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DNA repair: Dynamic defenders against cancer and aging

Description: You probably weren't thinking about your body's cellular DNA repair systems the last time you sat on the beach in the bright sunshine. Fortunately, however, while you were subjecting your DNA to the harmful effects of ultraviolet light, your cells were busy repairing the damage. The idea that our genetic material could be damaged by the sun was not appreciated in the early days of molecular biology. When Watson and Crick discovered the structure of DNA in 1953 [1], it was assumed that DNA is fundamentally stable since it carries the blueprint of life. However, over 50 years of research have revealed that our DNA is under constant assault by sunlight, oxygen, radiation, various chemicals, and even our own cellular processes. Cleverly, evolution has provided our cells with a diverse set of tools to repair the damage that Mother Nature causes. DNA repair processes restore the normal nucleotide sequence and DNA structure of the genome after damage [2]. These responses are highly varied and exquisitely regulated. DNA repair mechanisms are traditionally characterized by the type of damage repaired. A large variety of chemical modifications can alter normal DNA bases and either lead to mutations or block transcription if not repaired, and three distinct pathways exist to remove base damage. Base excision repair (BER) corrects DNA base alterations that do not distort the overall structure of the DNA helix such as bases damaged by oxidation resulting from normal cellular metabolism. While BER removes single damaged bases, nucleotide excision repair (NER) removes short segments of nucleotides (called oligonucleotides) containing damaged bases. NER responds to any alteration that distorts the DNA helix and is the mechanism responsible for repairing bulky base damage caused by carcinogenic chemicals such as benzo [a]pyrene (found in cigarette smoke and automobile exhaust) as well as covalent linkages between ...
Date: April 1, 2006
Creator: Fuss, Jill O. & Cooper, Priscilla K.
Partner: UNT Libraries Government Documents Department

The Beginning of Kinesin's Force-Generating Cycle Visualized at 9Angstrom Resolution

Description: We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesin's crucial nucleotide response elements, switch I and the switch II helix, in kinesin's poorly understood nucleotide-free state. Both of the switch elements undergo conformational change relative to the microtubule-free state. The changes in switch I suggest a role for it in ''ejecting'' adenosine diphosphate when kinesin initially binds to the microtubule. The switch II helix has an N-terminal extension, apparently stabilized by conserved microtubule contacts, implying a microtubule activation mechanism that could convey the state of the bound nucleotide to kinesin's putative force-delivering element (the ''neck linker''). In deriving this structure, we have adapted an image-processing technique, single-particle reconstruction, for analyzing decorated microtubules. The resulting reconstruction visualizes the asymmetric seam present in native, 13-protofilament microtubules, and this method will provide an avenue to higher-resolution characterization of a variety of microtubule- binding proteins, as well as the microtubule itself.
Date: June 20, 2007
Creator: Sindelar, Charles V. & Downing, Kenneth H.
Partner: UNT Libraries Government Documents Department

Behavioral versus genetic determination of lipoproteins andidentical twins discordant for exercise

Description: Lipoprotein and weight differences between vigorously active and sedentary MZ twins are used to: (1) estimate the effects of training while controlling for genotype; (2) estimate genetic concordance in the presence of divergent lifestyles.
Date: June 1, 2004
Creator: Williams, Paul T.; Blanche, Patricia J. & Krauss, Ronald M.
Partner: UNT Libraries Government Documents Department

1,2-HOIQO--A highly versatile 1,2-HOPO analog

Description: A cyclic, bidentate hydroxamic acid binding unit based on an isoquinoline scaffold has been utilized for the synthesis of a hexadentate tripodal ligand based on the TREN backbone. This prototype for a new class of multidentate chelators forms mononuclear iron(III) complexes and one-dimensional coordination polymers with lanthanide(III) cations. The latter has been determined by single crystal X-ray analysis of the cerium species. The solid state structure in the monoclinic space group P2{sub 1}/c (C{sub 36}H{sub 34}CeN{sub 7}O{sub 11}, a = 12.341(2){angstrom}, b = 26.649(4){angstrom}, c = 10.621(2){angstrom}, {alpha} = {gamma} = 90{sup o}, {beta} = 96.753(3){sup o}, V = 3468.6(9) {angstrom}{sup 3}, Z = 4) exhibits a trigonal-dodecahedral environment around the cerium cation. The proof of concept for the versatility of the new scaffold has been shown by the modification of the crucial precursor 3-carboxyiso-coumarin through electrophilic aromatic substitutions to yield the corresponding chlorosulfonated and nitrated analogs.
Date: August 7, 2006
Creator: Seitz, Michael; Pluth, Michael D. & Raymond, Kenneth N.
Partner: UNT Libraries Government Documents Department

Independence of replisomes in Escherichia coli chromosomalreplication

Description: In Escherichia coli DNA replication is carried out by the coordinated action of the proteins within a replisome. After replication initiation, the two bidirectionally oriented replisomes from a single origin are colocalized into higher-order structures termed replication factories. The factory model postulated that the two replisomes are also functionally coupled. We tested this hypothesis by using DNA combing and whole-genome microarrays. Nascent DNA surrounding oriC in single, combed chromosomes showed instead that one replisome, usually the leftward one, was significantly ahead of the other 70% of the time. We next used microarrays to follow replication throughout the genome by measuring DNA copy number. We found in multiple E. coli strains that the replisomes are independent, with the leftward replisome ahead of the rightward one. The size of the bias was strain-specific, varying from 50 to 130 kb in the array results. When we artificially blocked one replisome, the other continued unabated, again demonstrating independence. We suggest an improved version of the factory model that retains the advantages of threading DNA through colocalized replisomes at about equal rates, but allows the cell flexibility to overcome obstacles encountered during elongation.
Date: March 13, 2005
Creator: Breier, Adam M.; Weier, Heinz-Ulrich G. & Cozzarelli, Nicholas R.
Partner: UNT Libraries Government Documents Department

Defining interactions between DNA-PK and ligase IV/XRCC4

Description: Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks in mammalian cells. DNA-dependent protein kinase (DNA-PK), ligase IV, and XRCC4 are all critical components of the NHEJ repair pathway. DNA-PK is composed of a heterodimeric DNA-binding component, Ku, and a large catalytic subunit, DNA-PKcs. Ligase IV and XRCC4 associate to form a multimeric complex that is also essential for NHEJ. DNA-PK and ligase IV/XRCC4 interact at DNA termini which results in stimulated ligase activity. Here we define interactions between the components of these two essential complexes, DNA-PK and ligase IV/XRCC4. We find that ligase IV/XRCC4 associates with DNA-PK in a DNA-independent manner. The specific protein-protein interactions that mediate the interaction between these two complexes are further identified. Direct physical interactions between ligase IV and Ku as well as between XRCC4 and DNA-PKcs are shown. No direct interactions are observed between ligase IV and DNA-PKcs or between XRCC4 and Ku. Our data defines the specific protein pairs involved in the association of DNA-PK and ligase IV/XRCC4, and suggests a molecular mechanism for coordinating the assembly of the DNA repair complex at DNA breaks.
Date: April 10, 2001
Creator: Hsu, Hsin-Ling; Yannone, Steven M. & Chen, David J.
Partner: UNT Libraries Government Documents Department

Synthesis ofN-(2-chloro-5-methylthiophenyl)-N'-(3-methyl-thiophenyl)-N'-[3H3]methylguanidine, l brace [3H3]CNS-5161 r brace

Description: The preparation of the title compound, [{sup 3}H{sub 3}]CNS-5161, was accomplished in three steps starting with the production of [{sup 3}H{sub 3}]iodomethane (CT{sub 3}I). The intermediate N-[{sup 3}H{sub 3}]methyl-3-(thiomethylphenyl)cyanamide was prepared in 77% yield by the addition of CT{sub 3}I to 3-(thiomethylphenyl)cyanamide, previously treated with sodium hydride. Reaction of this tritiated intermediate with 2-chloro-5-thiomethylaniline hydrochloride formed the guanidine compound [{sup 3}H{sub 3}]CNS-5161. Purification by HPLC gave the desired labeled product in an overall yield of 9% with greater than 96% radiochemical purity and a final specific activity of 66 Ci mmol{sup -1}.
Date: September 28, 2001
Creator: Gibbs, Andrew R.; Morimoto, Hiromi; VanBrocklin, Henry F.; Williams, Philip G. & Biegon, Anat
Partner: UNT Libraries Government Documents Department

Factors influencing timing resolution in a commercial LSO PETcamera

Description: The CPS Accel is a commercial PET camera based on a block detector with 64 LSO scintillator crystals (each 6.75 x 6.75 x 25 mm)read out with 4 photomultiplier tubes. The excellent timing resolution of LSO suggests that this camera might be used for time-of-flight (TOF) PET, thereby reducing the statistical noise significantly. Although the Accel achieves 3 ns coincidence resolution (a factor of two better than BGO-based PET cameras), its timing resolution is nearly an order of magnitude worse than that demonstrated with individual LSO crystals. This paper quantifies the effect on the timing of each component in the Accel timing chain to identify which components most limit the camera's timing resolution. The components in the timing chain are: the scintillator crystal, the photomultiplier tube (PMT), the constant fraction discriminator (CFD), and the time to digital converter (TDC). To measure the contribution of each component, we construct a single crystal test system with high-performance versions of these components. This system achieves 221 ps fwhm coincidence timing resolution, which is used as a baseline measurement. One of the high-performance components is replaced by a production component, the coincidence timing resolution is re-measured, and the difference between measurements is the contribution of that (production) component. We find that the contributions of the TDC, CFD, PMT, and scintillator are 2000 ps, 1354 ps, 422 ps, and 326 psfwhm respectively, and that the overall timing resolution scales like the square root of the amount of scintillation light detected by the PMT. Based on these measurements we predict that the limit for the coincidence timing resolution in a practical, commercial, LSO-based PET camera is 528ps fwhm.
Date: October 23, 2004
Creator: Moses, William W. & Ullisch, Marcus
Partner: UNT Libraries Government Documents Department

The single-strand DNA binding activity of human PC4 preventsmutagenesis and killing by oxidative DNA damage

Description: Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Yeast mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub l{Delta} mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide-resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show XPG recruits PC4 to a bubble-containing DNA substrate with resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.
Date: February 1, 2004
Creator: Wang, Jen-Yeu; Sarker, Altaf Hossain; Cooper, Priscilla K. & Volkert, Michael R.
Partner: UNT Libraries Government Documents Department

Calibration of a PEM detector with depth of interactionmeasurement

Description: We present an in situ calibration technique for the LBNL positron emission mammography (PEM) detector module that is capable of measuring depth of interaction (DOI). The detector module consists of 64LSO crystals coupled on one end to a single photomultiplier tube (PMT) and on the opposite end to a 64 pixel array of silicon photodiodes (PD). The PMT provides an accurate timing pulse, the PDs identify the crystal of interaction, the sum provides a total energy signal and the /splGamma/=PD/(PD+PMT) ratio determines the depth of interaction. We calibrate using the /sup 176/Lu natural background radiation of the LSO crystals. We determine the relative gain (K) of the PMT and PD by minimizing the asymmetry of the /spl Gamma/ distribution. We determine the depth dependence from the width of the /spl Gamma/ distribution with optimal K. The performance of calibrated detector modules is evaluated by averaging results from 12 modules. The energy resolution is a function of depth ranging from 24 percent FWHM at the PD end to 51 percent FWHM at the PMT end, and the DOI resolution ranges from 6 mm FWHM at the PD end to 11 mm FWHM at the PMT end.
Date: June 3, 2004
Creator: Wang, G.-C.; Huber, J.S.; Moses, W.W.; Choong, W.-S. & Maltz, J.S.
Partner: UNT Libraries Government Documents Department

Crystallization of Mitochondrial Respiratory Complex II fromChicken Heart: A Membrane-Protein Complex Diffracting to 2.0Angstrom

Description: Procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Angstrom with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites.
Date: December 17, 2004
Creator: Huang, Li-shar; Borders, Toni M.; Shen, John T.; Wang, Chung-Jen & Berry, Edward A.
Partner: UNT Libraries Government Documents Department

Linkage and association of haplotypes at the APOA1/C3/A4/A5 genecluster to familial combined hyperlipidemia

Description: Combined hyperlipidemia (CHL) is a common disorder of lipidmetabolism that leads to an increased risk of cardiovascular disease. Thelipid profile of CHL is characterised by high levels of atherogeniclipoproteins and low levels of high-density-lipoprotein-cholesterol.Apolipoprotein (APO) A5 is a newly discovered gene involved in lipidmetabolism located within 30kbp of the APOA1/C3/A4 gene cluster. Previousstudies have indicated that sequence variants in this cluster areassociated with increased plasma lipid levels. To establish whethervariation at the APOA5 gene contributes to the transmission of CHL, weperformed linkage and linkage disequilibrium (LD) tests on a large cohortof families (n=128) with familial CHL (FCHL). The linkage data producedevidence for linkage of the APOA1/C3/A4/A5 genomic interval to FCHL (NPL= 1.7, P = 0.042). The LD studies substantiated these data. Twoindependent rare alleles, APOA5c.56G and APOC3c.386G of this gene clusterwere over-transmitted in FCHL (P = 0.004 and 0.007, respectively), andthis was associated with a reduced transmission of the most commonAPOA1/C3/A4/A5 haplotype (frequency 0.4425) to affected subjects (P =0.013). The APOA5c.56G allele was associated with increased plasmatriglyceride levels in FCHL probands, whereas the second, andindependent, APOC3c.386G allele was associated with increased plasmatriglyceride levels in FCHL pedigree founders. Thus, this allele (or anallele in LD) may mark a quantitative trait associated with FCHL, as wellas representing a disease susceptibility locus for the condition. Thisstudy establishes that sequence variation in the APOA1/C3/A4/A5 genecluster contributes to the transmission of FCHL in a substantialproportion of affected families, and that these sequence variants mayalso contribute to the lipid abnormalities of the metabolic syndrome,which is present in up to 40 percent of persons with cardiovasculardisease.
Date: September 15, 2002
Creator: Eichenbaum-Voline, Sophie; Olivier, Michael; Jones, Emma L.; Naoumova, Rossitza P.; Jones, Bethan; Gau, Brian et al.
Partner: UNT Libraries Government Documents Department

An in-situ cell for characterization of solids by soft X-rayabsorption

Description: An in-situ cell using ''lab-on-a-chip'' technologies has been designed and tested for characterization of catalysts and environmental materials using soft X-ray absorption spectroscopy and spectromicroscopy at photon energies above 250 eV. The sample compartment is 1.0 mm in diameter with a gas path length of 0.8 mm to minimize X-ray absorption in the gas phase. The sample compartment can be heated to 533 K by an Al resistive heater and gas flows up to 5.0 cm{sup 3} min{sup -1} can be supplied to the sample compartment through microchannels. The performance of the cell was tested by acquiring Cu L{sub 3}-edge XANES data during the reduction and oxidation of a silica-supported Cu catalyst using the beam line 11.0.2 Scanning Transmission X-ray Microscope (STXM) at the Advanced Light Source of LBNL. Two-dimensional images of individual catalyst particles were recorded at photon energies between 926 eV and 937 eV, the energy range in which the Cu(II) and Cu(I) L{sub 3} absorption edges are observed. Oxidation state specific images of the catalyst clearly show the disappearance of Cu(II) species during the exposure of the oxidized sample to 4% CO in He while increasing the temperature from 308 K to 473 K. Reoxidation restores the intensity of the image associated with Cu(II). L-edge XANES spectra obtained from stacks of STXM images show that with increasing temperature the Cu(II) peak intensity decreases as the Cu(I) peak intensity increases.
Date: January 4, 2007
Creator: Drake, Ian J.; Liu, Teris C.N.; Gilles, Mary; Tyliszczak, Tolek; Kilcoyne, A.L. David; Shuh, David K. et al.
Partner: UNT Libraries Government Documents Department

Targeted Gene Deletion Demonstrates that Cell Adhesion MoleculeICAM-4 is Critical for Erythroblastic Island Formation

Description: Erythroid progenitors differentiate in erythroblastic islands, bone marrow niches composed of erythroblasts surrounding a central macrophage. Evidence suggests that within islands adhesive interactions regulate erythropoiesis and apoptosis. We are exploring whether erythroid intercellular adhesion molecule-4 (ICAM-4), animmunoglobulin superfamily member, participates in island formation. Earlier, we identified alpha V integrins as ICAM-4 counter receptors. Since macrophages express alpha V, ICAM-4 potentially mediates island attachments. To test this, we generated ICAM-4 knockout mice and developed quantitative, live cell techniques for harvesting intact islands and for reforming islands in vitro. We observed a 47 percent decrease in islands reconstituted from ICAM-4 null marrow compared to wild type. We also found a striking decrease in islands formed in vivo in knockout mice. Further, peptides that block ICAM-4 alpha V adhesion produced a 53-57 percent decrease in reconstituted islands, strongly suggesting that ICAM-4 binding to macrophage alpha V functions in island integrity. Importantly, we documented that alpha V integrin is expressed in macrophages isolated from erythro blastic islands. Collectively, these data provide convincing evidence that ICAM-4 is critical in erythroblastic island formation via ICAM-4/alpha V adhesion and also demonstrate that the novel experimental strategies we developed will be valuable in exploring molecular mechanisms of erythroblastic island formation and their functional role in regulating erythropoiesis.
Date: February 15, 2006
Creator: Lee, Gloria; Lo, Annie; Short, Sarah A.; Mankelow, Tosti J.; Spring, Frances; Parsons, Stephen F. et al.
Partner: UNT Libraries Government Documents Department

Dialkoxyquinazolines: Screening Epidermal Growth Factor ReceptorTyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

Description: The epidermal growth factor receptor (EGFR), a long-standingdrug development target, is also a desirable target for imaging. Sixteendialkoxyquinazoline analogs, suitable for labeling with positron-emittingisotopes, have been synthesized and evaluated in a battery of in vitroassays to ascertain their chemical and biological properties. Thesecharacteristics provided the basis for the adoption of a selection schemato identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of thecompounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFRtyrosine kinase. All of the analogs inhibited ligand-induced EGFRtyrosine phosphorylation (IC50 = 0.8 - 20 nM). The HPLC-estimatedoctanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline aswell as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the bestcombination of characteristics that warrant radioisotope labeling andfurther evaluation in tumor-bearing mice.
Date: September 1, 2005
Creator: VanBrocklin, Henry F.; Lim, John K.; Coffing, Stephanie L.; Hom,Darren L.; Negash, Kitaw; Ono, Michele Y. et al.
Partner: UNT Libraries Government Documents Department