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Telomere dysfunction and cell survival: Roles for distinct TIN2-containing complexes

Description: Telomeres are maintained by three DNA binding proteins (TRF1, TRF2 and POT1), and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. Along with two other proteins, TPP1 and hRap1, these form a soluble complex that may be the core telomere maintenance complex. It is not clear whether sub-complexes also exist in vivo. We provide evidence for two TIN2 sub-complexes with distinct functions in human cells. We isolated these two TIN2 sub-complexes from nuclear lysates of unperturbed cells and cells expressing TIN2 mutants TIN2-13, TIN2-15C, which cannot bind TRF2 or TRF1, respectively. In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere dysfunction and cell death. Our findings suggest that distinct TIN2 complexes exist, and that TIN2-15C-sensitive subcomplexes are particularly important for cell survival in the absence of functional p53.
Date: October 2, 2007
Creator: Kim, Sahn-ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Zou, Ying; Beausejour, Christian et al.
Partner: UNT Libraries Government Documents Department

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP

Description: Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP.
Date: April 29, 2008
Creator: Stenerlöw, Bo; Karlsson, Karin H.; Radulescu, Irina; Rydberg, Bjorn & Stenerlow, Bo
Partner: UNT Libraries Government Documents Department

Particle Suspension Mechanisms - Supplemental Material

Description: This supplemental material provides a brief introduction to particle suspension mechanisms that cause exfoliated skin cells to become and remain airborne. The material presented here provides additional context to the primary manuscript and serves as background for designing possible future studies to assess the impact of skin cells as a source of infectious aerosols. This introduction is not intended to be comprehensive and interested readers are encouraged to consult the references cited.
Date: March 3, 2011
Creator: Dillon, M B
Partner: UNT Libraries Government Documents Department

Molecular Recognition of DNA Damage Sites by Apurinic/Apyrimidinic Endonucleases

Description: The DNA repair/redox factor AP endonuclease 1 (APE1) is a multifunctional protein which is known to to be essential for DNA repair activity in human cells. Structural/functional analyses of the APE activity is thus been an important research field to assess cellular defense mechanisms against ionizing radiation.
Date: July 28, 2005
Creator: Braun, W. A.
Partner: UNT Libraries Government Documents Department

Effects of Irradiation on the Intestinal Cell Population

Description: The radiation syndrome that kills mice in 3.5 days was found to be mainly due to irradiation of the small intestine. Radiation injuries were defined by anatomical fractionation following irradiation first of the entire body region, then of smaller areas, and finally of surgically exposed individual organs. Irradiation with 1200 rad or more of any region that includes the entire small intestine resulted in death at precisely the same time as irradiation of the whole body. Irradiation of any major fraction of the bowel alone resulted in death under the same circumstances but at a slightly later time, and irradiation of the entire body minus the protected surgically exposed small intestine did not cause a comparable syndrome. The sequence of histological changes in the intestinal epithelium after irradiation is described and cell population kinetics in the irradiated animal is discussed. (C.H.)
Date: January 1, 1963
Creator: Quastler, H.
Partner: UNT Libraries Government Documents Department

Telomere dysfunction and cell survival: roles for distinctTIN2-containing complexes

Description: Telomeres are maintained by three DNA binding proteins, TRF1, TRF2 and POT1, and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. These and two other proteins form a soluble complex that may be the core telomere-maintenance complex. It is not clear whether subcomplexes exist or function in vivo. Here, we provide evidence for two TIN2 subcomplexes with distinct functions in human cells. TIN2 ablation by RNA interference caused telomere uncapping and p53-independent cell death in all cells tested. However, we isolated two TIN2 complexes from cell lysates, each selectively sensitive to a TIN2 mutant (TIN2-13, TIN2-15C). In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN215C more than TIN2-13 caused genomic instability and cell death. Thus, TIN2 subcomplexes likely have distinct functions in telomere maintenance, and may provide selective targets for eliminating cells with mutant p53.
Date: November 7, 2006
Creator: Kim, Sahn-Ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Beausejour, Christian; Kaminker, Patrick et al.
Partner: UNT Libraries Government Documents Department

Promotion of Homologous Recombination and Genomic Stability byRAD51AP1 via RAD51 Recombinase Enhancement

Description: Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds dsDNA and RAD51, and it greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide the first evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.
Date: April 11, 2007
Creator: Wiese, Claudia; Dray, Eloise; Groesser, Torsten; San Filippo,Joseph; Shi, Idina; Collins, David W. et al.
Partner: UNT Libraries Government Documents Department

BEST: Bilingual environmental science training: Kindergarten level

Description: This booklet is one of a series of bilingual guides to environmental-science learning activities for students to do at home. Lesson objectives, materials required, procedure, vocabulary, and subjects integrated into the lesson are described in English for each lesson. A bilingual glossary, alphabetized by English entries, with Spanish equivalents in both English and Spanish, follows the lesson descriptions, and is itself followed by a bibliography of English-language references. This booklet includes descriptions of six lessons covering the senses of touch and sight, the sense of smell, how to distinguish living and non-living things, cell structures, the skeletal system, and the significance of food groups. 8 figs.
Date: March 1, 1996
Partner: UNT Libraries Government Documents Department

Direct methods for dynamic monitoring of secretions from single cells by capillary electrophoresis and microscopy with laser-induced native fluorescence detection

Description: Microscale separation and detection methods for real-time monitoring of dynamic cellular processes (e.g., secretion) by capillary electrophoresis (CE) and microscopic imaging were developed. Ultraviolet laser-induced native fluorescence (LINF) provides simple, sensitive and direct detection of neurotransmitters and proteins without any derivatization. An on-column CE-LINF protocol for quantification of the release from single cell was demonstrated. Quantitative measurements of both the amount of insulin released from and the amount remaining in the cell ({beta}TC3) were achieved simultaneously. Secretion of catecholamines (norepinephrine (NE) and epinephrine (E)) from individual bovine adrenal chromaffin cells was determined using the on-column CE-LINF. Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved by LINF imaging microscopy with high temporal and spatial resolution. The secretion of serotonin from individual leech Retzius neurons was directly characterized by LINF microscopy with high spatial resolution.
Date: October 8, 1997
Creator: Tong, W.
Partner: UNT Libraries Government Documents Department

Ionizing Radiation-Induced DNA Damage and Its Repair in Human Cells

Description: DNA damage in mammalian chromatin in vitro and in cultured mammalian cells including human cells was studied. In the first phase of these studies, a cell culture laboratory was established. Necessary equipment including an incubator, a sterile laminar flow hood and several centrifuges was purchased. We have successfully grown several cell lines such as murine hybridoma cells, V79 cells and human K562 leukemia cells. This was followed by the establishment of a methodology for the isolation of chromatin from cells. This was a very important step, because a routine and successful isolation of chromatin was a prerequisite for the success of the further studies in this project, the aim of which was the measurement of DNA darnage in mammalian chromatin in vitro and in cultured cells. Chromatin isolation was accomplished using a slightly modified procedure of the one described by Mee & Adelstein (1981). For identification and quantitation of DNA damage in cells, analysis of chromatin was preferred over the analysis of "naked DNA" for the following reasons: i. DNA may not be extracted efficiently from nucleoprotein in exposed cells, due to formation of DNA-protein cross-links, ii. the extractability of DNA is well known to decrease with increasing doses of radiation, iii. portions of DNA may not be extracted due to fragmentation, iv. unextracted DNA may contain a significant portion of damaged DNA bases and DNA-protein cross-links. The technique of gas chromatography/mass spectrometry (GC/MS), which was used in the present project, permits the identification and quantitation of modified DNA bases in chromatin in the presence of proteins without the necessity of first isolating DNA from chromatin. This has been demonstrated previously by the results from our laboratory and by the results obtained during the course of the present project. The quality of isolated chromatin was tested by measurement of its ...
Date: May 12, 1999
Creator: Dizdaroglu, Miral
Partner: UNT Libraries Government Documents Department

1999 Gordon Research Conference on Mammalian DNA Repair. Final Progress Report

Description: This Conference will examine DNA repair as the key component in genomic surveillance that is so crucial to the overall integrity and function of mammalian cells. Recent discoveries have catapulted the field of DNA repair into a pivotal position for fundamental investigations into oncology, aging, environmental health, and developmental biology. We hope to highlight the most promising and exciting avenues of research in robust discussions at this conference. This Mammalian DNA Repair Gordon Conference differs from the past conferences in this series, in which the programs were broader in scope, with respect to topics and biological systems covered. A conference sponsored by the Genetics Society in April 1998 emphasized recombinational mechanisms for double-strand break repair and the role of mismatch repair deficiency in colorectal cancer. These topics will therefore receive somewhat less emphasis in the upcoming Conference. In view of the recent mechanistic advances in mammalian DNA repair, an upcoming comprehensive DNA repair meeting next autumn at Hilton Head; and the limited enrollment for Gordon Conferences we have decided to focus session-by-session on particular areas of controversy and/or new developments specifically in mammalian systems. Thus, the principal presentations will draw upon results from other cellular systems only to the extent that they impact our understanding of mammalian DNA repair.
Date: February 12, 1999
Partner: UNT Libraries Government Documents Department

Opto-acoustic cell permeation

Description: Optically generated acoustic waves have been used to temporarily permeate biological cells. This technique may be useful for enhancing transfection of DNA into cells or enhancing the absorption of locally delivered drugs. A diode-pumped frequency-doubled Nd:YAG laser operating at kHz repetition rates was used to produce a series of acoustic pulses. An acoustic wave was formed via thermoelastic expansion by depositing laser radiation into an absorbing dye. Generated pressures were measured with a PVDF hydrophone. The acoustic waves were transmitted to cultured and plated cells. The cell media contained a selection of normally- impermeable fluorescent-labeled dextran dyes. Following treatment with the opto-acoustic technique, cellular incorporation of dyes, up to 40,000 Molecular Weight, was noted. Control cells that did not receive opto-acoustic treatment had unremarkable dye incorporation. Uptake of dye was quantified via fluorescent microscopic analysis. Trypan Blue membrane exclusion assays and fluorescent labeling assays confirmed the vitality of cells following treatment. This method of enhanced drug delivery has the potential to dramatically reduce required drug dosages and associated side effects and enable revolutionary therapies.
Date: March 9, 2000
Creator: Visuri, S R & Heredia, N
Partner: UNT Libraries Government Documents Department

Regulation of cell division in higher plants. Final technical report

Description: Research in the latter part of the grant period was divided into two parts: (1) expansion of the macromolecular tool kit for studying plant cell division; (2) experiments in which the roles played by plant cell cycle regulators were to be cast in the light of the emerging yeast and animal cell paradigm for molecular control of the mitotic cycle. The first objectives were accomplished to a very satisfactory degree. With regard to the second part of the project, we were driven to change our objectives for two reasons. First, the families of cell cycle control genes that we cloned encoded such closely related members that the prospects for success at raising distinguishing antisera against each were sufficiently dubious as to be impractical. Epitope tagging is not feasible in Pisum sativum, our experimental system, as this species is not realistically transformable. Therefore, differentiating the roles of diverse cyclins and cyclin-dependent kinases was problematic. Secondly, our procedure for generating mitotically synchronized pea root meristems for biochemical studies was far too labor intensive for the proposed experiments. We therefore shifted our objectives to identifying connections between the conserved proteins of the cell cycle engine and factors that interface it with plant physiology and development. In this, we have obtained some very exciting results.
Date: February 29, 2000
Creator: Jacobs, Thomas W.
Partner: UNT Libraries Government Documents Department

CHO/HGPRT mutagenicity assay. III. Adaptation for mutagen screening

Description: This assay system has been employed for a variety of quantitative studies of mutation induction in mammalian cells (Hise et al., 1978). The development of interest in utilizing this system for mutagen screening has prompted us to evaluate those aspects of the system which necessitate the largest time and monetary investments, and to investigate the possibilities of applying insights gained from our basic research program to optimize use of the system for routine testing procedures. This paper describe our progress in the areas of phenotypic expression time and the density dependent recovery of mutant colonies in selective medium.
Date: January 1, 1979
Creator: O'Neill, J. P. & Hsie, A. W.
Partner: UNT Libraries Government Documents Department

A High-Throughput Microenvironment for Single-Cell Operations

Description: This project was conducted as a feasibility study, in preparation for including this work in the forthcoming ''Instrumented Cell'' (IC) Strategic Initiative. The goal of the IC is to study individual cells; the goal of this feasibility study was to determine the best method for isolating large numbers of individual cells in a way that facilitates various types of environmental changes and intracellular measurements. We have the capability to do this with one cell, and sought to expand the number of cells that we could study simultaneously. Our specific goal for this feasibility study was to discover a way to isolate individual cells, and impale them on a nanopipette. This would enable samples to be introduced into and removed from a cell.
Date: January 7, 2003
Creator: Christian, A T; Buckley, P & Miles, R R
Partner: UNT Libraries Government Documents Department