, Soundarapandian, E-mail: sk_protein@buc.edu.in

    2013-09-01

    Graphical abstract: - Highlights: • Molecular approach of synthesis of Fe{sub 2}O{sub 3}-NC using goat blood as a bio-precursor. • The method is simple, efficient and environment friendly. • Synthesized nanocrystals were characterized by UV–vis spectroscopy, XRD, SEM, TEM, DLS and EDS. • Nanocrystals exhibited potent cytotoxicity against A549 cancer cell. • Nuclear targeting with expression of caspase-3, caspase-7 and Bcl-2 in A549 cancer cells. - Abstract: In this study, we synthesised iron oxide nanocrystals (Fe{sub 2}O{sub 3}-NC) from goat blood (bio-precursor) using red blood cells (RBC) lysis method (a molecular level approach) for the first time. The formation of Fe{sub 2}O{sub 3}-NC was achieved through a single-phase chemical reduction method. The size distribution of Fe{sub 2}O{sub 3}-NC falls between 20–30 nm for pellet and 100–200 nm for lysate and were found to be crystalline. Fe{sub 2}O{sub 3}-NC demonstrated significant cytotoxicity on A549. We report the direct visualization of interactions between Fe{sub 2}O{sub 3}-NC and the cancer cell nucleus. The active transport of Fe{sub 2}O{sub 3}-NC to the nucleus induces major changes to nuclear phenotype via nuclear envelope invaginations. We further examined the root cause for the involvement of Fe{sub 2}O{sub 3}-NC on the expression of caspase-3, caspase-7 and Bcl-2 in A549 cancer cells. This functional proteomic analysis clearly implies that the lung cancer cell proliferation is perfectly targeted by the biosynthesised Fe{sub 2}O{sub 3}-NC which could provide new insight for nuclear-targeted cancer therapy.

  8. Rhinovirus 3C protease facilitates specific nucleoporin cleavage and mislocalisation of nuclear proteins in infected host cells.

    Directory of Open Access Journals (Sweden)

    Erin J Walker

    Full Text Available Human Rhinovirus (HRV infection results in shut down of essential cellular processes, in part through disruption of nucleocytoplasmic transport by cleavage of the nucleoporin proteins (Nups that make up the host cell nuclear pore. Although the HRV genome encodes two proteases (2A and 3C able to cleave host proteins such as Nup62, little is known regarding the specific contribution of each. Here we use transfected as well as HRV-infected cells to establish for the first time that 3C protease is most likely the mediator of cleavage of Nup153 during HRV infection, while Nup62 and Nup98 are likely to be targets of HRV2A protease. HRV16 3C protease was also able to elicit changes in the appearance and distribution of the nuclear speckle protein SC35 in transfected cells, implicating it as a key mediator of the mislocalisation of SC35 in HRV16-infected cells. In addition, 3C protease activity led to the redistribution of the nucleolin protein out of the nucleolus, but did not affect nuclear localisation of hnRNP proteins, implying that complete disruption of nucleocytoplasmic transport leading to relocalisation of hnRNP proteins from the nucleus to the cytoplasm in HRV-infected cells almost certainly requires 2A in addition to 3C protease. Thus, a specific role for HRV 3C protease in cleavage and mislocalisation of host cell nuclear proteins, in concert with 2A, is implicated for the first time in HRV pathogenesis.

  9. Heat conduction through geological mattresses from cells storing mean activity and long life nuclear wastes

    International Nuclear Information System (INIS)

    Lajoie, D.; Raffourt, C.; Wendling, J.

    2010-01-01

    Document available in extended abstract form only. ANDRA ordered in 2008 a campaign of numerical simulations to assess the efficiency of the ventilation system designed for cells storing mean activity and long life nuclear wastes. Numerical models were performed by ACRIIN as research engineering office. The main objectives were to assess the risks of atmospheric explosions due to high rate of hydrogen and to determine the efficiency of the system to evacuate released heat from storage packages. Further calculations have been carried out to evaluate temperature gradients in the surrounding geological medium. Three-dimensional numerical models of a reference cell were built to simulate the air flow injected at the cell entrance and retrieved and the other extremity. The reference case is based on a cell full of storage packages, with rows and columns of packages methodically ordered. Analytic and numerical calculations have been performed introducing progressively each complex physical phenomenon in order to dissociate origins of transport of released mass or heat. Three kinds of flows have been physically distinguished: 1) Ventilation in a cell with storage package that are thermally inert, i.e. no heat release, but with hydrogen release. 2) Flow in a cell with storage packages that emit heat and warm the injected air, supposing that no heat were lost towards the surrounding concrete walls of the cell. 3) Air Flow warmed by the storage packages with heat losses towards concrete walls and geological medium. Simulations with absence of thermal effects allowed the knowledge of main topics of the ventilation air flows that may be synthesized as follows: - Flows infiltrate clearances between piles and rows of storage packages. Such apertures are a few centimetres wide. The flow is disorganised between the first rows, with distribution in both transversal and longitudinal directions. After a few tens of rows, the flow reaches its hydraulic equilibrium, with a nearly pure

  10. Genetic Basis for Developmental Homeostasis of Germline Stem Cell Niche Number: A Network of Tramtrack-Group Nuclear BTB Factors

    Science.gov (United States)

    Chalvet, Fabienne; Netter, Sophie; Dos Santos, Nicolas; Poisot, Emilie; Paces-Fessy, Mélanie; Cumenal, Delphine; Peronnet, Frédérique; Pret, Anne-Marie; Théodore, Laurent

    2012-01-01

    The potential to produce new cells during adult life depends on the number of stem cell niches and the capacity of stem cells to divide, and is therefore under the control of programs ensuring developmental homeostasis. However, it remains generally unknown how the number of stem cell niches is controlled. In the insect ovary, each germline stem cell (GSC) niche is embedded in a functional unit called an ovariole. The number of ovarioles, and thus the number of GSC niches, varies widely among species. In Drosophila, morphogenesis of ovarioles starts in larvae with the formation of terminal filaments (TFs), each made of 8–10 cells that pile up and sort in stacks. TFs constitute organizers of individual germline stem cell niches during larval and early pupal development. In the Drosophila melanogaster subgroup, the number of ovarioles varies interspecifically from 8 to 20. Here we show that pipsqueak, Trithorax-like, batman and the bric-à-brac (bab) locus, all encoding nuclear BTB/POZ factors of the Tramtrack Group, are involved in limiting the number of ovarioles in D. melanogaster. At least two different processes are differentially perturbed by reducing the function of these genes. We found that when the bab dose is reduced, sorting of TF cells into TFs was affected such that each TF contains fewer cells and more TFs are formed. In contrast, psq mutants exhibited a greater number of TF cells per ovary, with a normal number of cells per TF, thereby leading to formation of more TFs per ovary than in the wild type. Our results indicate that two parallel genetic pathways under the control of a network of nuclear BTB factors are combined in order to negatively control the number of germline stem cell niches. PMID:23185495

  11. Inner nuclear layer thickening is inversley proportional to retinal ganglion cell loss in optic neuritis.

    Directory of Open Access Journals (Sweden)

    Megha Kaushik

    Full Text Available AIM: To examine the relationship between retinal ganglion cell loss and changes in the inner nuclear layer (INL in optic neuritis (ON. METHODS: 36 multiple sclerosis (MS patients with a history of ON and 36 age and sex-matched controls underwent Optical Coherence Tomography. The paramacular retinal nerve fiber layer (RNFL, combined ganglion cell and inner plexiform layers (GCL/IPL and inner nuclear layer (INL thickness were measured at 36 points around the fovea. To remove inter-subject variability, the difference in thickness of each layer between the ON and fellow eye of each patient was calculated. A topographic analysis was conducted. RESULTS: The INL of the ON patients was thicker than the controls (42.9µm versus 39.6µm, p=0.002. ON patients also had a thinner RNFL (27.8µm versus 32.2µm, p<0.001 and GCL/IPL (69.3µm versus 98.1µm, p<0.001. Among the controls, there was no correlation between RNFL and GCL/IPL as well as RNFL and INL, but a positive correlation was seen between GCL/IPL and INL (r=0.65, p<0.001. In the ON group, there was a positive correlation between RNFL and GCL/IPL (r=0.80, p<0.001 but a negative correlation between RNFL and INL (r=-0.61, p<0.001 as well as GCL/IPL and INL (r=-0.44, p=0.007. The negative correlation between GCL/IPL and INL strengthened in the ON group when inter-subject variability was removed (r=-0.75, p<0.001. Microcysts within the INL were present in 5 ON patients, mainly in the superior and infero-nasal paramacular regions. While patients with microcysts lay at the far end of the correlation curve between GCL/IPL and INL (i.e. larger INL and smaller GCL/IPL compared to other patients, their exclusion did not affect the correlation (r= -0.76, p<0.001. CONCLUSIONS: INL enlargement in MS-related ON is associated with the severity of GCL loss. This is a continuous relationship and patients with INL microcysts may represent the extreme end of the scale.

  12. DNA level and stereologic estimates of nuclear volume in squamous cell carcinomas of the uterine cervix. A comparative study with analysis of prognostic impact

    DEFF Research Database (Denmark)

    Sørensen, Flemming Brandt; Bichel, P; Jakobsen, A

    1992-01-01

    Grading of malignancy in squamous cell carcinomas of the uterine cervix is based on qualitative, morphologic examination and suffers from poor reproducibility. Using modern stereology, unbiased estimates of the three-dimensional, volume-weighted mean nuclear volume (nuclear vv), were obtained...... with highly distinguishing capacity between prognostically poor and favorable cases was constructed (2P = 1.9 x 10(-7)). It is concluded that realistic estimates of nuclear volume are independent of nuclear DNA content and are of prognostic value for objective malignancy grading in patients with squamous cell...

  13. Biochemical characterization of nuclear receptors for vitamin D3 and glucocorticoids in prostate stroma cell microenvironment

    International Nuclear Information System (INIS)

    Hidalgo, Alejandro A.; Montecinos, Viviana P.; Paredes, Roberto; Godoy, Alejandro S.; McNerney, Eileen M.; Tovar, Heribelt; Pantoja, Diego; Johnson, Candace; Trump, Donald; Onate, Sergio A.

    2011-01-01

    Highlights: → Fibroblasts from benign and carcinoma-associated stroma were biochemically characterized for VDR and GR function as transcription factors in prostate stroma cell microenvironment. → Decreased SRC-1/CBP coactivators recruitment to VDR and GR may result in hormone resistance to 1,25D 3 in stromal cell microenvironment prostate cancer. → 1a,25-Dyhidroxyvitamin D 3 (1,25D 3 ) and glucocorticoids, either alone or in combination, may not be an alternative for 'some' advanced prostate cancers that fails androgen therapies. -- Abstract: The disruption of stromal cell signals in prostate tissue microenvironment influences the development of prostate cancer to androgen independence. 1α,25-Dihydroxyvitamin D 3 (1,25D 3 ) and glucocorticoids, either alone or in combination, have been investigated as alternatives for the treatment of advanced prostate cancers that fails androgen therapies. The effects of glucocorticoids are mediated by the intracellular glucocorticoid receptor (GR). Similarly, the effect of 1,25D 3 is mediated by the 1,25D 3 nuclear receptor (VDR). In this study, fibroblasts from benign- (BAS) and carcinoma-associated stroma (CAS) were isolated from human prostates to characterize VDR and GR function as transcription factors in prostate stroma. The VDR-mediated transcriptional activity assessed using the CYP24-luciferase reporter was limited to 3-fold induction by 1,25D 3 in 9 out of 13 CAS (70%), as compared to >10-fold induction in the BAS clinical sample pair. Expression of His-tagged VDR (Ad-his-VDR) failed to recover the low transcriptional activity of the luciferase reporter in 7 out of 9 CAS. Interestingly, expression of Ad-his-VDR successfully recovered receptor-mediated induction in 2 out of the 9 CAS analyzed, suggesting that changes in the receptor protein itself was responsible for decreased response and resistance to 1,25D 3 action. Conversely, VDR-mediated transcriptional activity was more efficient in 4 out of 13 CAS (30

  14. In situ titanium dioxide nanoparticles quantitative microscopy in cells and in C. elegans using nuclear microprobe analysis

    Energy Technology Data Exchange (ETDEWEB)

    Le Trequesser, Quentin [Université de Bordeaux, CENBG, Chemin du solarium, 33175 Gradignan (France); CNRS, UMR 5797, CENBG, Chemin du solarium, 33175 Gradignan (France); CNRS, Université de Bordeaux, ICMCB, 87 avenue du Dr. A. Schweitzer, Pessac F-33608 (France); Saez, Gladys; Devès, Guillaume; Michelet, Claire; Barberet, Philippe [Université de Bordeaux, CENBG, Chemin du solarium, 33175 Gradignan (France); CNRS, UMR 5797, CENBG, Chemin du solarium, 33175 Gradignan (France); Delville, Marie-Hélène [CNRS, Université de Bordeaux, ICMCB, 87 avenue du Dr. A. Schweitzer, Pessac F-33608 (France); Seznec, Hervé, E-mail: herve.seznec@cenbg.in2p3.fr [Université de Bordeaux, CENBG, Chemin du solarium, 33175 Gradignan (France); CNRS, UMR 5797, CENBG, Chemin du solarium, 33175 Gradignan (France)

    2014-12-15

    Detecting and tracking nanomaterials in biological systems is challenging and essential to understand the possible interactions with the living. In this context, in situ analyses were conducted on human skin cells and a multicellular organism (Caenorhabditiselegans) exposed to titanium dioxide nanoparticles (TiO{sub 2} NPs) using nuclear microprobe. Coupled to conventional methods, nuclear microprobe was found to be suitable for accurate description of chemical structure of biological systems and also for detection of native TiO{sub 2} NPs. The method presented herein opens the field to NPs exposure effects analyses and more generally to toxicological analyses assisted by nuclear microprobe. This method will show applications in key research areas where in situ imaging of chemical elements is essential.

  15. Nuclear networking.

    Science.gov (United States)

    Xie, Wei; Burke, Brian

    2017-07-04

    Nuclear lamins are intermediate filament proteins that represent important structural components of metazoan nuclear envelopes (NEs). By combining proteomics and superresolution microscopy, we recently reported that both A- and B-type nuclear lamins form spatially distinct filament networks at the nuclear periphery of mouse fibroblasts. In particular, A-type lamins exhibit differential association with nuclear pore complexes (NPCs). Our studies reveal that the nuclear lamina network in mammalian somatic cells is less ordered and more complex than that of amphibian oocytes, the only other system in which the lamina has been visualized at high resolution. In addition, the NPC component Tpr likely links NPCs to the A-type lamin network, an association that appears to be regulated by C-terminal modification of various A-type lamin isoforms. Many questions remain, however, concerning the structure and assembly of lamin filaments, as well as with their mode of association with other nuclear components such as peripheral chromatin.

  16. Cloning missy: obtaining multiple offspring of a specific canine genotype by somatic cell nuclear transfer.

    Science.gov (United States)

    Hossein, Mohammad Shamim; Jeong, Yeon Woo; Park, Sun Woo; Kim, Joung Joo; Lee, Eugine; Ko, Kyeong Hee; Kim, Huen Suk; Kim, Yeun Wook; Hyun, Sang Hwan; Shin, Taeyoung; Hawthorne, Lou; Hwang, Woo Suk

    2009-03-01

    The present study was undertaken to evaluate two activation methods for somatic cell nuclear transfer (SCNT), namely, fusion and simultaneous activation (FSA, fusion medium contains calcium), versus fusion followed by chemical activation (F+CA, fusion medium does not contain calcium), and to evaluate the effects of parity of recipient dogs on the success of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells collected from an 11-year-old female dog named Missy. In the FSA method, oocytes were fused and activated at the same time using two DC pulses of 1.75 kV/cm for 15 microsec. In the F+CA method, oocytes were fused with two DC pulses of 1.75 kV/cm for 15 microsec, and then activated 1 h after fusion by 10 microM calcium ionophore for 4 m and cultured for 4 h in 1.9 mM 6-dimethylaminopurine for postactivation. Activation method had a significant impact on the production efficiency of cloned dogs. There was a significant difference in full-term pregnancy rate and percentage of live puppies between the two methods (6.3% and 38.5% for FSA and F+CA, respectively). In our study, four out of five live offspring produced by F+CA survived versus FSA, which did not result in any surviving puppies. Overall, as few as 14 dogs and 54 reconstructed embryos were needed to produce a cloned puppy. In addition, the parity of recipient bitches had no effect on the success of SCNT in canine species. Both the nullipara and multipara bitches produced live puppies following SCNT-ET.

  17. Effects of nuclear receptor transactivation on steroid hormone synthesis and gene expression in porcine Leydig cells.

    Science.gov (United States)

    Gray, Matthew A; Squires, E James

    2013-01-01

    Male pigs are routinely castrated at a young age to prevent the formation of androstenone, a 16-androstene testicular steroid that is a major component of boar taint. The practice of castration has been increasingly viewed as unfavorable, due to both economic considerations and animal welfare concerns. Other means of controlling boar taint, including reducing the synthesis of androstenone in the testes, would eliminate the need for castration. In this study, we determined the effects of transactivation of three nuclear receptors, the constitutive androstane receptor (CAR), pregnane X receptor (PXR), and farnesoid X receptor (FXR), on gene expression and steroid hormone metabolism in primary porcine Leydig cells. Primary cells were isolated from mature boars, and transcript expression levels were assayed using real-time PCR. The transcripts of interest included porcine orthologs of common phase I and phase II metabolic enzymes, enzymes involved in steroidogenesis, and transcripts previously shown to be differentially expressed in boars with high androstenone and boar taint levels. Transactivation of CAR, PXR, or FXR increased the expression of several genes involved in steroidogenesis, including cytochrome B5A (CYB5A) and cytochrome B5 reductase 1 (CYB5R1), as well as hydroxysteroid (17-beta) dehydrogenase 4 (HSD17B4) and retinol dehydrogenase 12 (RDH12). Treatment with (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO), a CAR agonist, or rifampicin (RIF), a PXR agonist, resulted in significantly (pnuclear receptors may lead to increased levels of 16-androstene steroids, likely by altering the activity of CYP17A1 through CYB5A and CYB5R1 to the andien-β synthase reaction and away from the 17α-hydroxylase and C17, 20 lyase reactions. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Characterisation of human embryonic stem cells conditioning media by 1H-nuclear magnetic resonance spectroscopy.

    Directory of Open Access Journals (Sweden)

    David A MacIntyre

    Full Text Available BACKGROUND: Cell culture media conditioned by human foreskin fibroblasts (HFFs provide a complex supplement of protein and metabolic factors that support in vitro proliferation of human embryonic stem cells (hESCs. However, the conditioning process is variable with different media batches often exhibiting differing capacities to maintain hESCs in culture. While recent studies have examined the protein complement of conditioned culture media, detailed information regarding the metabolic component of this media is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Using a (1H-Nuclear Magnetic Resonance ((1H-NMR metabonomics approach, 32 metabolites and small compounds were identified and quantified in media conditioned by passage 11 HFFs (CMp11. A number of metabolites were secreted by HFFs with significantly higher concentration of lactate, alanine, and formate detected in CMp11 compared to non-conditioned media. In contrast, levels of tryptophan, folate and niacinamide were depleted in CMp11 indicating the utilisation of these metabolites by HFFs. Multivariate statistical analysis of the (1H-NMR data revealed marked age-related differences in the metabolic profile of CMp11 collected from HFFs every 24 h over 72 h. Additionally, the metabolic profile of CMp11 was altered following freezing at -20°C for 2 weeks. CM derived from passage 18 HFFs (CMp18 was found to be ineffective at supporting hESCs in an undifferentiated state beyond 5 days culture. Multivariate statistical comparison of CMp11 and CMp18 metabolic profiles enabled rapid and clear discrimination between the two media with CMp18 containing lower concentrations of lactate and alanine as well as higher concentrations of glucose and glutamine. CONCLUSIONS/SIGNIFICANCE: (1H-NMR-based metabonomics offers a rapid and accurate method of characterising hESC conditioning media and is a valuable tool for monitoring, controlling and optimising hESC culture media preparation.

  19. Growth and hematologic characteristics of cloned dogs derived from adult somatic cell nuclear transfer.

    Science.gov (United States)

    Park, Jung Eun; Kim, Min Kyu; Kang, Jung Taek; Oh, Hyun Ju; Hong, So Gun; Kim, Dae Young; Jang, Goo; Lee, Byeong Chun

    2010-04-01

    Three viable female dogs, which have the same genotype, have been successfully produced by somatic cell nuclear transfer (SCNT); however, data on the growth pattern of cloned dogs are lacking. Thus, the aim of this study was (1) to assess growth parameters among those cloned dogs with measurement of body weight, height, and radiographic analysis of skull size and bone plate, and (2) to compare hematologic characteristics among the donor dog, cloned dogs, and age-matched control dogs. The cloned dogs were kept in the same environmental conditions. The body weight increased from 0.52, 0.46, and 0.52 kg at birth to 21.9, 22.9, and 20.4 kg at 68 weeks of age for individual cloned dogs, respectively. The withers height increased from 34.5, 32.6, and 35.2 cm at 8 weeks of age to 67.1 cm at 68 weeks of age in the three clones. The radiographic data demonstrated that patterns of bone growth were similar among cloned dogs, and all measured parameters of matured cloned dogs were similar with that of the fully grown donor dog. An age-specific pattern was identified on hematologic and serum biochemical measurements in both cloned dogs and age-matched controls. The parameters examined were within the normal reference ranges for healthy dogs. In conclusion, three genetically identical cloned dogs showed similar growth characteristics and had normal hematological and serum biochemical parameters.

  20. Cell-specific integration of nuclear receptor function at the genome.

    Science.gov (United States)

    Everett, Logan J; Lazar, Mitchell A

    2013-01-01

    Nuclear receptors (NRs) encompass a family of regulatory proteins that directly couple small-molecule signaling to transcriptional regulation. Initial studies of specific NR targets led to a model in which NRs bind highly specific DNA motifs in proximal promoter regions and strongly induce gene transcription in response to ligand binding. More recently, genome-wide studies have added to the complexity of this classic model of NR function. In particular, binding of NRs at weaker or alternate motifs is common in the context of DNA assembled into chromatin, and ligand responsiveness varies at different NR target genes. Such findings have led to proposed modifications to the classic view of NR regulation, including the 'assisted loading' model in which NRs assist in opening chromatin rather than compete for binding sites, and context-specific models in which genomic and epigenomic features influence the NR function locally at each binding site. Further elucidation of these mechanisms will be particularly important for understanding cell-specific and ligand-specific functions of each NR. Emerging genomic technologies such as ChIP-seq and GRO-seq provide insights on a larger scale leading to deeper understanding of the complexities of transcriptional regulation by NRs. Copyright © 2013 Wiley Periodicals, Inc.

  1. Quantitative analysis of chemical elements in single cells using nuclear microprobe and nano-probe

    International Nuclear Information System (INIS)

    Deves, Guillaume

    2010-01-01

    The study of the role of trace elements at cellular level requires the use of state-of-the-art analytical tools that could achieve enough sensitivity and spatial resolution. We developed a new methodology for the accurate quantification of chemical element distribution in single cells based on a combination of ion beam analysis techniques STIM, PIXE and RBS. The quantification procedure relies on the development of a STIM data analysis software (Paparamborde). Validity of this methodology and limits are discussed here. The method allows the quantification of trace elements (μg/g) with a 19.8 % uncertainty in cellular compartments with mass below 0.1 ng. The main limit of the method lies in the poor number of samples that can be analyzed, due to long irradiation times required and limited access to ion beam analysis facilities. This is the reason why we developed a database for cellular chemical composition capitalization (BDC4). BDC4 has been designed in order to use cellular chemical composition as a tracer for biological activities and is expected to provide in the future reference chemical compositions for any cellular type or compartment. Application of the STIM-PIXE-RBS methodology to the study of nuclear toxicology of cobalt compounds is presented here showing that STIM analysis is absolutely needed when organic mass loss appears during PIXE-RBS irradiation. (author)

  2. Crystal structure of an archaeal DNA sliding clamp: proliferating cell nuclear antigen from Pyrococcus furiosus.

    Science.gov (United States)

    Matsumiya, S; Ishino, Y; Morikawa, K

    2001-01-01

    The proliferating cell nuclear antigen (PCNA) is now recognized as one of the key proteins in DNA metabolic events because of its direct interactions with many proteins involved in important cellular processes. We have determined the crystal structure of PCNA from a hyperthermophilic archaeon, Pyrococcus furiosus (pfuPCNA), at 2.1 A resolution. pfuPCNA forms a toroidal, ring-shaped structure consisting of homotrimeric molecules, which is also observed in the PCNA crystals from human and yeast. The overall structure of pfuPCNA is highly conserved with other PCNA proteins, as well as with the bacterial ss clamp and the bacteriophage gp45. This result shows that the three-dimensional structure of the sliding clamp is conserved in the three domains of life. pfuPCNA has two remarkable features compared with the human and yeast PCNA molecules: it has more ion pairs and fewer intermolecular main chain hydrogen bonds. The former may contribute to the thermal stability of pfuPCNA, and the latter may be the cause of the stimulatory effect of pfuPCNA on the DNA synthesizing activity of P. furiosus DNA polymerases in the absence of the clamp loader replication factor C in vitro.

  3. Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA).

    Science.gov (United States)

    Seo, Jae Sung; Kim, Ha Na; Kim, Sun-Jick; Bang, Jiyoung; Kim, Eun-A; Sung, Ki Sa; Yoon, Hyun-Joo; Yoo, Hae Yong; Choi, Cheol Yong

    2014-01-03

    Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain.

    Directory of Open Access Journals (Sweden)

    Miklos Halmai

    Full Text Available Proliferating cell nuclear antigen (PCNA plays a key role in many cellular processes and due to that it interacts with a plethora of proteins. The main interacting surfaces of Saccharomyces cerevisiae PCNA have been mapped to the interdomain connecting loop and to the carboxy-terminal domain. Here we report that the subunit interface of yeast PCNA also has regulatory roles in the function of several DNA damage response pathways. Using site-directed mutagenesis we engineered mutations at both sides of the interface and investigated the effect of these alleles on DNA damage response. Genetic experiments with strains bearing the mutant alleles revealed that mutagenic translesion synthesis, nucleotide excision repair, and homologous recombination are all regulated through residues at the subunit interface. Moreover, genetic characterization of one of our mutants identifies a new sub-branch of nucleotide excision repair. Based on these results we conclude that residues at the subunit boundary of PCNA are not only important for the formation of the trimer structure of PCNA, but they constitute a regulatory protein domain that mediates different DNA damage response pathways, as well.

  5. The nuclear aryl hydocarbon receptor is involved in regulation of DNA repair and cell survival following treatment with ionizing radiation.

    Science.gov (United States)

    Dittmann, K H; Rothmund, M C; Paasch, A; Mayer, C; Fehrenbacher, B; Schaller, M; Frauenstein, K; Fritsche, E; Haarmann-Stemmann, T; Braeuning, A; Rodemann, H P

    2016-01-05

    In the present study, we explored the role of the aryl hydrocarbon receptor (AhR) for γ-H2AX associated DNA repair in response to treatment with ionizing radiation. Ionizing radiation was able to stabilize AhR protein and to induce a nuclear translocation in a similar way as described for exposure to aromatic hydrocarbons. A comparable AhR protein stabilization was obtained by treatment with hydroxyl-nonenal-generated by radiation-induced lipid peroxidation. AhR knockdown resulted in significant radio-sensitization of both A549- and HaCaT cells. Under these conditions an increased amount of residual γ-H2AX foci and a delayed decline of γ-H2AX foci was observed. Knockdown of the co-activator ARNT, which is essential for transcriptional activation of AhR target genes, reduced AhR-dependent CYP1A expression in response to irradiation, but was without effect on the amount of residual γ-H2AX foci. Nuclear AhR was found in complex with γ-H2AX, DNA-PK, ATM and Lamin A. AhR and γ-H2AX form together nuclear foci, which disappear during DNA repair. Presence of nuclear AhR protein is associated with ATM activation and chromatin relaxation indicated by acetylation of histone H3. Taken together, we could show, that beyond the function as a transcription factor the nuclear AhR is involved in the regulation of DNA repair. Reduction of nuclear AhR inhibits DNA-double stand repair and radiosensitizes cells. First hints for its molecular mechanism suggest a role during ATM activation and chromatin relaxation, both essential for DNA repair. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. DJ-1 Modulates Nuclear Erythroid 2-Related Factor-2-Mediated Protection in Human Primary Alveolar Type II Cells in Smokers.

    Science.gov (United States)

    Bahmed, Karim; Messier, Elise M; Zhou, Wenbo; Tuder, Rubin M; Freed, Curt R; Chu, Hong Wei; Kelsen, Steven G; Bowler, Russell P; Mason, Robert J; Kosmider, Beata

    2016-09-01

    Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2-related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases.

  7. Effects of stress, circadian rhythms, and dietary sodium on brain cell-nuclear uptake of aldosterone and corticosterone

    International Nuclear Information System (INIS)

    Yongue, B.G.

    1985-01-01

    The binding of the adrenal steroid hormones aldosterone (ALD) and corticosterone (CORT) in brain cell-nuclei has been implicated as a necessary step in the behavioral and physiological actions of these hormones. In vivo uptake of radioactively labeled ALD and CORT in adrenalectomized (ADX) rats indicates a strong cell-nuclear localization of both of these hormones in limbic brain regions (such as hippocampus, septum and amygdala). Research using sub-cellular fractionation and radioimmunoassay (RIA), has confirmed both the presence of endogenously secreted CORT in cell-nuclei and its limbic localization in the brains of adrenal-intact rats. In this study, environmental and dietary factors were manipulated to induce variation in serum ALD and CORT. A series of experiments employing sub-cellular fractionation and RIA were performed, which reveal that: (1) endogenously secreted ALD and CORT, are concentrated by cell-nuclei of the brain in adrenal-intact rats, (2) the majority of the corticosteroids measured in ethanol extracts of brain cell-nuclei are associated with receptor molecules, and (3) the regional distribution of endogenously secreted ALD differs markedly from the predominantly limbic pattern predicted from in vivo uptake of labeled ALD in ADX rats. Instead, brain cell-nuclear ALD is heavily concentrated in the hypothalamus, which supports the hypothesized relationship between the interaction of ALD and angiotensin in the brain and the behavioral regulation of fluid/electrolyte balance

  8. Nuclear factor κB and cyclooxygenase-2 immunoexpression in oral dysplasia and oral squamous cell carcinoma.

    Science.gov (United States)

    Pontes, Hélder Antônio Rebelo; Pontes, Flávia Sirotheau Corrêa; Fonseca, Felipe Paiva; de Carvalho, Pedro Luiz; Pereira, Erika Martins; de Abreu, Michelle Carvalho; de Freitas Silva, Brunno Santos; dos Santos Pinto, Décio

    2013-02-01

    Oral leukoplakia is the main potentially malignant oral lesion, and oral squamous cell carcinoma accounts for more than 95% of all malignant neoplasms in the oral cavity. Therefore, the aim of this study was to verify the immunoexpression of nuclear factor κB (NF-κB) and cyclooxygenase-2 (COX-2) proteins in dysplastic oral lesions and oral squamous cell carcinoma. Immunohistochemical reactions were performed on 6 inflammatory fibrous hyperplasia, 28 oral leukoplakia, and 15 oral squamous cell carcinoma paraffin-embedded samples. Immunoperoxidase reaction for NF-κB and COX-2 was applied on the specimens, and the positivity of the reactions was calculated for 1000 epithelial cells. Using the analysis of variance and the Tukey post hoc statistical analyses, a significantly increased immunoexpression for NF-κB was observed when oral squamous cell carcinoma samples were compared with the other groups studied. However, using the Kruskal-Wallis and the Dunn post hoc tests, a statistically significant result for COX-2 expression was obtained only when the moderate dysplasia group was compared with the inflammatory fibrous hyperplasia group. Nuclear factor κB may participate in the malignant phenotype acquisition process of the oral squamous cell carcinoma in its late stages, whereas COX-2 may be involved in the early stages of oral carcinogenesis process. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Proliferating cell nuclear antigen as a molecular biomarker for spermatogenesis in PTU-induced hypothyroidism of rats.

    Science.gov (United States)

    Tousson, Ehab; Ali, Ehab M M; Ibrahim, Wafaa; Mansour, Mohammed A

    2011-07-01

    The thyroid hormone has few serious effects on the testes except during the neonatal stage. There is little knowledge concerning the prolonged effect of thyroid hormone deficiency throughout the rat's life span and its effect on spermatogenesis. Proliferating cell nuclear antigen (PCNA) is a nuclear matrix protein, which is essential for multiple cell cycle pathways. Here we used PCNA immunohistochemistry as a marker to differentiate between the testes of control and hypothyroid rats. About 20 rats were equally divided into 2 groups; the first group was the control group, while the second group was the experimental group in which rats were fed 0.05% 6-n-propyl thiouracil (PTU) in drinking water for 6 weeks. Immunohistochemistry, using an antibody against PCNA, showed at least 3 differences in the pattern of PCNA immunoreactivity (PCNA-ir). First, PCNA-ir was not detected in Sertoli and Leydig cells in the testes of control rats and detected in some of the hypothyroid rats. Second, in the control group more than 96% of spermatogonia were PCNA-positive cells; however, hypothyroidism caused the reduction to approximately 25% PCNA staining in spermatogonia. The third difference was in the abnormal distribution of spermatogonia seen in the hypothyroid rat testis, not in the control one. These results suggest that prepubertal hypothyroidism affects the proliferation of spermatogenic cells leading to impaired spermatogenesis and that PCNA index is a useful marker for assessing germ cell kinetics and spermatogenesis in prepubertal hypothyroidism.

  10. Biologic activity of the novel orally bioavailable selective inhibitor of nuclear export (SINE) KPT-335 against canine melanoma cell lines

    Science.gov (United States)

    2014-01-01

    Background Exportin 1 (XPO1, also known as CRM1), is a chaperone protein responsible for the export of over 200 target proteins out of the nucleus. The expression and activity of XPO1 is upregulated in several human cancers and its expression is also linked to the development of chemotherapy resistance. Recent studies using both human and murine cancer cell lines have demonstrated that XPO1 is a relevant target for therapeutic intervention. The present study sought to characterize the biologic activity of an orally bioavailable selective inhibitor of nuclear export (SINE), KPT-335, against canine melanoma cell lines as a prelude to future clinical trials in dogs with melanoma. Results We evaluated the effects of KPT-335 on 4 canine malignant melanoma cell lines and found that KPT-335 inhibited proliferation, blocked colony formation, and induced apoptosis of treated cells at biologically relevant concentrations of drug. Additionally, KPT-335 downregulated XPO1 protein while inducing a concomitant increase in XPO1 messenger RNA. Lastly, KPT-335 treatment of cell lines upregulated the expression of both protein and mRNA for the tumor suppressor proteins p53 and p21, and promoted their nuclear localization. Conclusions KPT-335 demonstrates biologic activity against canine melanoma cell lines at physiologically relevant doses, suggesting that KPT-335 may represent a viable treatment option for dogs with malignant melanoma. PMID:25022346

  11. Dynamic Changes in the Protein Localization in the Nuclear Environment in Pancreatic β-Cell after Brief Glucose Stimulation

    DEFF Research Database (Denmark)

    Kang, Taewook; Jensen, Pia; Solovyeva, Vita

    2018-01-01

    Characterization of molecular mechanisms underlying pancreatic β-cell function in relation to glucose-stimulated insulin secretion is incomplete, especially with respect to global response in the nuclear environment. We focus on the characterization of proteins in the nuclear environment of β...... or export to/from the nucleus to the cytoplasm. Putative insulin mRNA transcription-associated factors were identified among the regulated proteins, and they were cross-validated by Western blotting and confocal immunofluorescence imaging. Collectively, our data suggest that protein translocation between...... the nucleus and the cytoplasm is an important process, highly involved in the initial molecular mechanism underlying glucose-stimulated insulin secretion in pancreatic β-cells....

  12. Nuclear abnormalities in cells from nasal epithelium: a promising assay to evaluate DNA damage related to air pollution in infants

    OpenAIRE

    Michelle Mergener; Cláudia R. Rhoden; Sérgio L. Amantéa

    2014-01-01

    OBJECTIVES: This study intends to provide a quick, easy, and inexpensive way to assess nuclear abnormalities such as micronuclei and bud frequencies; binucleated, karyorrhectic, karyolytic, pycnotic, and condensed chromatin cells in nasal scrapings of infants, which are particularly important for conducting genotoxic studies related to the inhaled atmosphere in pediatric populations. METHODS: Nasal swab samples were collected from 40 infants under 12 months of age using a small cytobrush...

  13. Nuclear receptor NHR-25 is required for cell-shape dynamics during epidermal differentiation in Caenorhabditis elegans

    Czech Academy of Sciences Publication Activity Database

    Šilhánková, Marie; Jindra, Marek; Asahina, Masako

    2005-01-01

    Roč. 118, č. 1 (2005), s. 223-232 ISSN 0021-9533 R&D Projects: GA AV ČR KJB5022303; GA ČR GD524/03/H133 Institutional research plan: CEZ:AV0Z60220518 Keywords : Caenorhabditis elegans * nuclear receptor * epidermal stem cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.543, year: 2005

  14. A Grading System Combining Tumor Budding and Nuclear Diameter Predicts Prognosis in Resected Lung Squamous Cell Carcinoma.

    Science.gov (United States)

    Kadota, Kyuichi; Miyai, Yumi; Katsuki, Naomi; Kushida, Yoshio; Matsunaga, Toru; Okuda, Masaya; Yokomise, Hiroyasu; Kanaji, Nobuhiro; Bandoh, Shuji; Haba, Reiji

    2017-06-01

    For lung squamous cell carcinomas, there are no histologic findings that have been universally accepted as prognostic factors. Tumor budding and nuclear grade have been recognized as prognostic factors in other carcinomas. In this study, we investigated whether pathologic findings could determine clinical outcome in Japanese patients with lung squamous cell carcinomas. Tumor slides from surgically resected lung squamous cell carcinomas (1999 to 2012) were reviewed (n=216). Tumors were evaluated for histologic subtypes, differentiation, tumor budding, nuclear diameter, and mitosis. Recurrence-free survival (RFS) and overall survival (OS) were analyzed using the log-rank test and the Cox proportional hazards model. Tumor budding and large nuclei were independent prognostic factors of a worse RFS (P<0.001 and P=0.002, respectively) and a worse OS (P<0.001 and P=0.038, respectively) on multivariate analysis after adjustment for pathologic stage and lymphatic invasion. However, histologic subtypes, differentiation, and mitotic count did not correlate with prognosis. A grading system combining tumor budding and nuclear diameter was an independent prognostic factors of a worse RFS (grade 2 vs. 1, hazard ratio [HR]=2.91; P<0.001, and grade 3 vs. 1, HR=7.60, P<0.001) and a worse OS (grade 2 vs. 1, HR=2.15; P=0.014, and grade 3 vs. 1, HR=4.54, P<0.001). We found that a grading system combining tumor budding and nuclear diameter was a significant prognostic factor among Japanese patients with resected lung squamous cell carcinoma.

  15. Upregulation of Nuclear Factor of Activated T-Cells by Nerve Injury Contributes to Development of Neuropathic Pain

    OpenAIRE

    Cai, You-Qing; Chen, Shao-Rui; Pan, Hui-Lin

    2013-01-01

    Nerve injury induces long-term changes in gene expression in the nociceptive circuitry and can lead to chronic neuropathic pain. However, the transcriptional mechanism involved in neuropathic pain is poorly understood. Nuclear factor of activated T-cells (NFATc) is a transcriptional factor regulated by the Ca2+-dependent protein phosphatase calcineurin. In this study, we determined nerve injury–induced changes in the expression of NFATc1–c4 in the dorsal root ganglia (DRG) and spinal cords an...

  16. Propagation of Nuclear Data Uncertainties in Deterministic Calculations: Application of Generalized Perturbation Theory and the Total Monte Carlo Method to a PWR Burnup Pin-Cell

    Science.gov (United States)

    Sabouri, P.; Bidaud, A.; Dabiran, S.; Lecarpentier, D.; Ferragut, F.

    2014-04-01

    The development of tools for nuclear data uncertainty propagation in lattice calculations are presented. The Total Monte Carlo method and the Generalized Perturbation Theory method are used with the code DRAGON to allow propagation of nuclear data uncertainties in transport calculations. Both methods begin the propagation of uncertainties at the most elementary level of the transport calculation - the Evaluated Nuclear Data File. The developed tools are applied to provide estimates for response uncertainties of a PWR cell as a function of burnup.

  17. Nuclear translocation of PKC-α is associated with cell cycle arrest and erythroid differentiation in myelodysplastic syndromes (MDSs).

    Science.gov (United States)

    Poli, Alessandro; Ratti, Stefano; Finelli, Carlo; Mongiorgi, Sara; Clissa, Cristina; Lonetti, Annalisa; Cappellini, Alessandra; Catozzi, Alessia; Barraco, Marilena; Suh, Pann-Ghill; Manzoli, Lucia; McCubrey, James A; Cocco, Lucio; Follo, Matilde Y

    2018-02-01

    PI-PLCβ1 is involved in cell proliferation, differentiation, and myelodysplastic syndrome (MDS) pathogenesis. Moreover, the increased activity of PI-PLCβ1 reduces the expression of PKC-α, which, in turn, delays the cell proliferation and is linked to erythropoiesis. Lenalidomide is currently used in low-risk patients with MDS and del(5q), where it can suppress the del(5q) clone and restore normal erythropoiesis. In this study, we analyzed the effect of lenalidomide on 16 patients with low-risk del(5q) MDS, as well as del(5q) and non-del(5q) hematopoietic cell lines, mainly focusing on erythropoiesis, cell cycle, and PI-PLCβ1/PKC-α signaling. Overall, 11 patients were evaluated clinically, and 10 (90%) had favorable responses; the remaining case had a stable disease. At a molecular level, both responder patients and del(5q) cells showed a specific induction of erythropoiesis, with a reduced γ/β-globin ratio, an increase in glycophorin A, and a nuclear translocation of PKC-α. Moreover, lenalidomide could induce a selective G 0 /G 1 arrest of the cell cycle in del(5q) cells, slowing down the rate proliferation in those cells. Altogether, our results could not only better explain the role of PI-PLCβ1/PKC-α signaling in erythropoiesis but also lead to a better comprehension of the lenalidomide effect on del(5q) MDS and pave the way to innovative, targeted therapies.-Poli, A., Ratti, S., Finelli, C., Mongiorgi, S., Clissa, C., Lonetti, A., Cappellini, A., Catozzi, A., Barraco, M., Suh, P.-G., Manzoli, L., McCubrey, J. A., Cocco, L., Follo, M. Y. Nuclear translocation of PKC-α is associated with cell cycle arrest and erythroid differentiation in myelodysplastic syndromes (MDSs).

  18. Nuclear abnormalities in buccal mucosa cells of patients with type I and II diabetes treated with folic acid.

    Science.gov (United States)

    Gómez-Meda, B C; Zamora-Perez, A L; Muñoz-Magallanes, T; Sánchez-Parada, M G; García Bañuelos, J J; Guerrero-Velázquez, C; Sánchez-Orozco, L V; Vera-Cruz, J M; Armendáriz-Borunda, J; Zúñiga-González, G M

    2016-02-01

    Diabetes mellitus (DM) is characterized by high blood glucose. Excessive production of free radicals may cause oxidative damage to DNA and other molecules, leading to complications of the disease. It may be possible to delay or reduce such damage by administration of antioxidants such as folic acid (FA). The objective of this study was to determine the effect of FA on nuclear abnormalities (NAs) in the oral mucosa of patients with DM. NAs (micronucleated cells, binucleated cells, pyknotic nuclei, karyorrhexis, karyolysis, abnormally condensed chromatin, and nuclear buds) were analyzed in 2000 cells from 45 healthy individuals (control group) and 55 patients with controlled or uncontrolled type I or II DM; 35 patients in the latter group were treated with FA. Samples were taken from the FA group before and after treatment. An increased rate of NAs was found in patients with DM in comparison with that of the control group (Pabnormally condensed chromatin, karyolysis, and nuclear buds produced by FA supplementation in DM patients (P<0.02) are consistent with the idea that free radicals are responsible for the increased frequency of NAs in DM patients. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. GAGE cancer-germline antigens bind DNA and are recruited to the nuclear envelope by Germ cell-less

    DEFF Research Database (Denmark)

    Gjerstorff, Morten; Rösner, Heike; Pedersen, Christina Bøg

    GAGE genes encode a highly similar, primate-specific protein family with unique primary structure and undefined roles in germ cells, various fetal cells and cancer cells. We report that GAGE proteins are intrinsically disordered proteins that provide novel interfaces between chromatin and the nuc......GAGE genes encode a highly similar, primate-specific protein family with unique primary structure and undefined roles in germ cells, various fetal cells and cancer cells. We report that GAGE proteins are intrinsically disordered proteins that provide novel interfaces between chromatin...... and the nuclear envelope. Structural analysis by NMR and CD spectroscopy showed GAGE proteins lack distinct secondary or tertiary structure and are therefore intrinsically disordered. In normal cells and cancer cells GAGE proteins localize predominantly in the nucleus; we found GAGE proteins formed stable...... complexes with dsDNA at sub-physiological concentrations. GAGE12I bound several different dsDNA fragments, suggesting sequence-independent binding. GAGE1, GAGE2B and GAGE12I associated directly or indirectly with Germ cell-less (GCL), which directly binds LEM-domain proteins (LAP2β, emerin, MAN1...

  20. Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

    Science.gov (United States)

    Don-Salu-Hewage, Ayesha S.; Chan, Siu Yuen; McAndrews, Kathleen M.; Chetram, Mahandranauth A.; Dawson, Michelle R.; Bethea, Danaya A.; Hinton, Cimona V.

    2013-01-01

    The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), ‘RPRK’, within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinβ1 and Transportinβ1-binding to CXCR4 promoted its nuclear translocation. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4. PMID:23468933

  1. DNA level and stereologic estimates of nuclear volume in squamous cell carcinomas of the uterine cervix. A comparative study with analysis of prognostic impact

    DEFF Research Database (Denmark)

    Sørensen, Flemming Brandt; Bichel, P; Jakobsen, A

    1992-01-01

    Grading of malignancy in squamous cell carcinomas of the uterine cervix is based on qualitative, morphologic examination and suffers from poor reproducibility. Using modern stereology, unbiased estimates of the three-dimensional, volume-weighted mean nuclear volume (nuclear vv), were obtained...... carcinoma of the uterine cervix....

  2. Nuclear Positioning

    Science.gov (United States)

    Gundersen, Gregg G.; Worman, Howard J.

    2013-01-01

    SUMMARY The nucleus is the largest organelle and is commonly depicted in the center of the cell. Yet during cell division, migration and differentiation, it frequently moves to an asymmetric position aligned with cell function. We consider the toolbox of proteins that move and anchor the nucleus within the cell and how forces generated by the cytoskeleton are coupled to the nucleus to move it. The significance of proper nuclear positioning is underscored by numerous diseases resulting from genetic alterations in the toolbox proteins. Finally, we discuss how nuclear position may influence cellular organization and signaling pathways. PMID:23498944

  3. SRS06, a new semisynthetic andrographolide derivative with improved anticancer potency and selectivity, inhibits nuclear factor-κB nuclear binding in the A549 non-small cell lung cancer cell line.

    Science.gov (United States)

    Lim, Jonathan Chee Woei; Jeyaraj, Ethel Jeyaseela; Sagineedu, Sreenivasa Rao; Wong, Wai Shiu Fred; Stanslas, Johnson

    2015-01-01

    Andrographolide has been reported with anticancer and anti-inflammatory properties through the inhibition of the activity of signaling molecules such as v-Src, nuclear factor-κB (NF-κB), STAT3, and PI3K. NF-κB has been proven to promote cancer cell survival, and targeting this pathway will halt the growth of cancer cells. Efforts have been made to produce semisynthetic derivatives of andrographolide with improved anticancer potency and selectivity. Subsequently, the effect of a selected derivative, 3,14,19-tripropionylandrographolide (SRS06), was tested for its action against NF-κB. Screening against 60 US National Cancer Institute (NCI) human cancer cell lines representing leukemia and non-small cell lung (NSCL), colon, CNS, melanoma, ovarian, renal, prostate, and breast cancers was performed to determine the tumor type selectivity and potency of SRS06. Microculture tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and sulforhodamine B assays were used to determine the in vitro anticancer activity, while Western blot studies were performed to ascertain the inhibitory effect of SRS06 on the NF-κB signaling cascade. The TransAM™ p65 assay kit was used to determine NF-κB p65 DNA binding activity in the NSCL cancer cell line A549. From the NCI screening, SRS06 was found to exhibit potent growth-inhibitory effects on multiple cancer cell lines with 10-fold lower 50% growth inhibition (GI50) compared with andrographolide. It was also discerned that the compound preferentially targeted melanoma, CNS, renal, colon, ovarian, prostate, and NSCL cancer cell lines. The DNA fragmentation assay indicated that the main mode of cell death of SRS06-treated A549 cells was via apoptosis. At 5 µmol/l the compound decreased NF-κB protein expression and caused a significant reduction in the nuclear p65 DNA binding activity. SRS06 displayed improved anticancer selectivity and potency when compared with andrographolide. We alluded its anticancer

  4. Vaccinia virus G8R protein: a structural ortholog of proliferating cell nuclear antigen (PCNA.

    Directory of Open Access Journals (Sweden)

    Melissa Da Silva

    Full Text Available BACKGROUND: Eukaryotic DNA replication involves the synthesis of both a DNA leading and lagging strand, the latter requiring several additional proteins including flap endonuclease (FEN-1 and proliferating cell nuclear antigen (PCNA in order to remove RNA primers used in the synthesis of Okazaki fragments. Poxviruses are complex viruses (dsDNA genomes that infect eukaryotes, but surprisingly little is known about the process of DNA replication. Given our previous results that the vaccinia virus (VACV G5R protein may be structurally similar to a FEN-1-like protein and a recent finding that poxviruses encode a primase function, we undertook a series of in silico analyses to identify whether VACV also encodes a PCNA-like protein. RESULTS: An InterProScan of all VACV proteins using the JIPS software package was used to identify any PCNA-like proteins. The VACV G8R protein was identified as the only vaccinia protein that contained a PCNA-like sliding clamp motif. The VACV G8R protein plays a role in poxvirus late transcription and is known to interact with several other poxvirus proteins including itself. The secondary and tertiary structure of the VACV G8R protein was predicted and compared to the secondary and tertiary structure of both human and yeast PCNA proteins, and a high degree of similarity between all three proteins was noted. CONCLUSIONS: The structure of the VACV G8R protein is predicted to closely resemble the eukaryotic PCNA protein; it possesses several other features including a conserved ubiquitylation and SUMOylation site that suggest that, like its counterpart in T4 bacteriophage (gp45, it may function as a sliding clamp ushering transcription factors to RNA polymerase during late transcription.

  5. Interaction of Proliferating Cell Nuclear Antigen With DNA at the Single Molecule Level

    KAUST Repository

    Raducanu, Vlad-Stefan

    2016-05-01

    Proliferating cell nuclear antigen (PCNA) is a key factor involved in Eukaryotic DNA replication and repair, as well as other cellular pathways. Its importance comes mainly from two aspects: the large numbers of interacting partners and the mechanism of facilitated diffusion along the DNA. The large numbers of interacting partners makes PCNA a necessary factor to consider when studying DNA replication, either in vitro or in vivo. The mechanism of facilitated diffusion along the DNA, i.e. sliding along the duplex, reduces the six degrees of freedom of the molecule, three degrees of freedom of translation and three degrees of freedom of rotation, to only two, translation along the duplex and rotational tracking of the helix. Through this mechanism PCNA can recruit its partner proteins and localize them to the right spot on the DNA, maybe in the right spatial orientation, more effectively and in coordination with other proteins. Passive loading of the closed PCNA ring on the DNA without free ends is a topologically forbidden process. Replication factor C (RFC) uses energy of ATP hydrolysis to mechanically open the PCNA ring and load it on the dsDNA. The first half of the introduction gives overview of PCNA and RFC and the loading mechanism of PCNA on dsDNA. The second half is dedicated to a diffusion model and to an algorithm for analyzing PCNA sliding. PCNA and RFC were successfully purified, simulations and a mean squared displacement analysis algorithm were run and showed good stability and experimental PCNA sliding data was analyzed and led to parameters similar to the ones in literature.

  6. The Nuclear Mitotic Apparatus (NuMA) protein is contributed by the donor cell nucleus in cloned porcine embryos.

    Science.gov (United States)

    Liu, Zhonghua; Schatten, Heide; Hao, Yanhong; Lai, Liangxue; Wax, David; Samuel, Melissa; Zhong, Zhi-Sheng; Sun, Qing-Yuan; Prather, Randall S

    2006-05-01

    The Nuclear Mitotic Apparatus (NuMA) protein is a multifunctional protein that is localized to the nucleus in interphase and to the poles of the mitotic apparatus during mitosis. In unfertilized porcine oocytes, NuMA is localized to the meiotic spindle. NuMA is removed along with the meiotic spindle during the enucleation process before reconstructing the egg by introducing the donor cell nucleus to produce cloned embryos. Questions have been raised regarding the source for NuMA in cloned embryos, as the enucleated oocyte does not contain detectable NuMA in the cytoplasm. To determine the source of NuMA in porcine nuclear transfer (NT) embryos, we conducted an immunofluorescence microscopy study with antibodies against NuMA to investigate the appearance and distribution of NuMA before and after reconstructing NT embryos with porcine skin fibroblasts. We used donor cells from a confluent culture with all cells in interphase. For comparative studies, we also determined the immunofluorescence pattern of NuMA, gamma-tubulin, and alpha-tubulin in porcine fibroblasts, parthenogenetic embryos and in vitro fertilized (IVF) embryos. Results show that NuMA was localized in nuclei of 33.5% (163/456) of the serum-deprived fibroblasts used as donor cells. No NuMA staining was detected in enucleated pig oocytes. Immediately after nuclear transfer, NuMA staining was absent in all donor cell fibroblast nuclei (0 h) but staining was detected by 6 h within the reconstructed eggs, at which time the transferred somatic cell nucleus swelled in most cells (19/27) and became a pronucleus-like structure. NuMA was localized exclusively within the pronucleus-like structures (15/27). At 25 h, NuMA was detected inside the nucleus (16/25) either in one-cell or in 2-cell stage embryos. Interestingly, in parthenogenetic embryos, NuMA staining was not detected in all 42 eggs examined at 1 h, and evident NuMA staining was only detected inside a few (4/51 at 6 h; 6/48 at 25 h) of the nuclei. In IVF

  7. Effective gene delivery into adipose-derived stem cells: transfection of cells in suspension with the use of a nuclear localization signal peptide-conjugated polyethylenimine.

    Science.gov (United States)

    Park, Eulsoon; Cho, Hong-Baek; Takimoto, Koichi

    2015-05-01

    Adipose-derived stem cells have the ability to turn into several clinically important cell types. However, it is difficult to transfect these cells with the use of conventional cationic lipid-based reagents. Polyethylenimine (PEI) is considered to be an inexpensive and effective tool for delivery of nucleic acids into mammalian cells. We used a linear PEI conjugated with the nuclear localization signal (NLS) peptide of Simian vacuolating virus 40 large T antigen (PEI-NLS) for transfection of plasmid DNA into adipose-derived cells. We also tested if transfection of cells in suspension might improve the degree and duration of exogenous gene expression. Transfection of cells in suspension with the use of a PEI conjugated with an NLS peptide resulted in high levels of reporter gene expression for an extended period of time in clonal 3T3-L1 preadipocytes and native human adipose-derived stem cells. The reporter gene expression increased for 3 days after the addition of the PEI-NLS peptide-DNA mixture in cell suspension and remained significant for at least 7 days. Cell density did not influence the level of reporter gene expression. Thus, the suspension method with the use of an NLS peptide-conjugated PEI leads to a robust and sustained expression of exogenous genes in adipose-derived cells. The devised transfection method may be useful for reprogramming of adipose-derived stem cells and cell-based therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  8. Protective role of nuclear factor of activated T cells 2 in CD8+ long-lived memory T cells in an allergy model.

    Science.gov (United States)

    Karwot, Roman; Maxeiner, Joachim H; Schmitt, Steffen; Scholtes, Petra; Hausding, Michael; Lehr, Hans A; Glimcher, Laurie H; Finotto, Susetta

    2008-04-01

    The transcriptional regulation of cytokines released and controlled by memory T cells is not well understood. Defective IFN-gamma production in allergic asthma correlates in human beings with the risk of wheezing in childhood. To understand the role of the transcription factor nuclear factor of activated T cells 2 (NFATc2) in memory and effector T cells in the airways in experimental allergic asthma. We used murine models of allergic asthma and adoptive cell transfer of fluorescence-activated sorted cells in a disease model. Mice lacking NFATc2 developed an increase in airwayhyperresponsiveness (AHR), remodeling, and serum IgE levelson ovalbumin sensitization. This phenotype was associated withCD81CD1222 T cells deficient in IFN-g production in theairways. The origin of this phenotype in NFATc2(2/2) mice wasrelated to an expanded population of lung CD81CD1221(IL-2Rb chain) CD127hi (IL-7 receptor [R] a chain1) long-livedmemory cells. Adoptive transfer of ovalbumin-specific CD81NFATc2(2/2) T cells enhanced the AHR generated byNFATc2(2/2) CD41 T cells in immunodeficient mice, increasedIL-17, and reduced IFN-g production in the reconstituted mice. Depletion of the memory CD81CD1221IL-7Rhigh T-cellpopulation corrected the defect in IFN-g production by lungNFATc2(2/2) CD81CD1222 cells and abrogated the increasedAHR observed in NFATc2(2/2) CD81 T-cell-reconstituted micewith a severe combined immunodeficiency disorder. Taken together, our results suggest that NFATc2 expression in long-lived memory CD8+ T cells controls IL-2 and IFN-gamma production in lung CD8+ T cells, which then limits TH17 and TH2 development in the airways during allergen challenge.

  9. Rapid Nongenomic Glucocorticoid Actions in Male Mouse Hypothalamic Neuroendocrine Cells Are Dependent on the Nuclear Glucocorticoid Receptor

    Science.gov (United States)

    Nahar, Jebun; Haam, Juhee; Chen, Chun; Jiang, Zhiying; Glatzer, Nicholas R.; Muglia, Louis J.; Dohanich, Gary P.; Herman, James P.

    2015-01-01

    Corticosteroids act classically via cognate nuclear receptors to regulate gene transcription; however, increasing evidence supports rapid, nontranscriptional corticosteroid actions via activation of membrane receptors. Using whole-cell patch clamp recordings in hypothalamic slices from male mouse genetic models, we tested for nongenomic glucocorticoid actions at glutamate and gamma aminobutyric acid (GABA) synapses in hypothalamic neuroendocrine cells, and for their dependence on the nuclear glucocorticoid receptor (GR). In enhanced green fluorescent protein-expressing CRH neurons of the paraventricular nucleus (PVN) and in magnocellular neurons of the PVN and supraoptic nucleus (SON), dexamethasone activated postsynaptic membrane-associated receptors and G protein signaling to elicit a rapid suppression of excitatory postsynaptic inputs, which was blocked by genetic deletion of type I cannabinoid receptors and a type I cannabinoid receptor antagonist. In magnocellular neurons, dexamethasone also elicited a rapid nitric oxide-dependent increase in inhibitory postsynaptic inputs. These data indicate a rapid, synapse-specific glucocorticoid-induced retrograde endocannabinoid signaling at glutamate synapses and nitric oxide signaling at GABA synapses. Unexpectedly, the rapid glucocorticoid effects on both excitatory and inhibitory synaptic transmission were lost with conditional deletion of GR in the PVN and SON in slices from a single minded-1-cre-directed conditional GR knockout mouse. Thus, the nongenomic glucocorticoid actions at glutamate and GABA synapses on PVN and SON neuroendocrine cells are dependent on the nuclear GR. The nuclear GR, therefore, is responsible for transducing the rapid steroid response at the membrane, or is either a critical component in the signaling cascade or regulates a critical component of the signaling cascade of a distinct membrane GR. PMID:26061727

  10. Cellular distribution of uranium after acute exposure of renal epithelial cells: SEM, TEM and nuclear microscopy analysis

    Energy Technology Data Exchange (ETDEWEB)

    Carriere, Marie [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France)]. E-mail: carriere@drecam.cea.fr; Gouget, Barbara [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France); Gallien, Jean-Paul [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France); Avoscan, Laure [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France); Gobin, Renee [Laboratoire d' imagerie cellulaire et moleculaire, DBJC/SBFM/LTMD, CEA/Saclay, 91191 Gif sur Yvette (France); Verbavatz, Jean-Marc [Laboratoire d' imagerie cellulaire et moleculaire, DBJC/SBFM/LTMD, CEA/Saclay, 91191 Gif sur Yvette (France); Khodja, Hicham [Laboratoire Pierre Suee, CEA-CNRS UMR 9956, CEA/Saclay, 91191 Gif-sur-Yvette (France)

    2005-04-01

    The major health effect of uranium exposure has been reported to be chemical kidney toxicity, functional and histological damages being mainly observed in proximal tubule cells. Uranium enters the proximal tubule as uranyl-bicarbonate or uranyl-citrate complexes. The aim of our research is to investigate the mechanisms of uranium toxicity, intracellular accumulation and repartition after acute intoxication of rat renal proximal tubule epithelial cells, as a function of its chemical form. Microscopic observations of renal epithelial cells after acute exposure to uranyl-bicarbonate showing the presence of intracellular precipitates as thin needles of uranyl-phosphate localized in cell lysosomes have been published. However the initial site of precipitates formation has not been identified yet: they could either be formed outside the cells before internalization, or directly inside the cells. Uranium solubility as a function and initial concentration was specified by ICP-MS analysis of culture media. In parallel, uranium uptake and distribution in cell monolayers exposed to U-bicarbonate was investigated by nuclear microprobe analyses. Finally, the presence of uranium precipitates was tested out by scanning electron microscopic observations (SEM), while extracellular and/or intracellular precipitates were observed on thin sections of cells by transmission electron microscopy (TEM)

  11. Influence of radiotherapy on expression of the proliferating cell nuclear antigen (PCNA) and c-fos in human cervical cancer

    International Nuclear Information System (INIS)

    Shi Mei; Wei Lichun; Sun Chaoyang; Ma Haixin; Guo Yan

    2001-01-01

    Objective: To investigate changes of proliferating cell nuclear antigen (PCNA) expression in human cervical cancer following irradiation. Methods: Immunohistochemical staining for PCNA was performed in frozen sections of formalin-fixed cervical cancer biopsy tissues. Results: The majority of the cancer cells showed PCNA-immunoreactivity before irradiation. Following irradiation (30-40 Gy/15-20 f) PCNA-immuno-positive staining was hardly detectable in most of the cancer cells. The PCNA-immunoreactivity, however, increased after radiotherapy, and moderate or heavy immuno-positive staining for PCNA was seen in irradiated mesenchymal tissue cells. On the other hand, after irradiation Fos-immunoreactivity decreased remarkably, and Fos-immuno-positive staining was hardly detectable in most of cancer cells. No obvious change in Fos-immuno-reactivity, however, was seen in mesenchymal connective tissue following irradiation. Conclusion: Irradiation inhibits PCNA and c-fos expression in cervical cancer cells whereas it induces the expression of PCNA in mesenchymal tissue cells. The present results suggest that expression of PCNA and c-fos may be regarded as a molecular marker for evaluating the cancer cell proliferation and mesenchymal tissue repair during radiotherapy of human cervical cancer

  12. Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells.

    Science.gov (United States)

    Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao; Alevizos, Ilias; Ambudkar, Indu S

    2016-04-15

    The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells*

    Science.gov (United States)

    Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao; Alevizos, Ilias; Ambudkar, Indu S.

    2016-01-01

    The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca2+] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca2+-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca2+ entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca2+-dependent up-regulation of AQP5. These important findings reveal that the Ca2+-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture. PMID:26903518

  14. Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells.

    Science.gov (United States)

    Shimura, Tsutomu; Hamada, Nobuyuki; Sasatani, Megumi; Kamiya, Kenji; Kunugita, Naoki

    2014-01-01

    Cyclin D1 is a mitogenic sensor that responds to growth signals from the extracellular environment and regulates the G 1-to-S cell cycle transition. When cells are acutely irradiated with a single dose of 10 Gy, cyclin D1 is degraded, causing cell cycle arrest at the G 1/S checkpoint. In contrast, cyclin D1 accumulates in human tumor cells that are exposed to long-term fractionated radiation (0.5 Gy/fraction of X-rays). In this study we investigated the effect of fractionated low-dose radiation exposure on cyclin D1 localization in 3 strains of normal human fibroblasts. To specifically examine the nuclear accumulation of cyclin D1, cells were treated with a hypotonic buffer containing detergent to remove cytoplasmic cyclin D1. Proliferating cell nuclear antigen (PCNA) immunofluorescence was used to identify cells in S phase. With this approach, we observed S-phase nuclear retention of cyclin D1 following low-dose fractionated exposures, and found that cyclin D1 nuclear retention increased with exposure time. Cells that retained nuclear cyclin D1 were more likely to have micronuclei than non-retaining cells, indicating that the accumulation of nuclear cyclin D1 was associated with genomic instability. Moreover, inhibition of the v-akt murine thymoma viral oncogene homolog (AKT) pathway facilitated cyclin D1 degradation and eliminated cyclin D1 nuclear retention in cells exposed to fractionated radiation. Thus, cyclin D1 may represent a useful marker for monitoring long-term effects associated with exposure to low levels of radiation.

  15. Modulation of both activator protein-1 and nuclear factor-kappa B signal transduction of human T cells by amiodarone.

    Science.gov (United States)

    Cheng, Shu-Meng; Lin, Wei-Hsiang; Lin, Chin-Sheng; Ho, Ling-Jun; Tsai, Tsung-Neng; Wu, Chun-Hsien; Lai, Jenn-Haung; Yang, Shih-Ping

    2015-01-01

    Amiodarone, a common and effective antiarrhythmic drug, has been reported to have anti-inflammatory effects such as reducing the activation and movement of neutrophils. However, its effects on human T cells remain unclear. The aim of this study was to elucidate the effects and possible underlying mechanisms of amiodarone on human T cells. We isolated human primary T cells from the peripheral blood of healthy volunteers and performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, electrophoretic mobility shift assay, luciferase assay, and Western blotting to evaluate the modulatory effects of amiodarone on human T cells. We found that amiodarone dose dependently inhibited the production of cytokines, including interleukin-2 (IL-2), IL-4, tumor necrosis factor-alpha, and interferon-gamma in activated human T cells. By flow cytometry, we demonstrated that amiodarone suppressed the expression of IL-2 receptor-alpha (CD25) and CD69, the cell surface markers of activated T cells. Moreover, molecular investigations revealed that amiodarone down-regulated activator protein-1 (AP-1) and nuclear factor kappa-B (NF-κB) DNA-binding activities in activated human T cells and also inhibited DNA binding and transcriptional activities of both AP-1 and NF-κB in Jurkat cells. Finally, by Western blotting, we showed that amiodarone reduced the activation of c-Jun NH(2)-terminal protein kinase and P38 mitogen-activated protein kinase, and suppressed stimuli-induced I-kappa B-alpha degradation in activated human T cells. Through regulation of AP-1 and NF-κB signaling, amiodarone inhibits cytokine production and T cell activation. These results show the pleiotropic effects of amiodarone on human T cells and suggest its therapeutic potential in inflammation-related cardiovascular disorders. © 2014 by the Society for Experimental Biology and Medicine.

  16. The role of cytochrome c on apoptosis induced by Anagrapha falcifera multiple nuclear polyhedrosis virus in insect Spodoptera litura cells.

    Directory of Open Access Journals (Sweden)

    Kaiyu Liu

    Full Text Available There are conflicting reports on the role of cytochrome c during insect apoptosis. Our previous studies have showed that cytochrome c released from the mitochondria was an early event by western blot analysis and caspase-3 activation was closely related to cytochrome c release during apoptosis induced by baculovirus in Spodoptera litura cells (Sl-1 cell line. In the present study, alteration in mitochondrial morphology was observed by transmission electron microscopy, and cytochrome c release from mitochondria in apoptotic Sl-1 cells induced with Anagrapha falcifera multiple nuclear polyhedrosis virus (AfMNPV has further been confirmed by immunofluoresence staining protocol, suggesting that structural disruption of mitochondria and the release of cytochrome c are important events during Lepidoptera insect cell apoptosis. We also used Sl-1 cell-free extract system and the technique of RNA interference to further investigate the role of cytochrome c in apoptotic Sl-1 cells induced by AfMNPV. Caspase-3 activity in cell-free extracts supplemented with exogenous cytochrome c was determined and showed an increase with the extension of incubation time. DsRNA-mediated silencing of cytochrome c resulted in the inhibition of apoptosis and protected the cells from AfMNPV-induced cell death. Silencing of expression of cytochrome c had a remarkable effect on pro-caspase-3 and pro-caspase-9 activation and resulted in the reduction of caspase-3 and caspase-9 activity in Sl-1 cells undergoing apoptosis. Caspase-9 inhibitor could inhibit activation of pro-caspase-3, and the inhibition of the function of Apaf-1 with FSBA blocked apoptosis, hinting that Apaf-1 could be involved in Sl-1 cell apoptosis induced by AfMNPV. Taken together, these results strongly demonstrate that cytochrome c plays an important role in apoptotic signaling pathways in Lepidopteran insect cells.

  17. SiRNA knockdown of the DEK nuclear protein mRNA enhances apoptosis and chemosensitivity of canine transitional cell carcinoma cells.

    Science.gov (United States)

    Yamazaki, Hiroki; Iwano, Tomomi; Otsuka, Saori; Kagawa, Yumiko; Hoshino, Yuki; Hosoya, Kenji; Okumura, Masahiro; Takagi, Satoshi

    2015-04-01

    Transitional cell carcinoma (TCC) in dogs is an aggressive malignant neoplasm, originating in the epithelium of the urinary bladder. The DEK nuclear protein is overexpressed in several types of human bladder cancer, where it is involved in chromatin reconstruction, gene transcription and apoptosis. Since DEK represents a potential therapeutic target for canine TCC, this study was designed to investigate DEK expression in canine TCC and to determine the effects of DEK mRNA silencing on TCC cells in vitro. The gene expression profiles of seven selected cancer-associated genes was assessed in four canine TCC cell lines and expression of DEK protein was evaluated in bladder tissue biopsies from healthy dogs and those affected with cystitis or TCC. After transfection of four canine TCC cell lines with DEK-specific or scrambled siRNA, annexin V staining was performed to evaluate apoptosis, and methylthiazole tetrazolium assays were performed to assess both cell viability and sensitivity to carboplatin. DEK mRNA expression was relatively high in canine TCC cells and expression of the DEK protein was significantly greater in TCC tumours compared with the other tissue samples. After transfection with DEK-specific siRNA, apoptosis, cell growth inhibition, and enhanced sensitivity to carboplatin were observed in all TCC cells assessed. These research findings suggest that DEK could be a potential therapeutic target for canine TCC. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. The Hsp90 Inhibitor, 17-AAG, Prevents the Ligand-Independent Nuclear Localization of Androgen Receptor in Refractory Prostate Cancer Cells

    Science.gov (United States)

    Saporita, Anthony J.; Ai, Junkui; Wang, Zhou

    2010-01-01

    BACKGROUND Androgen receptor (AR) is the key molecule in androgen-refractory prostate cancer. Despite androgen ablative conditions, AR remains active and is necessary for the growth of androgen-refractory prostate cancer cells. Nuclear localization of AR is a prerequisite for its transcriptional activation. We examined AR localization in androgen-dependent and androgen-refractory prostate cancer cells. METHODS AND RESULTS We demonstrate increased nuclear localization of a GFP-tagged AR in the absence of hormone in androgen-refractory C4-2 cells compared to parental androgen-sensitive human prostate cancer LNCaP cells. Analysis of AR mutants impaired in ligand-binding indicates that the nuclear localization of AR in C4-2 cells is truly androgen-independent. The hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), inhibits basal PSA expression and disrupts the ligand-independent nuclear localization of AR at doses much lower than required to inhibit androgen-induced nuclear import. CONCLUSIONS Hsp90 is a key regulator of ligand-independent nuclear localization and activation of AR in androgen-refractory prostate cancer cells. PMID:17221841

  19. Escin, a Pentacyclic Triterpene, Chemosensitizes Human Tumor Cells through Inhibition of Nuclear Factor-κB Signaling Pathway

    Science.gov (United States)

    Harikumar, Kuzhuvelil B.; Sung, Bokyung; Pandey, Manoj K.; Guha, Sushovan; Krishnan, Sunil

    2010-01-01

    Agents that can enhance tumor cell apoptosis and inhibit invasion have potential for the treatment of cancer. Here, we report the identification of escin, a pentacyclic triterpenoid from horse chestnut that exhibits antitumor potential against leukemia and multiple myeloma. Whether examined by esterase staining, phosphatidyl-serine staining, DNA breakage, or caspase-mediated poly(ADP-ribose) polymerase cleavage, escin potentiated tumor necrosis factor (TNF)-induced apoptosis but inhibited tumor cell invasion. This correlated with the down-regulation of bcl-2, cellular inhibitor of apoptosis protein-2, cyclin D1, cyclooxygenase-2, intercellular adhesion molecule-1, matrix metalloproteinase-9, and vascular endothelial growth factor, which are all regulated by the activation of the transcription factor NF-κB. When examined by electrophoretic mobility shift assay, the triterpenoid suppressed nuclear factor-κB (NF-κB) activation induced by TNF and other inflammatory agents, and this correlated with the inhibition of IκBα phosphorylation and degradation, inhibition of IκB kinase complex (IKK) activation, suppression of p65 phosphorylation and nuclear translocation, and abrogation of NF-κB-dependent reporter activity. Overall, our results demonstrate that escin inhibits activation of NF-κB through inhibition of IKK, leading to down-regulation of NF-κB-regulated cell survival and metastatic gene products and thus resulting in sensitization of cells to cytokines and chemotherapeutic agents. PMID:20103608

  20. Escin, a pentacyclic triterpene, chemosensitizes human tumor cells through inhibition of nuclear factor-kappaB signaling pathway.

    Science.gov (United States)

    Harikumar, Kuzhuvelil B; Sung, Bokyung; Pandey, Manoj K; Guha, Sushovan; Krishnan, Sunil; Aggarwal, Bharat B

    2010-05-01

    Agents that can enhance tumor cell apoptosis and inhibit invasion have potential for the treatment of cancer. Here, we report the identification of escin, a pentacyclic triterpenoid from horse chestnut that exhibits antitumor potential against leukemia and multiple myeloma. Whether examined by esterase staining, phosphatidyl-serine staining, DNA breakage, or caspase-mediated poly(ADP-ribose) polymerase cleavage, escin potentiated tumor necrosis factor (TNF)-induced apoptosis but inhibited tumor cell invasion. This correlated with the down-regulation of bcl-2, cellular inhibitor of apoptosis protein-2, cyclin D1, cyclooxygenase-2, intercellular adhesion molecule-1, matrix metalloproteinase-9, and vascular endothelial growth factor, which are all regulated by the activation of the transcription factor NF-kappaB. When examined by electrophoretic mobility shift assay, the triterpenoid suppressed nuclear factor-kappaB (NF-kappaB) activation induced by TNF and other inflammatory agents, and this correlated with the inhibition of IkappaBalpha phosphorylation and degradation, inhibition of IkappaB kinase complex (IKK) activation, suppression of p65 phosphorylation and nuclear translocation, and abrogation of NF-kappaB-dependent reporter activity. Overall, our results demonstrate that escin inhibits activation of NF-kappaB through inhibition of IKK, leading to down-regulation of NF-kappaB-regulated cell survival and metastatic gene products and thus resulting in sensitization of cells to cytokines and chemotherapeutic agents.

  1. Loss of nuclear BRCA1 protein staining in normal tissue cells derived from BRCA1 and BRCA2 mutation carriers

    Energy Technology Data Exchange (ETDEWEB)

    Brakeleer, Sylvia de [Laboratory of Molecular Oncology (Department of Medical Oncology), UZ Brussel, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels (Belgium); Bogdani, Marika [Department of Experimental Pathology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels (Belgium); Greve, Jacques de [Laboratory of Molecular Oncology (Department of Medical Oncology), UZ Brussel, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels (Belgium); Familial Cancer Clinic, UZ Brussel, Laarbeeklaan 101, 1090 Brussels (Belgium); Decock, Julie [Laboratory of Molecular Oncology (Department of Medical Oncology), UZ Brussel, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels (Belgium); Sermijn, Erica [Familial Cancer Clinic, UZ Brussel, Laarbeeklaan 101, 1090 Brussels (Belgium); Bonduelle, Maryse [Familial Cancer Clinic, UZ Brussel, Laarbeeklaan 101, 1090 Brussels (Belgium); Goelen, Guido [Familial Cancer Clinic, UZ Brussel, Laarbeeklaan 101, 1090 Brussels (Belgium); Teugels, Erik [Laboratory of Molecular Oncology (Department of Medical Oncology), UZ Brussel, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels (Belgium) and Familial Cancer Clinic, UZ Brussel, Laarbeeklaan 101, 1090 Brussels (Belgium)]. E-mail: eteugels@uzbrussel.be

    2007-06-01

    Enhanced genomic instability has been recently reported in normal cells derived from BRCA1/2 mutation carriers when placed in vitro in non-physiological stress conditions. We present here original data which help to explain the observed genomic instability. Leucocytes from BRCA1/2 mutation carriers, sporadic breast cancer patients and controls were prepared for BRCA1 immunocytochemistry. We show that BRCA1 containing nuclear dot like structures are detectable in about 80% of the leucocytes from controls and sporadic breast cancer patients, but are absent in the majority of normal cells from BRCA1 as well as BRCA2 mutation carriers (also in their normal breast cells). Our results thus indicate that the genomic instability observed in normal cells from BRCA1 and BRCA2 mutation carriers is associated with a down-regulation of nuclear BRCA1 protein accumulation in the dot like structures. These results suggest in addition that immunocytochemical or alternative molecular screening strategies might help to identify women with a high risk for breast (ovarian) cancer even when the underlying genetic defect remains undetectable.

  2. Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

    Directory of Open Access Journals (Sweden)

    Raabe Timothy D

    2003-04-01

    Full Text Available Abstract Background Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97 have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. Results Using confocal microscopy and western analysis techniques, we determined 1 the immunolabeled structures are truly within the nucleus; 2 the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs and it can be identified on other intermediate filament proteins (IFs in other cell types; and 3 there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. Conclusion We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

  3. A Nuclear Role for miR-9 and Argonaute Proteins in Balancing Quiescent and Activated Neural Stem Cell States

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    Shauna Katz

    2016-10-01

    Full Text Available Throughout life, adult neural stem cells (NSCs produce new neurons and glia that contribute to crucial brain functions. Quiescence is an essential protective feature of adult NSCs; however, the establishment and maintenance of this state remain poorly understood. We demonstrate that in the adult zebrafish pallium, the brain-enriched miR-9 is expressed exclusively in a subset of quiescent NSCs, highlighting a heterogeneity within these cells, and is necessary to maintain NSC quiescence. Strikingly, miR-9, along with Argonaute proteins (Agos, is localized to the nucleus of quiescent NSCs, and manipulating their nuclear/cytoplasmic ratio impacts quiescence. Mechanistically, miR-9 permits efficient Notch signaling to promote quiescence, and we identify the RISC protein TNRC6 as a mediator of miR-9/Agos nuclear localization in vivo. We propose a conserved non-canonical role for nuclear miR-9/Agos in controlling the balance between NSC quiescence and activation, a key step in maintaining adult germinal pools.

  4. The nuclear chaperone nucleophosmin escorts an Epstein-Barr Virus nuclear antigen to establish transcriptional cascades for latent infection in human B cells.

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    Cheng-Der Liu

    Full Text Available Epstein-Barr Virus (EBV is an oncogenic γ-herpesvirus that capably establishes both latent and lytic modes of infection in host cells and causes malignant diseases in humans. Nuclear antigen 2 (EBNA2-mediated transcription of both cellular and viral genes is essential for the establishment and maintenance of the EBV latency program in B lymphocytes. Here, we employed a protein affinity pull-down and LC-MS/MS analysis to identify nucleophosmin (NPM1 as one of the cellular proteins bound to EBNA2. Additionally, the specific domains that are responsible for protein-protein interactions were characterized as EBNA2 residues 300 to 360 and the oligomerization domain (OD of NPM1. As in c-MYC, dramatic NPM1 expression was induced in EBV positively infected B cells after three days of viral infection, and both EBNA2 and EBNALP were implicated in the transactivation of the NPM1 promoter. Depletion of NPM1 with the lentivirus-expressed short-hairpin RNAs (shRNAs effectively abrogated EBNA2-dependent transcription and transformation outgrowth of lymphoblastoid cells. Notably, the ATP-bound state of NPM1 was required to induce assembly of a protein complex containing EBNA2, RBP-Jκ, and NPM1 by stabilizing the interaction of EBNA2 with RBP-Jκ. In a NPM1-knockdown cell line, we demonstrated that an EBNA2-mediated transcription defect was fully restored by the ectopic expression of NPM1. Our findings highlight the essential role of NPM1 in chaperoning EBNA2 onto the latency-associated membrane protein 1 (LMP1 promoters, which is coordinated with the subsequent activation of transcriptional cascades through RBP-Jκ during EBV infection. These data advance our understanding of EBV pathology and further imply that NPM1 can be exploited as a therapeutic target for EBV-associated diseases.

  5. Morphological changes of nuclear and chromatin architecture after microwave electromagnetic field exposure in 3T3 fibroblast cell cultures

    International Nuclear Information System (INIS)

    Mircea, D.; Chirila, Lavinia; Ciurea, A. V.; Helm, G.; Hankins, G.; Redrick, Jan; Gavrila, L.; Sheppard, B.; Bloodgoog, R.; Pallin, I.; Nitu, Rozalia; Rusu, I.

    2001-01-01

    It is already demonstrated in the literature that electromagnetic fields, particularly the microwave irradiation could be a powerful weapon against human tumors , but also against human body itself, depending on the wave parameters and irradiation time. The effects of microwave electromagnetic fields on living systems were studied in detail all over the world and, furthermore, the potential of intracellular damages by cytoskeleton, nuclear, chromatin and DNA alterations were carefully evaluated. In this study, the authors emphasize the morphological changes of nucleus and chromatin in fibroblast cell line 3T3 after microwave exposure with progressive increasing powers and times of irradiation. It was used a pulsed wave with 915 MHz frequency, with forward power ranging between 3 - 10 W, emitted by a helical microwave antenna placed into the cell culture medium, close to the cell monolayer. The authors tried to define certain severity stages of nuclear material alterations following different wave intensities and to compare these effects with other cytoplasmic organelle alterations. It was found that the nuclear material is the most sensitive intracellular structure in microwave electromagnetic field exposure. Also the authors tried to establish a well-defined protocol of irradiation with microwave electromagnetic fields in order to destroy the microtubule system of cytoskeleton in different types of cellular lines, in vitro. The cytoskeleton structure was evaluated by immunofluorescence methods. In non-muscle cells the cytoskeleton stability is achieved by interaction between microtubule system and actin filaments. Microtubule depolymerization by microwave exposure produces a secondary instability of cytoskeleton, the actin filaments coupling and cell contractility. The increasing of fibroblast contractility allows a more efficient treatment of the wounds with low spontaneous healing. Electromagnetic therapy could be an alternative therapy in plastic surgery

  6. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  7. Abortive replication of Bombyx mori nucleopolyhedrovirus in Sf9 and High Five cells: Defective nuclear transport of the virions

    International Nuclear Information System (INIS)

    Katou, Yasuhiro; Ikeda, Motoko; Kobayashi, Michihiro

    2006-01-01

    Despite close genetic relationship, Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multicapsid NPV (AcMNPV) display a distinct host range property. Here, BmNPV replication was examined in Sf9 and High Five cells that were nonproductive for BmNPV infection but supported high titers of AcMNPV replication. Recombinant BmNPV, vBm/gfp/lac, containing bm-ie1 promoter-driven egfp showed that few Sf9 and High Five cells infected with vBm/gfp/lac expressed EGFP, while large proportion of EGFP-expressing cells was observed when transfected with vBm/gfp/lac DNA. Immunocytochemical analysis showed that BmNPV was not imported into the nucleus of these two cell lines, while recombinant BmNPV, vBmΔ64/ac-gp64 possessing AcMNPV gp64 was imported into the nucleus, yielding progeny virions in High Five cells, but not Sf9 cells. These results indicate that the defective nuclear import of infected virions due to insufficient BmNPV GP64 function is involved in the restricted BmNPV replication in Sf9 and High Five cells

  8. A highly efficient method for generation of therapeutic quality human pluripotent stem cells by using naive induced pluripotent stem cells nucleus for nuclear transfer

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    Madhusudana Girija Sanal

    2014-09-01

    Full Text Available Even after several years since the discovery of human embryonic stem cells and induced pluripotent stem cells (iPSC, we are still unable to make any significant therapeutic benefits out of them such as cell therapy or generation of organs for transplantation. Recent success in somatic cell nuclear transfer (SCNT made it possible to generate diploid embryonic stem cells, which opens up the way to make high-quality pluripotent stem cells. However, the process is highly inefficient and hence expensive compared to the generation of iPSC. Even with the latest SCNT technology, we are not sure whether one can make therapeutic quality pluripotent stem cell from any patient’s somatic cells or by using oocytes from any donor. Combining iPSC technology with SCNT, that is, by using the nucleus of the candidate somatic cell which got reprogrammed to pluripotent state instead that of the unmodified nucleus of the candidate somatic cell, would boost the efficiency of the technique, and we would be able to generate therapeutic quality pluripotent stem cells. Induced pluripotent stem cell nuclear transfer (iPSCNT combines the efficiency of iPSC generation with the speed and natural reprogramming environment of SCNT. The new technique may be called iPSCNT. This technique could prove to have very revolutionary benefits for humankind. This could be useful in generating organs for transplantation for patients and for reproductive cloning, especially for childless men and women who cannot have children by any other techniques. When combined with advanced gene editing techniques (such as CRISPR-Cas system this technique might also prove useful to those who want to have healthy children but suffer from inherited diseases. The current code of ethics may be against reproductive cloning. However, this will change with time as it happened with most of the revolutionary scientific breakthroughs. After all, it is the right of every human to have healthy offspring and it is

  9. DNA level and stereologic estimates of nuclear volume in squamous cell carcinomas of the uterine cervix. A comparative study with analysis of prognostic impact

    DEFF Research Database (Denmark)

    Sørensen, Flemming Brandt; Bichel, P; Jakobsen, A

    1992-01-01

    Grading of malignancy in squamous cell carcinomas of the uterine cervix is based on qualitative, morphologic examination and suffers from poor reproducibility. Using modern stereology, unbiased estimates of the three-dimensional, volume-weighted mean nuclear volume (nuclear vv), were obtained...... clinical stages of disease did not differ with regard to nuclear vv (2P = 0.99) and DI (2P = 0.56). No relationship was disclosed between MGS and nuclear vv (2P = 0.85). Single-factor analysis showed prognostic impact of clinical stage of disease (2P = 0.0001) and DI (2P = 0.04), whereas estimates...... of nuclear vv were only of marginal prognostic significance (2P = 0.07). However, Cox multivariate regression analysis showed independent prognostic value of patient age and nuclear vv along with clinical stage and DI. All other investigated variables were rejected from the model. A prognostic index...

  10. Nuclear glutathione.

    Science.gov (United States)

    García-Giménez, José Luis; Markovic, Jelena; Dasí, Francisco; Queval, Guillaume; Schnaubelt, Daniel; Foyer, Christine H; Pallardó, Federico V

    2013-05-01

    Glutathione (GSH) is a linchpin of cellular defences in plants and animals with physiologically-important roles in the protection of cells from biotic and abiotic stresses. Moreover, glutathione participates in numerous metabolic and cell signalling processes including protein synthesis and amino acid transport, DNA repair and the control of cell division and cell suicide programmes. While it is has long been appreciated that cellular glutathione homeostasis is regulated by factors such as synthesis, degradation, transport, and redox turnover, relatively little attention has been paid to the influence of the intracellular partitioning on glutathione and its implications for the regulation of cell functions and signalling. We focus here on the functions of glutathione in the nucleus, particularly in relation to physiological processes such as the cell cycle and cell death. The sequestration of GSH in the nucleus of proliferating animal and plant cells suggests that common redox mechanisms exist for DNA regulation in G1 and mitosis in all eukaryotes. We propose that glutathione acts as "redox sensor" at the onset of DNA synthesis with roles in maintaining the nuclear architecture by providing the appropriate redox environment for the DNA replication and safeguarding DNA integrity. In addition, nuclear GSH may be involved in epigenetic phenomena and in the control of nuclear protein degradation by nuclear proteasome. Moreover, by increasing the nuclear GSH pool and reducing disulfide bonds on nuclear proteins at the onset of cell proliferation, an appropriate redox environment is generated for the stimulation of chromatin decompaction. This article is part of a Special Issue entitled Cellular functions of glutathione. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Stereological quantification of tumor volume, mean nuclear volume and total number of melanoma cells correlated with morbidity and mortality

    DEFF Research Database (Denmark)

    Bønnelykke-Behrndtz, Marie Louise; Sørensen, Flemming Brandt; Damsgaard, Tine Engberg

    2008-01-01

    potential indicators of prognosis. Sixty patients who underwent surgery at the Department of Plastic Surgery, Aarhus University Hospital, from 1991 to 1994 were included in the study. Total tumor volume was estimated by the Cavalieri technique, total number of tumor cells by the optical dissector principle...... showed a significant impact on both disease-free survival (p=0.001) and mortality (p=0.009). In conclusion, tumor volume and total number of cancer cells were highly reproducible but did not add additional, independent prognostic information regarding the study population.......Stereological quantification of tumor volume, total number of tumor cells and mean nuclear volume provides unbiased data, regardless of the three-dimensional shape of the melanocytic lesion. The aim of the present study was to investigate whether these variables are reproducible and may represent...

  12. Nuclear halo from Bradysia hygida (Diptera:Sciaridae salivary gland polytene cells

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    Celso Aparecido Polinarski

    2005-05-01

    Full Text Available A protocol for recovered nuclear halos from insect polytene nuclei after the extraction of the nuclear proteins using LIS detergent is reported in this work. Analysis was carried out using fluorescence and confocal laser scan microscopy. The extraction of nuclear halos was possible only with nuclei-fraction isolation in hypotonic buffer without spermine and spermidine. The recovered nuclear halos from Bradysia hygida salivary gland polytene nuclei, contributed greatly to the study of the structure and function of these special organelles.Observações à microscopia eletrônica e estudos bioquímicos de cromossomos e núcleos sem histonas tem suportado a hipótese que o DNA de eucariotos é organizado em alças associadas com o esqueleto cromossômico ou à matriz nuclear. A observação da matriz nuclear sem a remoção do DNA, através da digestão com enzimas de restrição, apresenta uma figura em halo que representa a liberação das alças de DNA. Um protocolo para a obtenção de halos nucleares de núcleos politênicos de insetos, através da extração de proteínas usando o detergente LIS, é reportado nesse trabalho. Foram realizadas análises utilizando-se microscopia de fluorescência e microscopia de varredura confocal a laser. A extração de halos nucleares foi possível somente com o isolamento da fração nuclear em tampão sem espermina e espermidina. A obtenção de halos nucleares de núcleos politênicos de glândula salivar de Bradysia hygida contribui significativamente para o estudo da estrutura e função dessas organelas tão especiais.

  13. Nuclear translocation of β-catenin during mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.

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    Carmen Herencia

    Full Text Available Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.

  14. Effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells

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    O. H. Minchenko

    2016-06-01

    Full Text Available We have studied the effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells under the inhibition of IRE1 (inositol requiring enzyme-1, which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. It was shown that hypoxia down-regulated gene expression of malate dehydrogenase 2 (MDH2, malic enzyme 2 (ME2, mitochondrial aspartate aminotransferase (GOT2, and subunit B of succinate dehydrogenase (SDHB in control (transfected by empty vector glioma cells in a gene specific manner. At the same time, the expression level of mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2 and subunit D of succinate dehydrogenase (SDHD genes in these cells does not significantly change in hypoxic conditions. It was also shown that the inhibition of ІRE1 signaling enzyme function in U87 glioma cells decreases the effect of hypoxia on the expression of ME2, GOT2, and SDHB genes and introduces the sensitivity of IDH2 gene to hypoxia. Furthermore, the expression of all studied genes depends on IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner, because ІRE1 knockdown significantly decreases their expression in normoxic conditions, except for IDH2 gene, which expression level is strongly up-regulated. Therefore, changes in the expression level of nuclear genes encoding ME2, MDH2, IDH2, SDHB, SDHD, and GOT2 proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function.

  15. NuMA and nuclear lamins are cleaved during viral infection--inhibition of caspase activity prevents cleavage and rescues HeLa cells from measles virus-induced but not from rhinovirus 1B-induced cell death.

    Science.gov (United States)

    Taimen, Pekka; Berghäll, Heidi; Vainionpää, Raija; Kallajoki, Markku

    2004-03-01

    Nuclear matrix is a structural framework of important nuclear processes. We studied the effect of two different types of viral infections on nuclear matrix. HeLa cells were infected with human rhinovirus 1B (HRV 1B) or measles virus (MV), and Nuclear Mitotic Apparatus protein (NuMA) and lamins A/C and B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. We show that NuMA, lamins, and poly(ADP-ribose) polymerase-1 are cleaved during viral infection in a virus family-specific manner suggesting that these viruses activate different sets of proteases. Morphologically, NuMA was excluded from the condensed chromatin, lamins showed a folded distribution, and both proteins finally remained around the nuclear fragments. A general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) prevented the nuclear disintegration and the cleavage of the proteins studied. Interestingly, z-VAD-FMK rescued MV-infected but not HRV 1B-infected cells from cell death. These results show for the first time that NuMA and lamins are specific target proteins during virus-induced programmed cell death.

  16. NuMA and nuclear lamins are cleaved during viral infection - inhibition of caspase activity prevents cleavage and rescues HeLa cells from measles virus-induced but not from rhinovirus 1B-induced cell death

    International Nuclear Information System (INIS)

    Taimen, Pekka; Berghaell, Heidi; Vainionpaeae, Raija; Kallajoki, Markku

    2004-01-01

    Nuclear matrix is a structural framework of important nuclear processes. We studied the effect of two different types of viral infections on nuclear matrix. HeLa cells were infected with human rhinovirus 1B (HRV 1B) or measles virus (MV), and Nuclear Mitotic Apparatus protein (NuMA) and lamins A/C and B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. We show that NuMA, lamins, and poly(ADP-ribose) polymerase-1 are cleaved during viral infection in a virus family-specific manner suggesting that these viruses activate different sets of proteases. Morphologically, NuMA was excluded from the condensed chromatin, lamins showed a folded distribution, and both proteins finally remained around the nuclear fragments. A general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) prevented the nuclear disintegration and the cleavage of the proteins studied. Interestingly, z-VAD-FMK rescued MV-infected but not HRV 1B-infected cells from cell death. These results show for the first time that NuMA and lamins are specific target proteins during virus-induced programmed cell death

  17. NUCLEAR TRANSFER IN TIER CELLS OF BOSTRYCHIA-RADICANS (RHODOMELACEAE, RHODOPHYTA)

    NARCIS (Netherlands)

    GOFF, LJ; WEST, JA; OLSEN, JL

    1992-01-01

    In the male gametophytes of the marine alga Bostrychia radicans (Montagne) Montagne (Rhodomelaceae, Rhodophyta), axial cells and the initial pericentral cells are uninucleate (4C DNA value). Each pericentral cell of axial segment 5 cuts off a uninucleate (2C) tier cell from the upper surface. In

  18. The C-terminal subunit of artificially truncated human cathepsin B mediates its nuclear targeting and contributes to cell viability

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    Dallner Claudia

    2005-04-01

    Full Text Available Abstract Background Splicing variants of human cathepsinB primary transcripts (CB(-2,3 result in an expression product product which lacks the signal peptide and parts of the propeptide. This naturally truncated Δ51CB is thus unable to follow the regular CB processing and sorting pathway. It is addressed to the mitochondria through an activated N-terminal mitochondrial targeting signal instead. Although Δ51CB is supposed to be devoid of the typical CB enzymatic activity, it might play a role in malignancies and trigger cell death/apoptosis independent from the function of the regular enzyme. Cytoplasmic presence of the mature CB might occur as a result of lysosomal damage. Results We investigated such "aberrant" proteins by artificial CB-GFP chimeras covering various sequence parts in respect to their enzymatic activity, their localization in different cell types, and the effects on the cell viability. Unlike the entire full length CB form, the artificial single chain form was not processed and did not reveal typical enzymatic CB activity during transient overexpression in large cell lung carcinoma cells. Δ51CB was found predominantly in mitochondria. In contrast, the shorter artificial CB constructs localized in the cytoplasm, inside the cell nucleus, and in the midbodies of dividing cells. Bleaching experiments revealed both mobile and immobile fractions of these constructs in the nucleus. Nuclear accumulation of artificially truncated CB variants led to disintegration of nuclei, followed by cell death. Conclusion We propose that cell death associated with CB is not necessarily triggered by its regular enzymatic activity but alternatively by a yet unknown activity profile of truncated CB. Cytoplasmic CB might be able to enter the cell nucleus. According to a mutational analysis, the part of CB that mediates its nuclear import is a signal patch within its heavy chain domain. The results suggest that besides the N-terminal signal peptide also

  19. In vitro angiogenesis and expression of nuclear factor κB and VEGF in high and low metastasis cell lines of salivary gland Adenoid Cystic Carcinoma

    International Nuclear Information System (INIS)

    Zhang, Jiali; Peng, Bin

    2007-01-01

    Adenoid cystic carcinoma is a high malignant carcinoma characterized by intensive local invasion and high incidence of distant metastasis. Although many reports have demonstrated that angiogenesis has played an important role in tumor metastasis, the relationship between metastasis characters and angiogenesis ability in high and low metastasis cell lines of Adenoid cystic carcinoma has rarely been reported. The present study aimed to compare the angiogenesis ability of ACC-M (high metastasis) and ACC-2 (low metastasis) cell lines in vitro. Furthermore, the activity of nuclear factor κappa B and the expression of vascular endothelial growth factor (VEGF) in ACC-2 and ACC-M were also detected. Electrophoretic mobility shift assay was used to detect nuclear factor κappa B activity. Semi-quantitative RT-PCR was used to quantify the mRNA level of VEGF. Immuofluorescence double staining and semi-quantitative confocal laser scanning analysis was carried out to detect nuclear factor κappa B nuclear localization and staining intensity of VEGF. The angiogenesis ability of ACC-M and ACC-2 was compared by an in vitro three-dimensional angiogenic model assay. The vector transfection assay was performed to transfect the PCMV-IκBαM vector into ACCs cell lines expressing the phosphorylation defective IκBαM. Nuclear factor κappa B activity and the rate of nuclear factor κappa B nuclear localization in ACC-M was significantly higher than that in ACC-2. Moreover, ACC-M exhibited higher mRNA and protein levels of vascular endothelial growth factor than ACC-2. VEGF mRNA expression was effectively decreased by inhibition of nuclear factor κappa B activity. Furthermore, ACC-M could remarkably stimulate the migration and tube formation of endothelial cells and induce The umbilical vein endothelial cells sprouting into the gel matrix. These results implicated that ACCs cells with higher metastasis feature might present greater angiogenesis ability

  20. Regulated Necrosis in HeLa Cells Induced by ZnPc Photodynamic Treatment: A New Nuclear Morphology

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    Jorge Soriano

    2014-12-01

    Full Text Available Photodynamic therapy (PDT is a cancer treatment modality based on the administration of a photosensitizer (PS, which accumulates preferentially in tumor cells. Subsequent irradiation of the neoplastic area triggers a cascade of photochemical reactions that leads to the formation of highly reactive oxygen species responsible for cell inactivation. Photodynamic treatments in vitro are performed with the PS, zinc-phthalocyanine (ZnPc. The PS is near the plasma membrane during uptake and internalization. Inactivation clearly occurs by a necrotic process, manifested by nuclear pyknosis, negative TUNEL and Annexin V assays and non-relocation of cytochrome c. In contrast, by increasing the incubation time, ZnPc is accumulated in the Golgi apparatus and produces cell inactivation with characteristics of apoptosis and necrosis: TUNEL positive, relocated cytochrome c and negative Annexin V assay. This type of death produces a still undescribed granulated nuclear morphology, which is different from that of necrosis or apoptosis. This morphology is inhibited by necrostatin-1, a specific inhibitor of regulated necrosis.

  1. Suppression of the Nuclear Factor Eny2 Increases Insulin Secretion in Poorly Functioning INS-1E Insulinoma Cells

    Directory of Open Access Journals (Sweden)

    P. Dames

    2012-01-01

    Full Text Available Eny2, the mammalian ortholog of yeast Sus1 and drosophila E(y2, is a nuclear factor that participates in several steps of gene transcription and in mRNA export. We had previously found that Eny2 expression changes in mouse pancreatic islets during the metabolic adaptation to pregnancy. We therefore hypothesized that the protein contributes to the regulation of islet endocrine cell function and tested this hypothesis in rat INS-1E insulinoma cells. Overexpression of Eny2 had no effect but siRNA-mediated knockdown of Eny2 resulted in markedly increased glucose and exendin-4-induced insulin secretion from otherwise poorly glucose-responsive INS-1E cells. Insulin content, cellular viability, and the expression levels of several key components of glucose sensing remained unchanged; however glucose-dependent cellular metabolism was higher after Eny2 knockdown. Suppression of Eny2 enhanced the intracellular incretin signal downstream of cAMP. The use of specific cAMP analogues and pathway inhibitors primarily implicated the PKA and to a lesser extent the EPAC pathway. In summary, we identified a potential link between the nuclear protein Eny2 and insulin secretion. Suppression of Eny2 resulted in increased glucose and incretin-induced insulin release from a poorly glucose-responsive INS-1E subline. Whether these findings extend to other experimental conditions or to in vivo physiology needs to be determined in further studies.

  2. Novel computer-aided diagnosis of mesothelioma using nuclear structure of mesothelial cells in effusion cytology specimens

    Science.gov (United States)

    Tosun, Akif Burak; Yergiyev, Oleksandr; Kolouri, Soheil; Silverman, Jan F.; Rohde, Gustavo K.

    2014-03-01

    diagnostic standard is a pleural biopsy with subsequent histologic examination of the tissue demonstrating invasion by the tumor. The diagnostic tissue is obtained through thoracoscopy or open thoracotomy, both being highly invasive procedures. Thoracocenthesis, or removal of effusion fluid from the pleural space, is a far less invasive procedure that can provide material for cytological examination. However, it is insufficient to definitively confirm or exclude the diagnosis of malignant mesothelioma, since tissue invasion cannot be determined. In this study, we present a computerized method to detect and classify malignant mesothelioma based on the nuclear chromatin distribution from digital images of mesothelial cells in effusion cytology specimens. Our method aims at determining whether a set of nuclei belonging to a patient, obtained from effusion fluid images using image segmentation, is benign or malignant, and has a potential to eliminate the need for tissue biopsy. This method is performed by quantifying chromatin morphology of cells using the optimal transportation (Kantorovich-Wasserstein) metric in combination with the modified Fisher discriminant analysis, a k-nearest neighborhood classification, and a simple voting strategy. Our results show that we can classify the data of 10 different human cases with 100% accuracy after blind cross validation. We conclude that nuclear structure alone contains enough information to classify the malignant mesothelioma. We also conclude that the distribution of chromatin seems to be a discriminating feature between nuclei of benign and malignant mesothelioma cells.

  3. Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage

    Directory of Open Access Journals (Sweden)

    F.D. Oliveira

    2015-12-01

    Full Text Available In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1 of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles.

  4. Nuclear Factor Erythroid 2 Regulates Human HSC Self-Renewal and T Cell Differentiation by Preventing NOTCH1 Activation

    Directory of Open Access Journals (Sweden)

    Alessandro Di Tullio

    2017-07-01

    Full Text Available Nuclear factor erythroid-derived 2 (NF-E2 has been associated with megakaryocyte maturation and platelet production. Recently, an increased in NF-E2 activity has been implicated in myeloproliferative neoplasms. Here, we investigate the role of NF-E2 in normal human hematopoiesis. Knockdown of NF-E2 in the hematopoietic stem and progenitor cells (HSPCs not only reduced the formation of megakaryocytes but also drastically impaired hematopoietic stem cell activity, decreasing human engraftment in immunodeficient (NSG mice. This phenotype is likely to be related to both increased cell proliferation (p21-mediated and reduced Notch1 protein expression, which favors HSPC differentiation over self-renewal. Strikingly, although NF-E2 silencing in HSPCs did not affect their myeloid and B cell differentiation in vivo, it almost abrogated T cell production in primary hosts, as confirmed by in vitro studies. This effect is at least partly due to Notch1 downregulation in NF-E2-silenced HSPCs. Together these data reveal that NF-E2 is an important driver of human hematopoietic stem cell maintenance and T lineage differentiation.

  5. Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

    International Nuclear Information System (INIS)

    Gao Yanhong; Yue Wen; Zhang Peng; Li Li; Xie Xiaoyan; Yuan Hongfeng; Chen Lin; Liu Daqing; Yan Fang; Pei Xuetao

    2005-01-01

    spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G 2 /M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis

  6. Conserved cell cycle regulatory properties within the amino terminal domain of the Epstein-Barr virus nuclear antigen 3C

    International Nuclear Information System (INIS)

    Sharma, Nikhil; Knight, Jason S.; Robertson, Erle S.

    2006-01-01

    The gammaherpesviruses Rhesus lymphocryptovirus (LCV) and Epstein-Barr virus (EBV) are closely related phylogenetically. Rhesus LCV efficiently immortalizes Rhesus B cells in vitro. However, despite a high degree of conservation between the Rhesus LCV and EBV genomes, Rhesus LCV fails to immortalize human B cells in vitro. This species restriction may, at least in part, be linked to the EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), known to be essential for B cell transformation. We compared specific properties of EBNA3C, a well-characterized and essential EBV protein, with its Rhesus counterpart to determine whether EBNA3C phenotypes which contribute to cell cycle regulation are conserved in the Rhesus LCV. We show that both EBNA3C and Rhesus EBNA3C bind to a conserved region of mammalian cyclins, regulate pRb stability, and modulate SCF Skp2 -dependent ubiquitination. These results suggest that Rhesus LCV restriction from human B cell immortalization is independent of the conserved cell cycle regulatory functions of the EBNA3C protein

  7. Nuclear Factor Erythroid 2 Regulates Human HSC Self-Renewal and T Cell Differentiation by Preventing NOTCH1 Activation.

    Science.gov (United States)

    Di Tullio, Alessandro; Passaro, Diana; Rouault-Pierre, Kevin; Purewal, Sukhveer; Bonnet, Dominique

    2017-07-11

    Nuclear factor erythroid-derived 2 (NF-E2) has been associated with megakaryocyte maturation and platelet production. Recently, an increased in NF-E2 activity has been implicated in myeloproliferative neoplasms. Here, we investigate the role of NF-E2 in normal human hematopoiesis. Knockdown of NF-E2 in the hematopoietic stem and progenitor cells (HSPCs) not only reduced the formation of megakaryocytes but also drastically impaired hematopoietic stem cell activity, decreasing human engraftment in immunodeficient (NSG) mice. This phenotype is likely to be related to both increased cell proliferation (p21-mediated) and reduced Notch1 protein expression, which favors HSPC differentiation over self-renewal. Strikingly, although NF-E2 silencing in HSPCs did not affect their myeloid and B cell differentiation in vivo, it almost abrogated T cell production in primary hosts, as confirmed by in vitro studies. This effect is at least partly due to Notch1 downregulation in NF-E2-silenced HSPCs. Together these data reveal that NF-E2 is an important driver of human hematopoietic stem cell maintenance and T lineage differentiation. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Nuclear reprogramming of somatic nucleus hybridized with embryonic stem cells by electrofusion.

    Science.gov (United States)

    Tada, Masako; Tada, Takashi

    2006-01-01

    Cell fusion is a powerful tool for understanding the molecular mechanisms of epigenetic reprogramming. In hybrid cells of somatic cells and pluripotential stem cells, including embryonic stem (ES) and embryonic germ cells, somatic nuclei acquire pluripotential competence. ES and embryonic germ cells retain intrinsic trans activity to induce epigenetic reprogramming. For generating hybrid cells, we have used the technique of electrofusion. Electrofusion is a highly effective, reproducible, and biomedically safe in vitro system. For successful cell fusion, two sequential steps of electric pulse stimulation are required for the alignment (pearl chain formation) of two different types of cells between electrodes in response to alternating current stimulation and for the fusion of cytoplasmic membranes by direct current stimulation. Optimal conditions for electrofusion with a pulse generator are introduced for ES and somatic cell fusion. Topics in the field of stem cell research include the successful production of cloned animals via the epigenetic reprogramming of somatic cells and contribution of spontaneous cell fusion to generating intrinsic plasticity of tissue stem cells. Cell fusion technology may make important contributions to the fields of epigenetic reprogramming and regenerative medicine.

  9. Ultrastructure of cytoplasmic and nuclear changes in Eimeria tenella during first-generation schizogony in cell culture.

    Science.gov (United States)

    Pacheco, N D; Vetterling, J M; Doran, D J

    1975-02-01

    Eimeria tenella sporozoites were inoculated into primary cultures of chick kidney cells. Cells fixed from 1 1/2 to 54 hr later were examined with the electron microscope. At 1 1/2 and 24 hr, most intracellular sporozoites were fusiform and retained organelles typical of extracellular sporozoites. However, at 35 hr, rounded trophozoites were present without these structures; only a refractile body, nucleus, mitochondria, and endoplasmic reticulum remained. Binucleate parasites were also present at that time, but at 48 hr many multinucleate schizonts were present. Nuclei, with adjacent conoids, were at the periphery of these schizonts. Partly developed merozoites, each containing a conoid and a nucleus, protruded into the parasitophorous vacuole. At 54 hr, fully developed merozoites were separated from the residual body. Merozoites resembled sporozoites but lacked the large refractile bodies seen in sporozoites. Linear inclusions were present near the merozoite nucleus and in the residual body. Round vacuoles and ribosomes were also found in the residuum. Nucleoli were first seen in sporozoite nuclei at 1 1/2 hr. They were also present in merozoites but were more prominent in trophozoites and schizonts. Peripheral and scattered nuclear heterochromatins were prominent in intracellular sporozoites and diminished in trophozoites, but increased after several nuclear divisions and were again prominent in the merozoite. Small, distinct interchromatin granules were found in all stages. Intranuclear spindles, centrocones, and centrioles were found in connection with nuclear divisions. Ultrastructure of first-generation schizogony in cell culture was similar to that described for second-generation E. tenella in the chicken and to schizogony of other species of Eimeria.

  10. Some nuclear chemical aspects of medical generator nuclide production at the Los Alamos hot cell facility

    CERN Document Server

    Fassbender, M; Heaton, R C; Jamriska, D J; Kitten, J J; Nortier, F M; Peterson, E J; Phillips, D R; Pitt, L R; Salazar, L L; Valdez, F O; 10.1524/ract.92.4.237.35596

    2004-01-01

    Generator nuclides constitute a convenient tool for applications in nuclear medicine. In this paper, some radiochemical aspects of generator nuclide parents regularly processed at Los Alamos are introduced. The bulk production of the parent nuclides /sup 68/Ge, /sup 82/Sr, /sup 109/Cd and /sup 88/Zr using charged particle beams is discussed. Production nuclear reactions for these radioisotopes, and chemical separation procedures are presented. Experimental processing yields correspond to 80%-98% of the theoretical thick target yield. Reaction cross sections are modeled using the code ALICE-IPPE; it is observed that the model largely disagrees with experimental values for the nuclear processes treated. Radionuclide production batches are prepared 1-6 times yearly for sales. Batch activities range from 40MBq to 75 GBq.

  11. GCR Transport in the Brain: Assessment of Self-Shielding, Columnar Damage, and Nuclear Reactions on Cell Inactivation Rates

    Science.gov (United States)

    Shavers, M. R.; Atwell, W.; Cucinotta, F. A.; Badhwar, G. D. (Technical Monitor)

    1999-01-01

    Radiation shield design is driven by the need to limit radiation risks while optimizing risk reduction with launch mass/expense penalties. Both limitation and optimization objectives require the development of accurate and complete means for evaluating the effectiveness of various shield materials and body-self shielding. For galactic cosmic rays (GCR), biophysical response models indicate that track structure effects lead to substantially different assessments of shielding effectiveness relative to assessments based on LET-dependent quality factors. Methods for assessing risk to the central nervous system (CNS) from heavy ions are poorly understood at this time. High-energy and charge (HZE) ion can produce tissue events resulting in damage to clusters of cells in a columnar fashion, especially for stopping heavy ions. Grahn (1973) and Todd (1986) have discussed a microlesion concept or model of stochastic tissue events in analyzing damage from HZE's. Some tissues, including the CNS, maybe sensitive to microlesion's or stochastic tissue events in a manner not illuminated by either conventional dosimetry or fluence-based risk factors. HZE ions may also produce important lateral damage to adjacent cells. Fluences of high-energy proton and alpha particles in the GCR are many times higher than HZE ions. Behind spacecraft and body self-shielding the ratio of protons, alpha particles, and neutrons to HZE ions increases several-fold from free-space values. Models of GCR damage behind shielding have placed large concern on the role of target fragments produced from tissue atoms. The self-shielding of the brain reduces the number of heavy ions reaching the interior regions by a large amount and the remaining light particle environment (protons, neutrons, deuterons. and alpha particles) may be the greatest concern. Tracks of high-energy proton produce nuclear reactions in tissue, which can deposit doses of more than 1 Gv within 5 - 10 cell layers. Information on rates of

  12. Cbf11 and Cbf12, the fission yeast CSL proteins, play opposing roles in cell adhesion and coordination of cell and nuclear division

    Energy Technology Data Exchange (ETDEWEB)

    Prevorovsky, Martin; Grousl, Tomas; Stanurova, Jana; Rynes, Jan [Department of Cell Biology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 43, Prague 2 (Czech Republic); Nellen, Wolfgang [Department of Genetics, Kassel University, Heinrich Plett Strasse 40, 34132 Kassel (Germany); Puta, Frantisek [Department of Cell Biology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 43, Prague 2 (Czech Republic); Folk, Petr, E-mail: folk@natur.cuni.cz [Department of Cell Biology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 43, Prague 2 (Czech Republic)

    2009-05-01

    The CSL (CBF1/RBP-J{kappa}/Suppressor of Hairless/LAG-1) family is comprised of transcription factors essential for metazoan development, mostly due to their involvement in the Notch receptor signaling pathway. Recently, we identified two novel classes of CSL genes in the genomes of several fungal species, organisms lacking the Notch pathway. In this study, we characterized experimentally cbf11{sup +} and cbf12{sup +}, the two CSL genes of Schizosaccharomyces pombe, in order to elucidate the CSL function in fungi. We provide evidence supporting their identity as genuine CSL genes. Both cbf11{sup +} and cbf12{sup +} are non-essential; they have distinct expression profiles and code for nuclear proteins with transcription activation potential. Significantly, we demonstrated that Cbf11 recognizes specifically the canonical CSL response element GTG{sup A}/{sub G}GAA in vitro. The deletion of cbf11{sup +} is associated with growth phenotypes and altered colony morphology. Furthermore, we found that Cbf11 and Cbf12 play opposite roles in cell adhesion, nuclear and cell division and their coordination. Disturbed balance of the two CSL proteins leads to cell separation defects (sep phenotype), cut phenotype, and high-frequency diploidization in heterothallic strains. Our data show that CSL proteins operate in an organism predating the Notch pathway, which should be of relevance to the understanding of (Notch-independent) CSL functions in metazoans.

  13. Cbf11 and Cbf12, the fission yeast CSL proteins, play opposing roles in cell adhesion and coordination of cell and nuclear division

    International Nuclear Information System (INIS)

    Prevorovsky, Martin; Grousl, Tomas; Stanurova, Jana; Rynes, Jan; Nellen, Wolfgang; Puta, Frantisek; Folk, Petr

    2009-01-01

    The CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) family is comprised of transcription factors essential for metazoan development, mostly due to their involvement in the Notch receptor signaling pathway. Recently, we identified two novel classes of CSL genes in the genomes of several fungal species, organisms lacking the Notch pathway. In this study, we characterized experimentally cbf11 + and cbf12 + , the two CSL genes of Schizosaccharomyces pombe, in order to elucidate the CSL function in fungi. We provide evidence supporting their identity as genuine CSL genes. Both cbf11 + and cbf12 + are non-essential; they have distinct expression profiles and code for nuclear proteins with transcription activation potential. Significantly, we demonstrated that Cbf11 recognizes specifically the canonical CSL response element GTG A / G GAA in vitro. The deletion of cbf11 + is associated with growth phenotypes and altered colony morphology. Furthermore, we found that Cbf11 and Cbf12 play opposite roles in cell adhesion, nuclear and cell division and their coordination. Disturbed balance of the two CSL proteins leads to cell separation defects (sep phenotype), cut phenotype, and high-frequency diploidization in heterothallic strains. Our data show that CSL proteins operate in an organism predating the Notch pathway, which should be of relevance to the understanding of (Notch-independent) CSL functions in metazoans.