WorldWideScience

Sample records for human nucleotide excision

  1. Both base excision repair and nucleotide excision repair in humans are influenced by nutritional factors.

    Science.gov (United States)

    Brevik, Asgeir; Karlsen, Anette; Azqueta, Amaya; Tirado, Anna Estaban; Blomhoff, Rune; Collins, Andrew

    2011-01-01

    Lack of reliable assays for DNA repair has largely prevented measurements of DNA repair from being included in human biomonitoring studies. Using newly developed modifications of the comet assay we tested whether a fruit- and antioxidant-rich plant-based intervention could affect base excision repair (BER) and nucleotide excision repair (NER) in a group of 102 male volunteers. BER and NER repair capacities were measured in lymphocytes before and after a dietary intervention lasting 8 weeks. The study had one control group, one group consuming three kiwifruits per day and one group consuming a variety of antioxidant-rich fruits and plant products in addition to their normal diet. DNA strand breaks were reduced following consumption of both kiwifruits (13%, p = 0.05) and antioxidant-rich plant products (20%, p = 0.02). Increased BER (55%, p = 0.01) and reduced NER (-39%, p plant products. Reduced NER was also observed in the kiwifruit group (-38%, p = 0.05), but BER was not affected in this group. Here we have demonstrated that DNA repair is affected by diet and that modified versions of the comet assay can be used to assess activity of different DNA repair pathways in human biomonitoring studies. Copyright © 2010 John Wiley & Sons, Ltd.

  2. Nucleotide excision repair in yeast

    NARCIS (Netherlands)

    Eijk, Patrick van

    2012-01-01

    Nucleotide Excision Repair (NER) is a conserved DNA repair pathway capable of removing a broad spectrum of DNA damage. In human cells a defect in NER leads to the disorder Xeroderma pigmentosum (XP). The yeast Saccharomyces cerevisiae is an excellent model organism to study the mechanism of NER. The

  3. Transcriptional and post-transcriptional regulation of nucleotide excision repair genes in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Lefkofsky, Hailey B. [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Veloso, Artur [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Bioinformatics Program, Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI (United States); Ljungman, Mats, E-mail: ljungman@umich.edu [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI (United States)

    2015-06-15

    Nucleotide excision repair (NER) removes DNA helix-distorting lesions induced by UV light and various chemotherapeutic agents such as cisplatin. These lesions efficiently block the elongation of transcription and need to be rapidly removed by transcription-coupled NER (TC-NER) to avoid the induction of apoptosis. Twenty-nine genes have been classified to code for proteins participating in nucleotide excision repair (NER) in human cells. Here we explored the transcriptional and post-transcriptional regulation of these NER genes across 13 human cell lines using Bru-seq and BruChase-seq, respectively. Many NER genes are relatively large in size and therefore will be easily inactivated by UV-induced transcription-blocking lesions. Furthermore, many of these genes produce transcripts that are rather unstable. Thus, these genes are expected to rapidly lose expression leading to a diminished function of NER. One such gene is ERCC6 that codes for the CSB protein critical for TC-NER. Due to its large gene size and high RNA turnover rate, the ERCC6 gene may act as dosimeter of DNA damage so that at high levels of damage, ERCC6 RNA levels would be diminished leading to the loss of CSB expression, inhibition of TC-NER and the promotion of cell death.

  4. DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.

    Science.gov (United States)

    de Laat, W L; Appeldoorn, E; Sugasawa, K; Weterings, E; Jaspers, N G; Hoeijmakers, J H

    1998-08-15

    The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.

  5. Studies on the molecular mechanism of nucleotide excision repair in human cells

    International Nuclear Information System (INIS)

    Friedberg, E.C.

    1987-01-01

    Studies in this laboratory have focused on attempts to define the mechanism of nucleotide excision repair of DNA in human cells, with a view to understanding the molecular pathogenesis of the disease XP. With the advent of recombinant DNA technology, they directed their efforts to the molecular cloning of human genes defective in XP, with a view to using the cloned genes to overexpress proteins of interest for biochemical investigations. Initial studies exploited the selectable phenotype of marked sensitivity to killing of XP group A cells by UV radiation and by other DNA damaging agents. However, except for a single report in 1982 there has been no reproducible demonstration of complementation of the UV sensitivity of XP cells by DNA-mediated transfection. The apparent difficulties associated with transfection of XP cells have been the subject of several recent studies. In view of the multiple problems associated with stable transfection of XP cells using total genomic DNA, they have embarked on an alternative strategy designed to facilitate the cloning of human XP genes. This strategy involves the transfer of single human chromosomes into XP cells and screening for this relatively high frequency event. The idea is to identify chromosomes on which particular XP genes reside and then to isolate non-complementing derivatives of these chromosomes so that highly enriched DNA pools containing genes of interest can be generated by employing one or more subtractive strategies

  6. Oxidative Damage to RPA Limits the Nucleotide Excision Repair Capacity of Human Cells.

    Science.gov (United States)

    Guven, Melisa; Brem, Reto; Macpherson, Peter; Peacock, Matthew; Karran, Peter

    2015-11-01

    Nucleotide excision repair (NER) protects against sunlight-induced skin cancer. Defective NER is associated with photosensitivity and a high skin cancer incidence. Some clinical treatments that cause photosensitivity can also increase skin cancer risk. Among these, the immunosuppressant azathioprine and the fluoroquinolone antibiotics ciprofloxacin and ofloxacin interact with UVA radiation to generate reactive oxygen species that diminish NER capacity by causing protein damage. The replication protein A (RPA) DNA-binding protein has a pivotal role in DNA metabolism and is an essential component of NER. The relationship between protein oxidation and NER inhibition was investigated in cultured human cells expressing different levels of RPA. We show here that RPA is limiting for NER and that oxidative damage to RPA compromises NER capability. Our findings reveal that cellular RPA is surprisingly vulnerable to oxidation, and we identify oxidized forms of RPA that are associated with impaired NER. The vulnerability of NER to inhibition by oxidation provides a connection between cutaneous photosensitivity, protein damage, and increased skin cancer risk. Our findings emphasize that damage to DNA repair proteins, as well as to DNA itself, is likely to be an important contributor to skin cancer risk.

  7. Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway.

    Directory of Open Access Journals (Sweden)

    Nathaniel Holcomb

    Full Text Available Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC, a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6-4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke.

  8. Substrate overlap and functional competition between human nucleotide excision repair and Escherichia coli photolyase and (A)BC excision nuclease

    International Nuclear Information System (INIS)

    Sibghat-Ullah; Sancar, Z.

    1990-01-01

    Human cell free extract prepared by the method of Manley et al. carries out repair synthesis on UV-irradiated DNA. Removal of pyrimidine dimers by photoreactivation with DNA photolyase reduces repair synthesis by about 50%. With excess enzyme in the reaction mixture photolyase reduced the repair signal by the same amount even in the absence of photoreactivating light, presumably by binding to pyrimidine dimers and interfering with the binding of human damage recognition protein. Similarly, the UvrB subunit of Escherichia coli (A)BC excinuclease when loaded onto UV-irradiated or psoralen-adducted DNA inhibited repair synthesis by cell-free extract by 75-80%. The opposite was true also as HeLa cell free extract specifically inhibited the photorepair of a thymine dimer by DNA photolyase and its removal by (A)BC excinuclease. Cell-free extracts from xeroderma pigmentosum (XP) complementation groups A and C were equally effective in blocking the E. coli repair proteins, while extracts from complementation groups D and E were ineffective in blocking the E. coli enzyme. These results suggest that XP-D and XP-E cells are defective in the damage recognition subunits(s) of human excision nuclease

  9. Nucleotide excision repair in the test tube.

    NARCIS (Netherlands)

    N.G.J. Jaspers (Nicolaas); J.H.J. Hoeijmakers (Jan)

    1995-01-01

    textabstractThe eukaryotic nucleotide excision-repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway.

  10. Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells.

    Science.gov (United States)

    Al-Serori, Halh; Ferk, Franziska; Kundi, Michael; Bileck, Andrea; Gerner, Christopher; Mišík, Miroslav; Nersesyan, Armen; Waldherr, Monika; Murbach, Manuel; Lah, Tamara T; Herold-Mende, Christel; Collins, Andrew R; Knasmüller, Siegfried

    2018-01-01

    Some epidemiological studies indicate that the use of mobile phones causes cancer in humans (in particular glioblastomas). It is known that DNA damage plays a key role in malignant transformation; therefore, we investigated the impact of the UMTS signal which is widely used in mobile telecommunications, on DNA stability in ten different human cell lines (six brain derived cell lines, lymphocytes, fibroblasts, liver and buccal tissue derived cells) under conditions relevant for users (SAR 0.25 to 1.00 W/kg). We found no evidence for induction of damage in single cell gel electrophoresis assays when the cells were cultivated with serum. However, clear positive effects were seen in a p53 proficient glioblastoma line (U87) when the cells were grown under serum free conditions, while no effects were found in p53 deficient glioblastoma cells (U251). Further experiments showed that the damage disappears rapidly in U87 and that exposure induced nucleotide excision repair (NER) and does not cause double strand breaks (DSBs). The observation of NER induction is supported by results of a proteome analysis indicating that several proteins involved in NER are up-regulated after exposure to UMTS; additionally, we found limited evidence for the activation of the γ-interferon pathway. The present findings show that the signal causes transient genetic instability in glioma derived cells and activates cellular defense systems.

  11. Nucleotide excision repair : complexes and complexities : a study of global genome repair in human cells

    NARCIS (Netherlands)

    Volker, Marcel

    2006-01-01

    Of all exogenous agents that damage genomic DNA and hence threaten its integrity, the ultraviolet B (UVB) component of sunlight is highly relevant because of its abundance. UVB induces predominantly cyclobutane pyrimidine dimers and 6-4 photoproducts. In humans, these photolesions are repaired by

  12. Histone displacement during nucleotide excision repair

    DEFF Research Database (Denmark)

    Dinant, C.; Bartek, J.; Bekker-Jensen, S.

    2012-01-01

    Nucleotide excision repair (NER) is an important DNA repair mechanism required for cellular resistance against UV light and toxic chemicals such as those found in tobacco smoke. In living cells, NER efficiently detects and removes DNA lesions within the large nuclear macromolecular complex called...... of histone variants and histone displacement (including nucleosome sliding). Here we review current knowledge, and speculate about current unknowns, regarding those chromatin remodeling activities that physically displace histones before, during and after NER....

  13. Nucleotide excision repair in differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Wees, Caroline van der [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Jansen, Jacob [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Laarse, Arnoud van der [Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Zeeland, Albert van [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Mullenders, Leon [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands)]. E-mail: l.mullenders@lumc.nl

    2007-01-03

    Nucleotide excision repair (NER) is the principal pathway for the removal of a wide range of DNA helix-distorting lesions and operates via two NER subpathways, i.e. global genome repair (GGR) and transcription-coupled repair (TCR). Although detailed information is available on expression and efficiency of NER in established mammalian cell lines, little is known about the expression of NER pathways in (terminally) differentiated cells. The majority of studies in differentiated cells have focused on repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4-photoproducts (6-4PP) because of the high frequency of photolesions at low level of toxicity and availability of sensitive technologies to determine photolesions in defined regions of the genome. The picture that emerges from these studies is blurred and rather complex. Fibroblasts and terminally differentiated myocytes of the rat heart display equally efficient GGR of 6-4PP but poor repair of CPD due to the absence of p48 expression. This repair phenotype is clearly different from human terminal differentiated neurons. Furthermore, both cell types were found to carry out TCR of CPD, thus mimicking the repair phenotype of established rodent cell lines. In contrast, in intact rat spermatogenic cells repair was very inefficient at the genome overall level and in transcriptionally active genes indicating that GGR and TCR are non-functional. Also, non-differentiated mouse embryonic stem (ES) cells exhibit low levels of NER after UV irradiation. However, the mechanisms that lead to low NER activity are clearly different: in differentiated spermatogenic cells differences in chromatin compaction and sequestering of NER proteins may underlie the lack of NER activity in pre-meiotic cells, whereas in non-differentiated ES cells NER is impaired by a strong apoptotic response.

  14. Removal of oxygen free-radical-induced 5′,8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells

    Science.gov (United States)

    Kuraoka, Isao; Bender, Christina; Romieu, Anthony; Cadet, Jean; Wood, Richard D.; Lindahl, Tomas

    2000-01-01

    Exposure of cellular DNA to reactive oxygen species generates several classes of base lesions, many of which are removed by the base excision-repair pathway. However, the lesions include purine cyclodeoxynucleoside formation by intramolecular crosslinking between the C-8 position of adenine or guanine and the 5′ position of 2-deoxyribose. This distorting form of DNA damage, in which the purine is attached by two covalent bonds to the sugar-phosphate backbone, occurs as distinct diastereoisomers. It was observed here that both diastereoisomers block primer extension by mammalian and microbial replicative DNA polymerases, using DNA with a site-specific purine cyclodeoxynucleoside residue as template, and consequently appear to be cytotoxic lesions. Plasmid DNA containing either the 5′R or 5′S form of 5′,8-cyclo-2-deoxyadenosine was a substrate for the human nucleotide excision-repair enzyme complex. The R diastereoisomer was more efficiently repaired than the S isomer. No correction of the lesion by direct damage reversal or base excision repair was detected. Dual incision around the lesion depended on the core nucleotide excision-repair protein XPA. In contrast to several other types of oxidative DNA damage, purine cyclodeoxynucleosides are chemically stable and would be expected to accumulate at a slow rate over many years in the DNA of nonregenerating cells from xeroderma pigmentosum patients. High levels of this form of DNA damage might explain the progressive neurodegeneration seen in XPA individuals. PMID:10759556

  15. Regulation of nucleotide excision repair through ubiquitination

    Institute of Scientific and Technical Information of China (English)

    Jia Li; Audesh Bhat; Wei Xiao

    2011-01-01

    Nucleotide excision repair (NER) is the most versatile DNA-repair pathway in all organisms.While bacteria require only three proteins to complete the incision step of NER,eukaryotes employ about 30 proteins to complete the same step.Here we summarize recent studies demonstrating that ubiquitination,a post-translational modification,plays critical roles in regulating the NER activity either dependent on or independent of ubiquitin-proteolysis.Several NER components have been shown as targets of ubiquitination while others are actively involved in the ubiquitination process.We argue through this analysis that ubiquitination serves to coordinate various steps of NER and meanwhile connect NER with other related pathways to achieve the efficient global DNA-damage response.

  16. Protective Effect of Diphlorethohydroxycarmalol against Ultraviolet B Radiation-Induced DNA Damage by Inducing the Nucleotide Excision Repair System in HaCaT Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Mei Jing Piao

    2015-09-01

    Full Text Available We investigated the protective properties of diphlorethohydroxycarmalol (DPHC, a phlorotannin, against ultraviolet B (UVB radiation-induced cyclobutane pyrimidine dimers (CPDs in HaCaT human keratinocytes. The nucleotide excision repair (NER system is the pathway by which cells identify and repair bulky, helix-distorting DNA lesions such as ultraviolet (UV radiation-induced CPDs and 6-4 photoproducts. CPDs levels were elevated in UVB-exposed cells; however, this increase was reduced by DPHC. Expression levels of xeroderma pigmentosum complementation group C (XPC and excision repair cross-complementing 1 (ERCC1, which are essential components of the NER pathway, were induced in DPHC-treated cells. Expression of XPC and ERCC1 were reduced following UVB exposure, whereas DPHC treatment partially restored the levels of both proteins. DPHC also increased expression of transcription factor specificity protein 1 (SP1 and sirtuin 1, an up-regulator of XPC, in UVB-exposed cells. DPHC restored binding of the SP1 to the XPC promoter, which is reduced in UVB-exposed cells. These results indicate that DPHC can protect cells against UVB-induced DNA damage by inducing the NER system.

  17. A human homolog of the yeast nucleotide excision repair gene MMS19 interacts with transcription repair factor TFIIH through the XPB and XPD helicases.

    NARCIS (Netherlands)

    T. Seroz; G.S. Winkler (Sebastiaan); J. Auriol; R.A. Verhage; W. Vermeulen (Wim); B. Smit (Bep); J. Brouwer (Jaap); A.P.M. Eker (André); G. Weeda (Geert); J-M. Egly (Jean-Marc); J.H.J. Hoeijmakers (Jan)

    2000-01-01

    textabstractNucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins have been identified which are dispensable for NER in vitro

  18. HHR23A, a human homolog of Saccharomyces cerevisiae Rad23, regulates xeroderma pigmentosum C protein and is required for nucleotide excision repair

    International Nuclear Information System (INIS)

    Hsieh, Hui-Chuan; Hsieh, Yi-Hsuan; Huang, Yu-Hsin; Shen, Fan-Ching; Tsai, Han-Ni; Tsai, Jui-He; Lai, Yu-Ting; Wang, Yu-Ting; Chuang, Woei-Jer; Huang, Wenya

    2005-01-01

    HHR23A and hHR23B are the human homologs of Saccharomyces cerevisiae Rad23. hHR23B is associated with the nucleotide excision repair (NER) factor xeroderma pigmentosum C (XPC) protein and is required for global genome repair. The function of hHR23A is not yet clear. In this study, the potential function of the hHR23A protein was investigated using RNA interference techniques. The hHR23A knock-down (KD) construct diminished the RNA level of hHR23A protein by approximately 60%, and it did not interfere with expression of the hHR23B gene. Based on Southwestern immunoblot and host-cell reactivation assays, hHR23A KD cells were found to be deficient in DNA repair activity against the DNA damage caused by UVC irradiation. In these hHR23A KD cells, the XPC gene was not normally induced by UVC irradiation, indicating that the hHR23A protein is involved in NER through regulation of the DNA damage recognition protein XPC. Co-immunoprecipitation experiments revealed that hHR23A was associated with a small portion of hHR23B and the majority of p53 protein, indicating that hHR23A regulates the function of XPC by its association with the NER activator p53

  19. HHR23B, a human RAD23 homolog, stimulates XPC protein in nucleotide excision repair in vitro.

    NARCIS (Netherlands)

    K. Sugasawa (Kaoru); C. Masutani (Chikahide); A. Uchida; T. Maekawa; P.J. van der Spek (Peter); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan); F. Hanaoka (Fumio)

    1996-01-01

    textabstractA protein complex which specifically complements defects of XP-C cell extracts in vitro was previously purified to near homogeneity from HeLa cells. The complex consists of two tightly associated proteins: the XPC gene product and HHR23B, one of two human homologs of the Saccharomyces

  20. Nucleotide excision repair II: From yeast to mammals

    NARCIS (Netherlands)

    J.H.J. Hoeijmakers (Jan)

    1993-01-01

    textabstractAn intricate network of repair systems safeguards the integrity of genetic material, by eliminating DNA lesions induced by numerous environmental and endogenous genotoxic agents. Nucleotide excision repair (NER) is one of the most versatile DNA repair systems. Deficiencies in this

  1. Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

    Science.gov (United States)

    Sugitani, Norie; Voehler, Markus W; Roh, Michelle S; Topolska-Woś, Agnieszka M; Chazin, Walter J

    2017-10-13

    Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Base Sequence Context Effects on Nucleotide Excision Repair

    Directory of Open Access Journals (Sweden)

    Yuqin Cai

    2010-01-01

    Full Text Available Nucleotide excision repair (NER plays a critical role in maintaining the integrity of the genome when damaged by bulky DNA lesions, since inefficient repair can cause mutations and human diseases notably cancer. The structural properties of DNA lesions that determine their relative susceptibilities to NER are therefore of great interest. As a model system, we have investigated the major mutagenic lesion derived from the environmental carcinogen benzo[a]pyrene (B[a]P, 10S (+-trans-anti-B[a]P-2-dG in six different sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups. We have obtained molecular structural data by NMR and MD simulations, bending properties from gel electrophoresis studies, and NER data obtained from human HeLa cell extracts for our six investigated sequence contexts. This model system suggests that disturbed Watson-Crick base pairing is a better recognition signal than a flexible bend, and that these can act in concert to provide an enhanced signal. Steric hinderance between the minor groove-aligned lesion and nearby guanine amino groups determines the exact nature of the disturbances. Both nearest neighbor and more distant neighbor sequence contexts have an impact. Regardless of the exact distortions, we hypothesize that they provide a local thermodynamic destabilization signal for repair.

  3. Effects of nucleotide pool imbalances on the excision repair of ultraviolet-induced damage in the DNA of human diploid fibroblasts

    International Nuclear Information System (INIS)

    Snyder, R.D.

    1985-01-01

    In an attempt to better understand the mechanism of repair inhibition by DNA polymerase inhibitors, and the nature of hydroxyurea enhancement, experiments were initiated in which the effects of a series of ribonucleotide reductase inhibitors on dNTP pools and on the DNA repair process were determined in both quiescent cultures and log-phase cultures of human fibroblasts. It was determined that hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone (TSC), pyrozoloimidazole (IMPY), 3,5-diamino-1,2,4-triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid (THBA) and 3,4-dihydroxy benzohydroxamic acid (DHBA) are all effective inhibitors of the DNA repair process in confluent cells but not in log-phase cells. Moreover, the effects of these inhibitors can be reversed by the addition of certain combinations of deoxynucleosides. These reversal studies and the direct analysis of dNTP pool modulation by these compounds in log phase and confluent cultures support the notion that specific pool depletions rather than general imbalance of pools gives rise to the inhibition of the DNA excision repair process

  4. Implication of SUMO E3 ligases in nucleotide excision repair.

    Science.gov (United States)

    Tsuge, Maasa; Kaneoka, Hidenori; Masuda, Yusuke; Ito, Hiroki; Miyake, Katsuhide; Iijima, Shinji

    2015-08-01

    Post-translational modifications alter protein function to mediate complex hierarchical regulatory processes that are crucial to eukaryotic cellular function. The small ubiquitin-like modifier (SUMO) is an important post-translational modification that affects transcriptional regulation, nuclear localization, and the maintenance of genome stability. Nucleotide excision repair (NER) is a very versatile DNA repair system that is essential for protection against ultraviolet (UV) irradiation. The deficiencies in NER function remarkably increase the risk of skin cancer. Recent studies have shown that several NER factors are SUMOylated, which influences repair efficiency. However, how SUMOylation modulates NER has not yet been elucidated. In the present study, we performed RNAi knockdown of SUMO E3 ligases and found that, in addition to PIASy, the polycomb protein Pc2 affected the repair of cyclobutane pyrimidine dimers. PIAS1 affected both the removal of 6-4 pyrimidine pyrimidone photoproducts and cyclobutane pyrimidine dimers, whereas other SUMO E3 ligases did not affect the removal of either UV lesion.

  5. Implication of Posttranslational Histone Modifications in Nucleotide Excision Repair

    Directory of Open Access Journals (Sweden)

    Shisheng Li

    2012-09-01

    Full Text Available Histones are highly alkaline proteins that package and order the DNA into chromatin in eukaryotic cells. Nucleotide excision repair (NER is a conserved multistep reaction that removes a wide range of generally bulky and/or helix-distorting DNA lesions. Although the core biochemical mechanism of NER is relatively well known, how cells detect and repair lesions in diverse chromatin environments is still under intensive research. As with all DNA-related processes, the NER machinery must deal with the presence of organized chromatin and the physical obstacles it presents. A huge catalogue of posttranslational histone modifications has been documented. Although a comprehensive understanding of most of these modifications is still lacking, they are believed to be important regulatory elements for many biological processes, including DNA replication and repair, transcription and cell cycle control. Some of these modifications, including acetylation, methylation, phosphorylation and ubiquitination on the four core histones (H2A, H2B, H3 and H4 or the histone H2A variant H2AX, have been found to be implicated in different stages of the NER process. This review will summarize our recent understanding in this area.

  6. Repair of DNA-polypeptide crosslinks by human excision nuclease

    Science.gov (United States)

    Reardon, Joyce T.; Sancar, Aziz

    2006-03-01

    DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair

  7. Nucleotide Excision Repair in Cellular Chromatin: Studies with Yeast from Nucleotide to Gene to Genome

    Directory of Open Access Journals (Sweden)

    Simon Reed

    2012-09-01

    Full Text Available Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes.

  8. Molecular cloning and characterization of genes required for nucleotide excision repair in yeast

    International Nuclear Information System (INIS)

    Friedberg, E.C.

    1987-01-01

    Nucleotide excision repair in the yeast S. cerevisiae is a complex process which involves a large number of genes. At least five of these genes (RAD1, RAD2, RAD3, RAD4 and RAD10) are absolutely required for this process and mutations in any of these genes result in no detectable excision repair in vivo. In order to understand the function of these genes in DNA repair, the authors isolated a number of them by screening a yeast genomic library for recombinant plasmids which complement the phentoype of sensitivity to ultraviolet (UV) radiation imparted to mutant strains. A plasmid containing the RAD4 gene was isolated by an alternative strategy which will be discussed. The cloned genes have been extensively characterized. It has been determined that the RAD3 gene is essential for the viability of haploid yeast cells in the absence of DNA damage. The RAD2 gene is inducible by treatment of cells with a variety of DNA-damaging agents, including UV radiation and ionizing radiation. The RAD10 gene shares considerable amino acid sequence homology with a cloned gene involved in nucleotide excision repair in human cells. Yeast is a particularly versatile organism for studying gene function by molecular and genetic approaches and emphasis is placed on many of the techniques used in the present studies

  9. The effect of DNA repair defects on reproductive performance in nucleotide excision repair (NER) mouse models: an epidemiological approach

    NARCIS (Netherlands)

    Tsai, P.S.; Nielen, M.; Horst, G.T.J. van der; Colenbrander, B.; Heesterbeek, J.A.P.; Fentener van Vlissingen, J.M.

    2005-01-01

    In this study, we used an epidemiological approach to analyze an animal database of DNA repair deficient mice on reproductive performance in five Nucleotide Excision Repair (NER) mutant mouse models on a C57BL/6 genetic background, namely CSA, CSB, XPA, XPC [models for the human DNA repair disorders

  10. In vitro Repair of Oxidative DNA Damage by Human Nucleotide Excision Repair System: Possible Explanation for Neurodegeneration in Xeroderma Pigmentosum Patients

    Science.gov (United States)

    Reardon, Joyce T.; Bessho, Tadayoshi; Kung, Hsiang Chuan; Bolton, Philip H.; Sancar, Aziz

    1997-08-01

    Xeroderma pigmentosum (XP) patients fail to remove pyrimidine dimers caused by sunlight and, as a consequence, develop multiple cancers in areas exposed to light. The second most common sign, present in 20-30% of XP patients, is a set of neurological abnormalities caused by neuronal death in the central and peripheral nervous systems. Neural tissue is shielded from sunlight-induced DNA damage, so the cause of neurodegeneration in XP patients remains unexplained. In this study, we show that two major oxidative DNA lesions, 8-oxoguanine and thymine glycol, are excised from DNA in vitro by the same enzyme system responsible for removing pyrimidine dimers and other bulky DNA adducts. Our results suggest that XP neurological disease may be caused by defective repair of lesions that are produced in nerve cells by reactive oxygen species generated as by-products of an active oxidative metabolism.

  11. Nucleotide excision repair- and p53-deficient mouse models in cancer research

    Energy Technology Data Exchange (ETDEWEB)

    Hoogervorst, Esther M. [Laboratory of Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven (Netherlands); Utrecht University, Department of Pathobiology, Utrecht (Netherlands); Steeg, Harry van [Laboratory of Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven (Netherlands); Vries, Annemieke de [Laboratory of Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven (Netherlands)]. E-mail: Annemieke.de.Vries@rivm.nl

    2005-07-01

    Cancer is caused by the loss of controlled cell growth due to mutational (in)activation of critical genes known to be involved in cell cycle regulation. Three main mechanisms are known to be involved in the prevention of cells from becoming cancerous; DNA repair and cell cycle control, important to remove DNA damage before it will be fixed into mutations and apoptosis, resulting in the elimination of cells containing severe DNA damage. Several human syndromes are known to have (partially) deficiencies in these pathways, and are therefore highly cancer prone. Examples are xeroderma pigmentosum (XP) caused by an inborn defect in the nucleotide excision repair (NER) pathway and the Li-Fraumeni syndrome, which is the result of a germ line mutation in the p53 gene. XP patients develop skin cancer on sun exposed areas at a relatively early age, whereas Li-Fraumeni patients spontaneously develop a wide variety of early onset tumors, including sarcomas, leukemia's and mammary gland carcinomas. Several mouse models have been generated to mimic these human syndromes, providing us information about the role of these particular gene defects in the tumorigenesis process. In this review, spontaneous phenotypes of mice deficient for nucleotide excision repair and/or the p53 gene will be described, together with their responses upon exposure to either chemical carcinogens or radiation. Furthermore, possible applications of these and newly generated mouse models for cancer will be given.

  12. Nucleotide Excision DNA Repair is Associated with Age-Related Vascular Dysfunction

    Science.gov (United States)

    Durik, Matej; Kavousi, Maryam; van der Pluijm, Ingrid; Isaacs, Aaron; Cheng, Caroline; Verdonk, Koen; Loot, Annemarieke E.; Oeseburg, Hisko; Musterd-Bhaggoe, Usha; Leijten, Frank; van Veghel, Richard; de Vries, Rene; Rudez, Goran; Brandt, Renata; Ridwan, Yanto R.; van Deel, Elza D.; de Boer, Martine; Tempel, Dennie; Fleming, Ingrid; Mitchell, Gary F.; Verwoert, Germaine C.; Tarasov, Kirill V.; Uitterlinden, Andre G.; Hofman, Albert; Duckers, Henricus J.; van Duijn, Cornelia M.; Oostra, Ben A.; Witteman, Jacqueline C.M.; Duncker, Dirk J.; Danser, A.H. Jan; Hoeijmakers, Jan H.; Roks, Anton J.M.

    2012-01-01

    Background Vascular dysfunction in atherosclerosis and diabetes, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction. Methods and Results In mice with genomic instability due to the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1d/− and XpdTTD mice), we explored age-dependent vascular function as compared to wild-type mice. Ercc1d/− mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness and elevated blood pressure at very young age. The vasodilator dysfunction was due to decreased endothelial eNOS levels as well as impaired smooth muscle cell function, which involved phosphodiesterase (PDE) activity. Similar to Ercc1d/− mice, age-related endothelium-dependent vasodilator dysfunction in XpdTTD animals was increased. To investigate the implications for human vascular disease, we explored associations between single nucleotide polymorphisms (SNPs) of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium, and found a significant association of a SNP (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity. Conclusions Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans, but with an accelerated progression, as compared to wild type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease. PMID:22705887

  13. Structural and Functional Studies on Nucleotide Excision Repair From Recognition to Incision.

    Energy Technology Data Exchange (ETDEWEB)

    Caroline Kisker

    2001-01-01

    Maintenance of the correct genetic information is crucial for all living organisms because mutations are the primary cause of hereditary diseases, as well as cancer and may also be involved in aging. The importance of genomic integrity is underscored by the fact that 80 to 90% of all human cancers are ultimately due to DNA damage. Among the different repair mechanisms that have evolved to protect the genome, nucleotide excision repair (NER) is a universal pathway found in all organisms. NER removes a wide variety of bulky DNA adducts including the carcinogenic cyclobutane pyrimidine dimers induced by UV radiation, benzo(a)pyrene-guanine adducts caused by smoking and the guanine-cisplatin adducts induced by chemotherapy. The importance of this repair mechanism is reflected by three severe inherited diseases in humans, which are due to defects in NER: xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.

  14. Nucleotide excision repair I: from E.coli to yeast.

    NARCIS (Netherlands)

    J.H.J. Hoeijmakers (Jan)

    1993-01-01

    textabstractGenetic information is constantly deteriorating, mainly as a consequence of the action of numerous genotoxic agents. In order to cope with this fundamental problem, all living organisms have acquired a complex network of DNA repair systems to safeguard their genetic integrity. Nucleotide

  15. The Fanconi anaemia components UBE2T and FANCM are functionally linked to nucleotide excision repair.

    Directory of Open Access Journals (Sweden)

    Ian R Kelsall

    Full Text Available The many proteins that function in the Fanconi anaemia (FA monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes--UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage. Comprehensive genetic dissection experiments indicate that both of these FA genes collaborate to promote nucleotide excision repair rather than translesion bypass to protect cells form UV genotoxicity. Furthermore, UBE2T deficiency impacts on the efficient removal of the UV-induced photolesion cyclobutane pyrimidine dimer. Therefore, this work reveals that the FA pathway shares two components with nucleotide excision repair, intimating not only crosstalk between the two major repair pathways, but also potentially identifying a UBE2T-mediated ubiquitin-signalling response pathway that contributes to nucleotide excision repair.

  16. SUMO and ubiquitin-dependent XPC exchange drives nucleotide excision repair

    DEFF Research Database (Denmark)

    Van Cuijk, Loes; Van Belle, Gijsbert J.; Turkyilmaz, Yasemin

    2015-01-01

    XPC recognizes UV-induced DNA lesions and initiates their removal by nucleotide excision repair (NER). Damage recognition in NER is tightly controlled by ubiquitin and SUMO modifications. Recent studies have shown that the SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation o...

  17. Dynamic interaction of TTDA with TFIIH is stabilized by nucleotide excision repair in living cells.

    NARCIS (Netherlands)

    G. Giglia-Mari (Giuseppina); C. Miquel (Catherine); A.F. Theil (Arjan); P.O. Mari (Pierre-Olivier); D. Hoogstraten (Deborah); J.M.Y. Ng (Jessica); C. Dinant (Christoffel); J.H.J. Hoeijmakers (Jan); W. Vermeulen (Wim)

    2006-01-01

    textabstractTranscription/repair factor IIH (TFIIH) is essential for RNA polymerase II transcription and nucleotide excision repair (NER). This multi-subunit complex consists of ten polypeptides, including the recently identified small 8-kDa trichothiodystrophy group A (TTDA)/ hTFB5 protein.

  18. KIN17, XPC, DNA-PKCS and XRCC4 proteins in the cellular response to DNA damages. Relations between nucleotide excision repair and non-homologous end joining in a human syn-genic model

    International Nuclear Information System (INIS)

    Despras, Emmanuelle

    2006-01-01

    The response to genotoxic stress involves many cellular factors in a complex network of mechanisms that aim to preserve the genetic integrity of the organism. These mechanisms enclose the detection and repair of DNA lesions, the regulation of transcription and replication and, eventually, the setting of cell death. Among the nuclear proteins involved in this response, kin17 proteins are zinc-finger proteins conserved through evolution and activated by ultraviolet (UV) or ionizing radiations (IR). We showed that human kin17 protein (HSAkin17) is found in the cell under a soluble form and a form tightly anchored to nuclear structures. A fraction of HSAkin17 protein is directly associated with chromatin. HSAkin17 protein is recruited to nuclear structures 24 hours after treatment with various agents inducing DNA double-strand breaks (DSB) and/or replication forks blockage. Moreover, the reduction of total HSAkin17 protein level sensitizes RKO cells to IR. We also present evidence for the involvement of HSAkin17 protein in DNA replication. This hypothesis was further confirmed by the biochemical demonstration of its belonging to the replication complex. HSAkin17 protein could link DNA replication and DNA repair, a defect in the HSAkin17 pathway leading to an increased radiosensitivity. In a second part, we studied the interactions between two DNA repair mechanisms: nucleotide excision repair (NER) and non-homologous end joining (NHEJ). NER repairs a wide variety of lesions inducing a distortion of the DNA double helix including UV-induced pyrimidine dimers. NHEJ allows the repair of DSB by direct joining of DNA ends. We used a syn-genic model for DNA repair defects based on RNA interference developed in the laboratory. Epstein-Barr virus-derived vectors (pEBV) allow long-term expression of siRNA and specific extinction of the targeted gene. The reduction of the expression of genes involved in NER (XPA and XPC) or NHEJ (DNA-PKcs and XRCC4) leads to the expected

  19. Deficiency of UV-induced excision repair in human thymocytes

    International Nuclear Information System (INIS)

    Gensler, H.L.; Lindberg, R.E.; Pinnas, J.L.; Jones, J.F.

    1985-01-01

    The capacity of human thymocytes and of differentiated lymphocytes circulating in peripheral blood to perform unscheduled DNA synthesis (a measure of nucleotide excision repair) after UV irradiation was measured by radioautographic analysis. Only 4% of immature T lymphocytes, but 68% of circulating lymphocytes exhibited unscheduled DNA synthesis. When UV sensitivity of peripheral blood lymphocytes and thymocytes from the same donor were compared, the thymocytes, in each case, were significantly more UV sensitive than were the circulating lymphocytes. Peripheral blood lymphocytes from subjects undergoing halothane and morphine anesthesia during surgery showed 56% less excision repair capacity than those from unanesthetized donors. The difference occurred in the number of cells capable of repair rather than in the extent of repair synthesis per cell. Ultraviolet-induced unscheduled DNA synthesis occurred in only 3% of the thymocytes removed from rats killed by cervical dislocation. Therefore, the deficiency of excision repair was observed in rat thymocytes which had not been affected by anesthesia or surgical trauma. The results indicate that immature T-cells are deficient in nucleotide excision repair whereas the majority of mature peripheral blood lymphocytes exhibit such repair. (author)

  20. Crystal structure of the FeS cluster-containing nucleotide excision repair helicase XPD.

    Directory of Open Access Journals (Sweden)

    Stefanie C Wolski

    2008-06-01

    Full Text Available DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an alpha-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster-containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.

  1. Chronic low-dose ultraviolet-induced mutagenesis in nucleotide excision repair-deficient cells.

    Science.gov (United States)

    Haruta, Nami; Kubota, Yoshino; Hishida, Takashi

    2012-09-01

    UV radiation induces two major types of DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidine photoproducts, which are both primarily repaired by nucleotide excision repair (NER). Here, we investigated how chronic low-dose UV (CLUV)-induced mutagenesis occurs in rad14Δ NER-deficient yeast cells, which lack the yeast orthologue of human xeroderma pigmentosum A (XPA). The results show that rad14Δ cells have a marked increase in CLUV-induced mutations, most of which are C→T transitions in the template strand for transcription. Unexpectedly, many of the CLUV-induced C→T mutations in rad14Δ cells are dependent on translesion synthesis (TLS) DNA polymerase η, encoded by RAD30, despite its previously established role in error-free TLS. Furthermore, we demonstrate that deamination of cytosine-containing CPDs contributes to CLUV-induced mutagenesis. Taken together, these results uncover a novel role for Polη in the induction of C→T transitions through deamination of cytosine-containing CPDs in CLUV-exposed NER deficient cells. More generally, our data suggest that Polη can act as both an error-free and a mutagenic DNA polymerase, depending on whether the NER pathway is available to efficiently repair damaged templates.

  2. Nrf1 CNC-bZIP protein promotes cell survival and nucleotide excision repair through maintaining glutathione homeostasis.

    Science.gov (United States)

    Han, Weinong; Ming, Mei; Zhao, Rui; Pi, Jingbo; Wu, Chunli; He, Yu-Ying

    2012-05-25

    Skin cancer is the most common cancer in the United States. Its major environmental risk factor is UVB radiation in sunlight. In response to UVB damage, epidermal keratinocytes activate a specific repair pathway, i.e. nucleotide excision repair, to remove UVB-induced DNA lesions. However, the regulation of UVB response is not fully understood. Here we show that the long isoform of the nuclear factor erythroid 2-related factor 1 (Nrf1, also called NFE2L1), a cytoprotective transcription factor critical for the expression of multiple antioxidant response element-dependent genes, plays an important role in the response of keratinocytes to UVB. Nrf1 loss sensitized keratinocytes to UVB-induced apoptosis by up-regulating the expression of the proapoptotic Bcl-2 family member Bik through reducing glutathione levels. Knocking down Bik reduced UVB-induced apoptosis in Nrf1-inhibited cells. In UVB-irradiated surviving cells, however, disruption of Nrf1 impaired nucleotide excision repair through suppressing the transcription of xeroderma pigmentosum C (XPC), a factor essential for initiating the global genome nucleotide excision repair by recognizing the DNA lesion and recruiting downstream factors. Nrf1 enhanced XPC expression by increasing glutathione availability but was independent of the transcription repressor of XPC. Adding XPC or glutathione restored the DNA repair capacity in Nrf1-inhibited cells. Finally, we demonstrate that Nrf1 levels are significantly reduced by UVB radiation in mouse skin and are lower in human skin tumors than in normal skin. These results indicate a novel role of Nrf1 in UVB-induced DNA damage repair and suggest Nrf1 as a tumor suppressor in the skin.

  3. Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers

    Directory of Open Access Journals (Sweden)

    Peter eRuethemann

    2016-04-01

    Full Text Available Global-genome nucleotide excision repair (GG-NER prevents genome instability by excising a wide range of structurally unrelated DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV radiation or intracellular metabolic by-products. As a versatile damage sensor, xeroderma pigmentosum group C (XPC protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4DDB2 and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4DDB2 or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin.

  4. Genetic instability associated with loop or stem–loop structures within transcription units can be independent of nucleotide excision repair

    Science.gov (United States)

    Burns, John A; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Scicchitano, David A

    2018-01-01

    Abstract Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem–loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them. PMID:29474673

  5. Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli

    Science.gov (United States)

    Isogawa, Asako; Fujii, Shingo

    2017-01-01

    It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80–100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication. PMID:28686598

  6. Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers

    International Nuclear Information System (INIS)

    Fonseca, A.S.; Campos, V.M.A.; Magalhaes, L.A.G.; Paoli, F.

    2015-01-01

    Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T 4 endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T 4 endonuclease V. Low-intensity lasers: i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells, ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, and iv) did not alter the electrophoretic profile of plasmids incubated with T 4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers. (author)

  7. Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers

    Energy Technology Data Exchange (ETDEWEB)

    Fonseca, A.S.; Campos, V.M.A.; Magalhaes, L.A.G., E-mail: adnfonseca@ig.com.br [Instituto de Biologia Roberto Alcantara Gomes, Rio de Janeiro, RJ (Brazil). Departamento de Biofisica e Biometria. Lab. de Ciencias Radiologicas; Paoli, F. [Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, MG (Brazil). Instituto de Ciencias Biologicas. Departamento de Morfologia

    2015-10-15

    Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T{sub 4} endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T{sub 4} endonuclease V. Low-intensity lasers: i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells, ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, and iv) did not alter the electrophoretic profile of plasmids incubated with T{sub 4} endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers. (author)

  8. E2F1 and p53 Transcription Factors as Accessory Factors for Nucleotide Excision Repair

    Directory of Open Access Journals (Sweden)

    David G. Johnson

    2012-10-01

    Full Text Available Many of the biochemical details of nucleotide excision repair (NER have been established using purified proteins and DNA substrates. In cells however, DNA is tightly packaged around histones and other chromatin-associated proteins, which can be an obstacle to efficient repair. Several cooperating mechanisms enhance the efficiency of NER by altering chromatin structure. Interestingly, many of the players involved in modifying chromatin at sites of DNA damage were originally identified as regulators of transcription. These include ATP-dependent chromatin remodelers, histone modifying enzymes and several transcription factors. The p53 and E2F1 transcription factors are well known for their abilities to regulate gene expression in response to DNA damage. This review will highlight the underappreciated, transcription-independent functions of p53 and E2F1 in modifying chromatin structure in response to DNA damage to promote global NER.

  9. Transcriptional and Posttranslational Regulation of Nucleotide Excision Repair: The Guardian of the Genome against Ultraviolet Radiation

    Directory of Open Access Journals (Sweden)

    Jeong-Min Park

    2016-11-01

    Full Text Available Ultraviolet (UV radiation from sunlight represents a constant threat to genome stability by generating modified DNA bases such as cyclobutane pyrimidine dimers (CPD and pyrimidine-pyrimidone (6-4 photoproducts (6-4PP. If unrepaired, these lesions can have deleterious effects, including skin cancer. Mammalian cells are able to neutralize UV-induced photolesions through nucleotide excision repair (NER. The NER pathway has multiple components including seven xeroderma pigmentosum (XP proteins (XPA to XPG and numerous auxiliary factors, including ataxia telangiectasia and Rad3-related (ATR protein kinase and RCC1 like domain (RLD and homologous to the E6-AP carboxyl terminus (HECT domain containing E3 ubiquitin protein ligase 2 (HERC2. In this review we highlight recent data on the transcriptional and posttranslational regulation of NER activity.

  10. Nucleotide excision repair is a potential therapeutic target in multiple myeloma

    Science.gov (United States)

    Szalat, R; Samur, M K; Fulciniti, M; Lopez, M; Nanjappa, P; Cleynen, A; Wen, K; Kumar, S; Perini, T; Calkins, A S; Reznichenko, E; Chauhan, D; Tai, Y-T; Shammas, M A; Anderson, K C; Fermand, J-P; Arnulf, B; Avet-Loiseau, H; Lazaro, J-B; Munshi, N C

    2018-01-01

    Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM. PMID:28588253

  11. True Lies: The Double Life of the Nucleotide Excision Repair Factors in Transcription and DNA Repair

    Directory of Open Access Journals (Sweden)

    Nicolas Le May

    2010-01-01

    Full Text Available Nucleotide excision repair (NER is a major DNA repair pathway in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation or bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to three genetic disorders that result in predisposition to cancers, accelerated aging, neurological and developmental defects. During NER, more than 30 polypeptides cooperate to recognize, incise, and excise a damaged oligonucleotide from the genomic DNA. Recent papers reveal an additional and unexpected role for the NER factors. In the absence of a genotoxic attack, the promoters of RNA polymerases I- and II-dependent genes recruit XPA, XPC, XPG, and XPF to initiate gene expression. A model that includes the growth arrest and DNA damage 45α protein (Gadd45α and the NER factors, in order to maintain the promoter of active genes under a hypomethylated state, has been proposed but remains controversial. This paper focuses on the double life of the NER factors in DNA repair and transcription and describes the possible roles of these factors in the RNA synthesis process.

  12. A multistep damage recognition mechanism for global genomic nucleotide excision repair.

    Science.gov (United States)

    Sugasawa, K; Okamoto, T; Shimizu, Y; Masutani, C; Iwai, S; Hanaoka, F

    2001-03-01

    A mammalian nucleotide excision repair (NER) factor, the XPC-HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free repair reaction. Here we describe a detailed analysis of its binding specificity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC-HR23B specifically binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at least two steps; XPC-HR23B first binds to a site that has a DNA helix distortion, and then the presence of injured bases is verified prior to dual incision. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC-HR23B, suggesting that additional factors may be required for CPD recognition. Although the presence of mismatched bases opposite a CPD potentiated XPC-HR23B binding, probably due to enhancement of the helix distortion, cell-free excision of such compound lesions was much more efficient than expected from the observed affinity for XPC-HR23B. This also suggests that additional factors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensuring the high level of damage discrimination that is required for global genomic NER.

  13. Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Wang, Z.; Wu, X.; Friedberg, E.C.

    1993-01-01

    A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg 2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae

  14. Removal of misincorporated ribonucleotides from prokaryotic genomes: an unexpected role for nucleotide excision repair.

    Directory of Open Access Journals (Sweden)

    Alexandra Vaisman

    2013-11-01

    Full Text Available Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER. We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8(th phosphodiester bond 5' and 4(th-5(th phosphodiester bonds 3' of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be

  15. Nucleotide sequence preservation of human mitochondrial DNA

    International Nuclear Information System (INIS)

    Monnat, R.J. Jr.; Loeb, L.A.

    1985-01-01

    Recombinant DNA techniques have been used to quantitate the amount of nucleotide sequence divergence in the mitochondrial DNA population of individual normal humans. Mitochondrial DNA was isolated from the peripheral blood lymphocytes of five normal humans and cloned in M13 mp11; 49 kilobases of nucleotide sequence information was obtained from 248 independently isolated clones from the five normal donors. Both between- and within-individual differences were identified. Between-individual differences were identified in approximately = to 1/200 nucleotides. In contrast, only one within-individual difference was identified in 49 kilobases of nucleotide sequence information. This high degree of mitochondrial nucleotide sequence homogeneity in human somatic cells is in marked contrast to the rapid evolutionary divergence of human mitochondrial DNA and suggests the existence of mechanisms for the concerted preservation of mammalian mitochondrial DNA sequences in single organisms

  16. Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair.

    Science.gov (United States)

    Mazloum, Nayef; Stegman, Melanie A; Croteau, Deborah L; Van Houten, Bennett; Kwon, Nyoun Soo; Ling, Yan; Dickinson, Caitlyn; Venugopal, Aditya; Towheed, Mohammad Atif; Nathan, Carl

    2011-03-01

    Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen's ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb's Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.

  17. PCAF/GCN5-Mediated Acetylation of RPA1 Promotes Nucleotide Excision Repair

    Directory of Open Access Journals (Sweden)

    Meimei Zhao

    2017-08-01

    Full Text Available The RPA complex can integrate multiple stress signals into diverse responses by activating distinct DNA repair pathways. However, it remains unclear how RPA1 elects to activate a specific repair pathway during different types of DNA damage. Here, we report that PCAF/GCN5-mediated K163 acetylation of RPA1 is crucial for nucleotide excision repair (NER but is dispensable for other DNA repair pathways. Mechanistically, we demonstrate that the acetylation of RPA1 is critical for the steady accumulation of XPA at damaged DNA sites and preferentially activates the NER pathway. DNA-PK phosphorylates and activates PCAF upon UV damage and consequently promotes the acetylation of RPA1. Moreover, the acetylation of RPA1 is tightly regulated by HDAC6 and SIRT1. Together, our results demonstrate that the K163 acetylation of RPA1 plays a key role in the repair of UV-induced DNA damage and reveal how the specific RPA1 modification modulates the choice of distinct DNA repair pathways.

  18. Physical interaction between components of DNA mismatch repair and nucleotide excision repair

    International Nuclear Information System (INIS)

    Bertrand, P.; Tishkoff, D.X.; Filosi, N.; Dasgupta, R.; Kolodner, R.D.

    1998-01-01

    Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as 'bait,' and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes

  19. The Differential Expression of Core Genes in Nucleotide Excision Repair Pathway Indicates Colorectal Carcinogenesis and Prognosis

    Directory of Open Access Journals (Sweden)

    Jingwei Liu

    2018-01-01

    Full Text Available Background. Nucleotide excision repair (NER plays a critical role in maintaining genome integrity. This study aimed to investigate the expression of NER genes and their associations with colorectal cancer (CRC development. Method. Expressions of NER genes in CRC and normal tissues were analysed by ONCOMINE. The Cancer Genome Atlas (TCGA data were downloaded to explore relationship of NER expression with clinicopathological parameters and survival of CRC. Results. ERCC1, ERCC2, ERCC5, and DDB2 were upregulated while ERCC4 was downregulated in CRC. For colon cancer, high ERCC3 expression was related to better T stage; ERCC5 expression indicated deeper T stage and distant metastasis; DDB2 expression suggested earlier TNM stage. For rectal cancer, ERCC2 expression correlated with favourable T stage; XPA expression predicted worse TNM stage. ERCC2 expression was associated with worse overall survival (OS in colon cancer (HR=1.53, P=0.043. Colon cancer patients with high ERCC4 expression showed favorable OS in males (HR=0.54, P=0.035. High XPC expression demonstrated decreased death hazards in rectal cancer (HR=0.40, P=0.026. Conclusion. ERCC1, ERCC2, ERCC4, ERCC5, and DDB2 were differently expressed in CRC and normal tissues; ERCC2, ERCC3, ERCC5, XPA, and DDB2 correlated with clinicopathological parameters of CRC, while ERCC2, ERCC4, and XPC might predict CRC prognosis.

  20. Conservation of the nucleotide excision repair pathway: characterization of hydra Xeroderma Pigmentosum group F homolog.

    Directory of Open Access Journals (Sweden)

    Apurva Barve

    Full Text Available Hydra, one of the earliest metazoans with tissue grade organization and nervous system, is an animal with a remarkable regeneration capacity and shows no signs of organismal aging. We have for the first time identified genes of the nucleotide excision repair (NER pathway from hydra. Here we report cloning and characterization of hydra homolog of xeroderma pigmentosum group F (XPF gene that encodes a structure-specific 5' endonuclease which is a crucial component of NER. In silico analysis shows that hydra XPF amino acid sequence is very similar to its counterparts from other animals, especially vertebrates, and shows all features essential for its function. By in situ hybridization, we show that hydra XPF is expressed prominently in the multipotent stem cell niche in the central region of the body column. Ectoderm of the diploblastic hydra was shown to express higher levels of XPF as compared to the endoderm by semi-quantitative RT-PCR. Semi-quantitative RT-PCR analysis also demonstrated that interstitial cells, a multipotent and rapidly cycling stem cell lineage of hydra, express higher levels of XPF mRNA than other cell types. Our data show that XPF and by extension, the NER pathway is highly conserved during evolution. The prominent expression of an NER gene in interstitial cells may have implications for the lack of senescence in hydra.

  1. The Role of Altered Nucleotide Excision Repair and UVB-Induced DNA Damage in Melanomagenesis

    Directory of Open Access Journals (Sweden)

    Timothy Budden

    2013-01-01

    Full Text Available UVB radiation is the most mutagenic component of the UV spectrum that reaches the earth’s surface and causes the development of DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts. UV radiation usually results in cellular death, but if left unchecked, it can affect DNA integrity, cell and tissue homeostasis and cause mutations in oncogenes and tumour-suppressor genes. These mutations, if unrepaired, can lead to abnormal cell growth, increasing the risk of cancer development. Epidemiological data strongly associates UV exposure as a major factor in melanoma development, but the exact biological mechanisms involved in this process are yet to be fully elucidated. The nucleotide excision repair (NER pathway is responsible for the repair of UV-induced lesions. Patients with the genetic disorder Xeroderma Pigmentosum have a mutation in one of eight NER genes associated with the XP complementation groups XP-A to XP-G and XP variant (XP-V. XP is characterized by diminished repair capacity, as well as a 1000-fold increase in the incidence of skin cancers, including melanoma. This has suggested a significant role for NER in melanoma development as a result of UVB exposure. This review discusses the current research surrounding UVB radiation and NER capacity and how further investigation of NER could elucidate the role of NER in avoiding UV-induced cellular death resulting in melanomagenesis.

  2. DNA excision repair in permeable human fibroblasts

    International Nuclear Information System (INIS)

    Kaufmann, W.K.; Bodell, W.J.; Cleaver, J.E.

    1983-01-01

    U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the [3H]DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells

  3. Decreased nucleotide excision repair in steatotic livers associates with myeloperoxidase-immunoreactivity

    International Nuclear Information System (INIS)

    Schults, Marten A.; Nagle, Peter W.; Rensen, Sander S.; Godschalk, Roger W.; Munnia, Armelle; Peluso, Marco; Claessen, Sandra M.; Greve, Jan W.; Driessen, Ann; Verdam, Froukje J.; Buurman, Wim A.; Schooten, Frederik J. van; Chiu, Roland K.

    2012-01-01

    Chronic inflammation is characterized by the influx of neutrophils and is associated with an increased production of reactive oxygen species that can damage DNA. Oxidative DNA damage is generally thought to be involved in the increased risk of cancer in inflamed tissues. We previously demonstrated that activated neutrophil mediated oxidative stress results in a reduction in nucleotide excision repair (NER) capacity, which could further enhance mutagenesis. Inflammation and oxidative stress are critical factors in the progression of nonalcoholic fatty liver disease that is linked with enhanced liver cancer risk. In this report, we therefore evaluated the role of neutrophils and the associated oxidative stress in damage recognition and DNA repair in steatotic livers of 35 severely obese subjects with either nonalcoholic steatohepatitis (NASH) (n = 17) or steatosis alone (n = 18). The neutrophilic influx in liver was assessed by myeloperoxidase (MPO) staining and the amount of oxidative DNA damage by measuring M 1 dG adducts. No differences in M 1 dG adduct levels were observed between patients with or without NASH and also not between individuals with high or low MPO immunoreactivity. However, we found that high expression of MPO in the liver, irrespective of disease status, reduced the damage recognition capacity as determined by staining for histone 2AX phosphorylation (γH2AX). This reduction in γH2AX formation in individuals with high MPO immunoreactivity was paralleled by a significant decrease in NER capacity as assessed by a functional repair assay, and was not related to cell proliferation. Thus, the observed reduction in NER capacity upon hepatic inflammation is associated with and may be a consequence of reduced damage recognition. These findings suggest a novel mechanism of liver cancer development in patients with nonalcoholic fatty liver disease.

  4. The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

    Science.gov (United States)

    Kozmin, Stanislav G.; Jinks-Robertson, Sue

    2013-01-01

    Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps. PMID:23307894

  5. Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing.

    Science.gov (United States)

    Calmels, Nadège; Greff, Géraldine; Obringer, Cathy; Kempf, Nadine; Gasnier, Claire; Tarabeux, Julien; Miguet, Marguerite; Baujat, Geneviève; Bessis, Didier; Bretones, Patricia; Cavau, Anne; Digeon, Béatrice; Doco-Fenzy, Martine; Doray, Bérénice; Feillet, François; Gardeazabal, Jesus; Gener, Blanca; Julia, Sophie; Llano-Rivas, Isabel; Mazur, Artur; Michot, Caroline; Renaldo-Robin, Florence; Rossi, Massimiliano; Sabouraud, Pascal; Keren, Boris; Depienne, Christel; Muller, Jean; Mandel, Jean-Louis; Laugel, Vincent

    2016-03-22

    Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS). Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies). We identified causative mutations in 17 out of the 40 patients (43%). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes. Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects.

  6. Decreased nucleotide excision repair in steatotic livers associates with myeloperoxidase-immunoreactivity

    Energy Technology Data Exchange (ETDEWEB)

    Schults, Marten A.; Nagle, Peter W. [Department of Toxicology, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Rensen, Sander S. [Department of Surgery, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Godschalk, Roger W. [Department of Toxicology, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Munnia, Armelle; Peluso, Marco [Cancer Risk Factor Branch, ISPO Cancer Prevention and Research Institute, Via Cosimo il Vecchio 2, 50139 Florence (Italy); Claessen, Sandra M. [Department of Toxicogenomics, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Greve, Jan W. [Department of Surgery, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Driessen, Ann [Department of Pathology, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Verdam, Froukje J.; Buurman, Wim A. [Department of Surgery, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Schooten, Frederik J. van [Department of Toxicology, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands); Chiu, Roland K., E-mail: r.k.chiu@med.umcg.nl [Department of Toxicology, NUTRIM-School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 616, 6200 MD Maastricht (Netherlands)

    2012-08-01

    Chronic inflammation is characterized by the influx of neutrophils and is associated with an increased production of reactive oxygen species that can damage DNA. Oxidative DNA damage is generally thought to be involved in the increased risk of cancer in inflamed tissues. We previously demonstrated that activated neutrophil mediated oxidative stress results in a reduction in nucleotide excision repair (NER) capacity, which could further enhance mutagenesis. Inflammation and oxidative stress are critical factors in the progression of nonalcoholic fatty liver disease that is linked with enhanced liver cancer risk. In this report, we therefore evaluated the role of neutrophils and the associated oxidative stress in damage recognition and DNA repair in steatotic livers of 35 severely obese subjects with either nonalcoholic steatohepatitis (NASH) (n = 17) or steatosis alone (n = 18). The neutrophilic influx in liver was assessed by myeloperoxidase (MPO) staining and the amount of oxidative DNA damage by measuring M{sub 1}dG adducts. No differences in M{sub 1}dG adduct levels were observed between patients with or without NASH and also not between individuals with high or low MPO immunoreactivity. However, we found that high expression of MPO in the liver, irrespective of disease status, reduced the damage recognition capacity as determined by staining for histone 2AX phosphorylation ({gamma}H2AX). This reduction in {gamma}H2AX formation in individuals with high MPO immunoreactivity was paralleled by a significant decrease in NER capacity as assessed by a functional repair assay, and was not related to cell proliferation. Thus, the observed reduction in NER capacity upon hepatic inflammation is associated with and may be a consequence of reduced damage recognition. These findings suggest a novel mechanism of liver cancer development in patients with nonalcoholic fatty liver disease.

  7. Nucleotide Excision Repair and Vitamin D--Relevance for Skin Cancer Therapy.

    Science.gov (United States)

    Pawlowska, Elzbieta; Wysokinski, Daniel; Blasiak, Janusz

    2016-04-06

    Ultraviolet (UV) radiation is involved in almost all skin cancer cases, but on the other hand, it stimulates the production of pre-vitamin D3, whose active metabolite, 1,25-dihydroxyvitamin D3 (1,25VD3), plays important physiological functions on binding with its receptor (vitamin D receptor, VDR). UV-induced DNA damages in the form of cyclobutane pyrimidine dimers or (6-4)-pyrimidine-pyrimidone photoproducts are frequently found in skin cancer and its precursors. Therefore, removing these lesions is essential for the prevention of skin cancer. As UV-induced DNA damages are repaired by nucleotide excision repair (NER), the interaction of 1,25VD3 with NER components can be important for skin cancer transformation. Several studies show that 1,25VD3 protects DNA against damage induced by UV, but the exact mechanism of this protection is not completely clear. 1,25VD3 was also shown to affect cell cycle regulation and apoptosis in several signaling pathways, so it can be considered as a potential modulator of the cellular DNA damage response, which is crucial for mutagenesis and cancer transformation. 1,25VD3 was shown to affect DNA repair and potentially NER through decreasing nitrosylation of DNA repair enzymes by NO overproduction by UV, but other mechanisms of the interaction between 1,25VD3 and NER machinery also are suggested. Therefore, the array of NER gene functioning could be analyzed and an appropriate amount of 1.25VD3 could be recommended to decrease UV-induced DNA damage important for skin cancer transformation.

  8. Deficiency in nucleotide excision repair family gene activity, especially ERCC3, is associated with non-pigmented hair fiber growth.

    Directory of Open Access Journals (Sweden)

    Mei Yu

    Full Text Available We conducted a microarray study to discover gene expression patterns associated with a lack of melanogenesis in non-pigmented hair follicles (HF by microarray. Pigmented and non-pigmented HFs were collected and micro-dissected into the hair bulb (HB and the upper hair sheaths (HS including the bulge region. In comparison to pigmented HS and HBs, nucleotide excision repair (NER family genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH exhibited statistically significantly lower expression in non- pigmented HS and HBs. Quantitative PCR verified microarray data and identified ERCC3 as highly differentially expressed. Immunohistochemistry confirmed ERCC3 expression in HF melanocytes. A reduction in ERCC3 by siRNA interference in human melanocytes in vitro reduced their tyrosinase production ability. Our results suggest that loss of NER gene function is associated with a loss of melanin production capacity. This may be due to reduced gene transcription and/or reduced DNA repair in melanocytes which may eventually lead to cell death. These results provide novel information with regard to melanogenesis and its regulation.

  9. Important role of the nucleotide excision repair pathway in Mycobacterium smegmatis in conferring protection against commonly encountered DNA-damaging agents.

    Science.gov (United States)

    Kurthkoti, Krishna; Kumar, Pradeep; Jain, Ruchi; Varshney, Umesh

    2008-09-01

    Mycobacteria are an important group of human pathogens. Although the DNA repair mechanisms in mycobacteria are not well understood, these are vital for the pathogen's persistence in the host macrophages. In this study, we generated a null mutation in the uvrB gene of Mycobacterium smegmatis to allow us to compare the significance of the nucleotide excision repair (NER) pathway with two important base excision repair pathways, initiated by uracil DNA glycosylase (Ung) and formamidopyrimidine DNA glycosylase (Fpg or MutM), in an isogenic strain background. The strain deficient in NER was the most sensitive to commonly encountered DNA-damaging agents such as UV, low pH, reactive oxygen species, hypoxia, and was also sensitive to acidified nitrite. Taken together with previous observations on NER-deficient M. tuberculosis, these results suggest that NER is an important DNA repair pathway in mycobacteria.

  10. The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells

    International Nuclear Information System (INIS)

    Abbasi, Rashda; Efferth, Thomas; Kuhmann, Christine; Opatz, Till; Hao, Xiaojiang; Popanda, Odilia; Schmezer, Peter

    2012-01-01

    Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC 50 values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of > 1000-fold in resistance between normal and NER-deficient cells (IC 50 values for cells with deficiency in ERCC6: 0.15 μM, XPC: 0.18 μM, and normal cells: > 180 μM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes. -- Highlights: ► Thousand-fold higher Ascaridol activity in NER-deficient versus proficient cells. ► Impaired repair of Ascaridol-induced oxidative DNA damage in NER-deficient cells. ► Selective activity of Ascaridol opens new therapy options in

  11. Gastroesophageal junction adenocarcinoma displays abnormalities in homologous recombination and nucleotide excision repair

    Directory of Open Access Journals (Sweden)

    Dewalt RI

    2014-02-01

    Full Text Available Robin I Dewalt,1 Kenneth A Kesler,2 Zane T Hammoud,3 LeeAnn Baldridge,4 Eyas M Hattab,4 Shadia I Jalal1,5 1Division of Hematology/Oncology, Department of Medicine, 2Cardiothoracic Division, Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA; 3Henry Ford Hospital, Detroit, MI, USA; 4Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; 5Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, USA Objective: Esophageal adenocarcinoma (EAC continues to be a disease associated with high mortality. Among the factors leading to poor outcomes are innate resistance to currently available therapies, advanced stage at diagnosis, and complex biology. Platinum and ionizing radiation form the backbone of treatment for the majority of patients with EAC. Of the multiple processes involved in response to platinum chemotherapy or ionizing radiation, deoxyribonucleic acid (DNA repair has been a major player in cancer sensitivity to these agents. DNA repair defects have been described in various malignancies. The purpose of this study was to determine whether alterations in DNA repair are present in EAC compared with normal gastroesophageal tissues. Methods: We analyzed the expression of genes involved in homologous recombination (HR, nonhomologous end-joining, and nucleotide excision repair (NER pathways in 12 EAC tumor samples with their matched normal counterparts. These pathways were chosen because they are the main pathways involved in the repair of platinum- or ionizing-radiation-induced damage. In addition, abnormalities in these pathways have not been well characterized in EAC. Results: We identified increased expression of at least one HR gene in eight of the EAC tumor samples. Alterations in the expression of EME1, a structure-specific endonuclease involved in HR, were the most prevalent, with messenger (mRNA overexpression in six of the EAC samples

  12. The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells

    Energy Technology Data Exchange (ETDEWEB)

    Abbasi, Rashda [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Efferth, Thomas [Institute of Pharmacy und Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany); Kuhmann, Christine [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Opatz, Till [Institute of Organic Chemistry, Johannes Gutenberg University, Duesbergweg 10-14, 55128 Mainz (Germany); Hao, Xiaojiang [Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204 (China); Popanda, Odilia, E-mail: o.popanda@dkfz.de [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Schmezer, Peter [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany)

    2012-03-15

    Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC{sub 50} values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of > 1000-fold in resistance between normal and NER-deficient cells (IC{sub 50} values for cells with deficiency in ERCC6: 0.15 μM, XPC: 0.18 μM, and normal cells: > 180 μM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes. -- Highlights: ► Thousand-fold higher Ascaridol activity in NER-deficient versus proficient cells. ► Impaired repair of Ascaridol-induced oxidative DNA damage in NER-deficient cells. ► Selective activity of Ascaridol opens new therapy

  13. Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay - A methodological overview

    Czech Academy of Sciences Publication Activity Database

    Azqueta, A.; Langie, S. A. S.; Slyšková, Jana; Collins, A. R.

    2013-01-01

    Roč. 12, č. 11 (2013), s. 1007-1010 ISSN 1568-7864 Grant - others:EU FP6(XE) LSHB-CT-2006-037575 Institutional support: RVO:68378041 Keywords : comet assay * base excision repair * nucleotide excision repair Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.362, year: 2013

  14. Silymarin protects epidermal keratinocytes from ultraviolet radiation-induced apoptosis and DNA damage by nucleotide excision repair mechanism.

    Directory of Open Access Journals (Sweden)

    Santosh K Katiyar

    Full Text Available Solar ultraviolet (UV radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum, inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells.

  15. A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of global genome nucleotide excision repair.

    Science.gov (United States)

    Wong, J M; Ingles, C J

    2001-02-01

    Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation. In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively. A RAD7/16-dependent reduction in survival after UV irradiation was also seen in the presence of mutations in RNA Pol II that confer a defect in its response to SII, as well as with other mutations which reside in regions of the largest subunit of Pol II not involved in SII interactions. Indeed, an increase in UV sensitivity was achieved by simply decreasing the steadystate level of RNA Pol II. Truncation of the C-terminal domain and other RNA Pol II mutations conferred sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and RNR2 mRNAs after UV irradiation was attenuated in these mutant cells. That UV sensitivity can be a consequence of mutations in the RNA Pol II machinery in yeast cells suggests that alterations in transcriptional programs could underlie some of the pathophysiological defects seen in the human disease Cockayne syndrome.

  16. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription*

    Science.gov (United States)

    Nadkarni, Aditi; Burns, John A.; Gandolfi, Alberto; Chowdhury, Moinuddin A.; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E.; Scicchitano, David A.

    2016-01-01

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. PMID:26559971

  17. Homology modeling, molecular docking and DNA binding studies of nucleotide excision repair UvrC protein from M. tuberculosis.

    Science.gov (United States)

    Parulekar, Rishikesh S; Barage, Sagar H; Jalkute, Chidambar B; Dhanavade, Maruti J; Fandilolu, Prayagraj M; Sonawane, Kailas D

    2013-08-01

    Mycobacterium tuberculosis is a Gram positive, acid-fast bacteria belonging to genus Mycobacterium, is the leading causative agent of most cases of tuberculosis. The pathogenicity of the bacteria is enhanced by its developed DNA repair mechanism which consists of machineries such as nucleotide excision repair. Nucleotide excision repair consists of excinuclease protein UvrABC endonuclease, multi-enzymatic complex which carries out repair of damaged DNA in sequential manner. UvrC protein is a part of this complex and thus helps to repair the damaged DNA of M. tuberculosis. Hence, structural bioinformatics study of UvrC protein from M. tuberculosis was carried out using homology modeling and molecular docking techniques. Assessment of the reliability of the homology model was carried out by predicting its secondary structure along with its model validation. The predicted structure was docked with the ATP and the interacting amino acid residues of UvrC protein with the ATP were found to be TRP539, PHE89, GLU536, ILE402 and ARG575. The binding of UvrC protein with the DNA showed two different domains. The residues from domain I of the protein VAL526, THR524 and LEU521 interact with the DNA whereas, amino acids interacting from the domain II of the UvrC protein included ARG597, GLU595, GLY594 and GLY592 residues. This predicted model could be useful to design new inhibitors of UvrC enzyme to prevent pathogenesis of Mycobacterium and so the tuberculosis.

  18. NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jeong-Min; Choi, Ji Ye [Department of Biological Science, Dong-A University, Busan (Korea, Republic of); Yi, Joo Mi [Research Center, Dongnam Institute of Radiological & Medical Sciences, Busan (Korea, Republic of); Chung, Jin Woong; Leem, Sun-Hee; Koh, Sang Seok [Department of Biological Science, Dong-A University, Busan (Korea, Republic of); Kang, Tae-Hong, E-mail: thkang@dau.ac.kr [Department of Biological Science, Dong-A University, Busan (Korea, Republic of)

    2015-06-05

    Nucleotide excision repair (NER) is the sole mechanism of UV-induced DNA lesion repair in mammals. A single round of NER requires multiple components including seven core NER factors, xeroderma pigmentosum A–G (XPA–XPG), and many auxiliary effector proteins including ATR serine/threonine kinase. The XPA protein helps to verify DNA damage and thus plays a rate-limiting role in NER. Hence, the regulation of XPA is important for the entire NER kinetic. We found that NDR1, a novel XPA-interacting protein, modulates NER by modulating the UV-induced DNA-damage checkpoint. In quiescent cells, NDR1 localized mainly in the cytoplasm. After UV irradiation, NDR1 accumulated in the nucleus. The siRNA knockdown of NDR1 delayed the repair of UV-induced cyclobutane pyrimidine dimers in both normal cells and cancer cells. It did not, however, alter the expression levels or the chromatin association levels of the core NER factors following UV irradiation. Instead, the NDR1-depleted cells displayed reduced activity of ATR for some set of its substrates including CHK1 and p53, suggesting that NDR1 modulates NER indirectly via the ATR pathway. - Highlights: • NDR1 is a novel XPA-interacting protein. • NDR1 accumulates in the nucleus in response to UV irradiation. • NDR1 modulates NER (nucleotide excision repair) by modulating the UV-induced DNA-damage checkpoint response.

  19. Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription.

    Science.gov (United States)

    Nadkarni, Aditi; Burns, John A; Gandolfi, Alberto; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Geacintov, Nicholas E; Scicchitano, David A

    2016-01-08

    DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. New insights in the removal of the hydantoins, oxidation product of pyrimidines, via the base excision and nucleotide incision repair pathways.

    Directory of Open Access Journals (Sweden)

    Modesto Redrejo-Rodríguez

    Full Text Available BACKGROUND: Oxidative damage to DNA, if not repaired, can be both miscoding and blocking. These genetic alterations can lead to mutations and/or cell death, which in turn cause cancer and aging. Oxidized DNA bases are substrates for two overlapping repair pathways: base excision (BER and nucleotide incision repair (NIR. Hydantoin derivatives such as 5-hydroxyhydantoin (5OH-Hyd and 5-methyl-5-hydroxyhydantoin (5OH-5Me-Hyd, major products of cytosine and thymine oxidative degradation pathways, respectively, have been detected in cancer cells and ancient DNA. Hydantoins are blocking lesions for DNA polymerases and excised by bacterial and yeast DNA glycosylases in the BER pathway. However little is known about repair of pyrimidine-derived hydantoins in human cells. METHODOLOGY/PRINCIPAL FINDINGS: Here, using both denaturing PAGE and MALDI-TOF MS analyses we report that the bacterial, yeast and human AP endonucleases can incise duplex DNA 5' next to 5OH-Hyd and 5OH-5Me-Hyd thus initiating the NIR pathway. We have fully reconstituted the NIR pathway for these lesions in vitro using purified human proteins. Depletion of Nfo in E. coli and APE1 in HeLa cells abolishes the NIR activity in cell-free extracts. Importantly, a number of redundant DNA glycosylase activities can excise hydantoin residues, including human NTH1, NEIL1 and NEIL2 and the former protein being a major DNA glycosylase activity in HeLa cells extracts. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that both BER and NIR pathways can compete and/or back-up each other to remove hydantoin DNA lesions in vivo.

  1. Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time.

    Science.gov (United States)

    Kong, Muwen; Beckwitt, Emily C; Springall, Luke; Kad, Neil M; Van Houten, Bennett

    2017-01-01

    Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein-DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair. © 2017 Elsevier Inc. All rights reserved.

  2. Both ATPase sites of Escherichia coli UvrA have functional roles in nucleotide excision repair

    International Nuclear Information System (INIS)

    Thiagalingam, S.; Grossman, L.

    1991-01-01

    The roles of the two tandemly arranged putative ATP binding sites of Escherichia coli UvrA in UvrABC endonuclease-mediated excision repair were analyzed by site-directed mutagenesis and biochemical characterization of the representative mutant proteins. Evidence is presented that UvrA has two functional ATPase sites which coincide with the putative ATP binding motifs predicted from its amino acid sequence. The individual ATPase sites can independently hydrolyze ATP. The C-terminal ATPase site has a higher affinity for ATP than the N-terminal site. The invariable lysine residues at the ends of the glycine-rich loops of the consensus Walker type A motifs are indispensable for ATP hydrolysis. However, the mutations at these lysine residues do not significantly affect ATP binding. UvrA, with bound ATP, forms the most favored conformation for DNA binding. The initial binding of UvrA to DNA is chiefly at the undamaged sites. In contrast to the wild type UvrA, the ATPase site mutants bind equally to damaged and undamaged sites. Dissociation of tightly bound nucleoprotein complexes from the undamaged sites requires hydrolysis of ATP by the C-terminal ATPase site of UvrA. Thus, both ATP binding and hydrolysis are required for the damage recognition step enabling UvrA to discriminate between damaged and undamaged sites on DNA

  3. Proteins of nucleotide and base excision repair pathways interact in mitochondria to protect from loss of subcutaneous fat, a hallmark of aging

    NARCIS (Netherlands)

    Y. Kamenisch (York); M.I. Fousteri (Maria); J. Knoch (Jennifer); A.K. Von Thaler (Anna Katherina); B. Fehrenbacher (Birgit); H. Kato (Hiroki); T. Becker (Tim); M.E.T. Dollé (Martijn); R. Kuiper (Ruud); M. Majora (Marc); M. Schaller (Martin); G.T.J. van der Horst (Gijsbertus); H. van Steeg (Harry); M. Röcken (Martin); D. Rapaport (Doron); J. Krutmann (Jean); L.H.F. Mullenders (Leon); M. Berneburg (Mark)

    2010-01-01

    textabstractDefects in the DNA repair mechanism nucleotide excision repair (NER) may lead to tumors in xeroderma pigmentosum (XP) or to premature aging with loss of subcutaneous fat in Cockayne syndrome (CS). Mutations of mitochondrial (mt)DNA play a role in aging, but a link between the

  4. Recombinant methods for screening human DNA excision repair proficiency

    International Nuclear Information System (INIS)

    Athas, W.F.

    1988-01-01

    A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period

  5. Pyrimidine dimer excision in human cells and skin cancer

    International Nuclear Information System (INIS)

    Regan, J.D.; Carrier, W.L.; Smith, D.P.; Waters, R.

    1977-01-01

    We have compared three different methods for estimating the induction and removal of uv induced pyrimidine dimers from the DNA of human fibroblasts. Results indicate that after uv doses of 5-20 J/m 2 50% of the dimers are removed by 24 hours after irradiation. Almost complete excision can be observed if the cells are incubated for periods not less than 72 hours after 5 J/m 2 . After higher doses it probably takes even longer fr such complete removal to be seen

  6. Bypass of a 5',8-cyclopurine-2'-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks.

    Science.gov (United States)

    Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E; Terzidis, Michael A; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

    2015-09-01

    5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair.

    Science.gov (United States)

    Krasikova, Yuliya S; Rechkunova, Nadejda I; Maltseva, Ekaterina A; Lavrik, Olga I

    2018-01-01

    Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.

  8. A UV-Induced Genetic Network Links the RSC Complex to Nucleotide Excision Repair and Shows Dose-Dependent Rewiring

    Directory of Open Access Journals (Sweden)

    Rohith Srivas

    2013-12-01

    Full Text Available Efficient repair of UV-induced DNA damage requires the precise coordination of nucleotide excision repair (NER with numerous other biological processes. To map this crosstalk, we generated a differential genetic interaction map centered on quantitative growth measurements of >45,000 double mutants before and after different doses of UV radiation. Integration of genetic data with physical interaction networks identified a global map of 89 UV-induced functional interactions among 62 protein complexes, including a number of links between the RSC complex and several NER factors. We show that RSC is recruited to both silenced and transcribed loci following UV damage where it facilitates efficient repair by promoting nucleosome remodeling. Finally, a comparison of the response to high versus low levels of UV shows that the degree of genetic rewiring correlates with dose of UV and reveals a network of dose-specific interactions. This study makes available a large resource of UV-induced interactions, and it illustrates a methodology for identifying dose-dependent interactions based on quantitative shifts in genetic networks.

  9. Relationship between polymorphisms of nucleotide excision repair genes and oral cancer risk in Taiwan: evidence for modification of smoking habit.

    Science.gov (United States)

    Bau, Da-Tian; Tsai, Ming-Hsui; Huang, Chih-Yang; Lee, Cheng-Chun; Tseng, Hsien-Chang; Lo, Yen-Li; Tsai, Yuhsin; Tsai, Fuu-Jen

    2007-12-31

    Inherited polymorphisms in DNA repair genes may be associated with differences in the repair capacity and contribute to individual's susceptibility to smoking-related cancers. Both XPA and XPD encode proteins that are part of the nucleotide excision repair (NER) pathway. In a hospital-based case-control study, we have investigated the influence of XPA A-23G and XPD Lys751Gln polymorphisms on oral cancer risk in a Taiwanese population. In total, 154 patients with oral cancer, and 105 age-matched controls recruited from the Chinese Medical Hospital in Central Taiwan were genotyped. No significant association was found between the heterozygous variant allele (AG), the homozygous variant allele (AA) at XPA A-23G, the heterozygous variant allele (AC), the homozygous variant allele (CC) at XPD Lys751Gln, and oral cancer risk. There was no significant joint effect of XPA A-23G and XPD Lys751Gln on oral cancer risk either. Since XPA and XPD are both NER genes, which are very important in removing tobacco-induced DNA adducts, further stratified analyses of both genotype and smoking habit were performed. We found a synergistic effect of variant genotypes of both XPA and XPD, and smoking status on oral cancer risk. Our results suggest that the genetic polymorphisms are modified by environmental carcinogen exposure status, and combined analyses of both genotype and personal habit record are a better access to know the development of oral cancer and useful for primary prevention and early intervention.

  10. Major Roles for Pyrimidine Dimers, Nucleotide Excision Repair, and ATR in the Alternative Splicing Response to UV Irradiation

    Directory of Open Access Journals (Sweden)

    Manuel J. Muñoz

    2017-03-01

    Full Text Available We have previously found that UV irradiation promotes RNA polymerase II (RNAPII hyperphosphorylation and subsequent changes in alternative splicing (AS. We show now that UV-induced DNA damage is not only necessary but sufficient to trigger the AS response and that photolyase-mediated removal of the most abundant class of pyrimidine dimers (PDs abrogates the global response to UV. We demonstrate that, in keratinocytes, RNAPII is the target, but not a sensor, of the signaling cascade initiated by PDs. The UV effect is enhanced by inhibition of gap-filling DNA synthesis, the last step in the nucleotide excision repair pathway (NER, and reduced by the absence of XPE, the main NER sensor of PDs. The mechanism involves activation of the protein kinase ATR that mediates the UV-induced RNAPII hyperphosphorylation. Our results define the sequence UV-PDs-NER-ATR-RNAPII-AS as a pathway linking DNA damage repair to the control of both RNAPII phosphorylation and AS regulation.

  11. Calcium-binding capacity of centrin2 is required for linear POC5 assembly but not for nucleotide excision repair.

    Directory of Open Access Journals (Sweden)

    Tiago J Dantas

    Full Text Available Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC, stabilising it, and its presence slightly increases nucleotide excision repair (NER activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.

  12. RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair.

    Directory of Open Access Journals (Sweden)

    Yuliya S Krasikova

    Full Text Available Replication protein A (RPA and the xeroderma pigmentosum group A (XPA protein are indispensable for both pathways of nucleotide excision repair (NER. Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.

  13. Inhibition of nucleotide excision repair by fludarabine in normal lymphocytes in vitro, measured by the alkaline single cell gel electrophoresis (comet) assay

    Energy Technology Data Exchange (ETDEWEB)

    Yamauchi, Takahiro; Kawai, Yasukazu; Ueda, Takanori [Fukui Medical Univ., Matsuoka (Japan)

    2002-05-01

    Alkylating agents or platinum analogues initiate several excision repair mechanisms, which involve incision of the DNA strand, excision of the damaged nucleotide, gap filling by DNA resynthesis, and rejoining by ligation. The previous study described that nucleotide excision repair permitted incorporation of fludarabine nucleoside (F-area-A) into the repair patch, thereby inhibiting the DNA resynthesis. In the present study, to clarify the repair kinetics in view of the inhibition by F-ara-A, normal lymphocytes were stimulated to undergo nucleotide excision repair by ultraviolet C (UV) irradiation in the presence or absence of F-ara-A. The repair kinetics were determined as DNA single strand breaks resulting from the incision and the rejoining using the alkaline single cell gel electrophoresis (comet) assay. DNA resynthesis was evaluated in terms of the uptake of tritiated thymidine into DNA. The lymphocytes initiated the incision step maximally at 1 h, and completed the rejoining process within 4 h after UV exposure. UV also initiated thymidine uptake, which increased time-dependently and reached a plateau at 4 h. A 2-h pre-incubation with F-ara-A inhibited the repair in a concentration-dependent manner, with the maximal inhibition by 5 {mu}M. This inhibitory effect was demonstrated by the reduction of the thymidine uptake and by the inhibition of the rejoining. A DNA polymerase inhibitor, aphidicolin, and a ribonucleotide reductase inhibitor, hydroxyurea, were not so inhibitory to the repair process as F-ara-A at equimolar concentrations. The present findings suggest that inhibition of nucleotide excision repair may represent a novel therapeutic strategy against cancer, especially in the context of resistant cells with an increased repair capacity. (author)

  14. Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo.

    Science.gov (United States)

    Bol, S A; van Steeg, H; van Oostrom, C T; Tates, A D; Vrieling, H; de Groot, A J; Mullenders, L H; van Zeeland, A A; Jansen, J G

    1999-05-01

    The butylating agent N-n-butyl-N-nitrosourea (BNU) was employed to study the role of nucleotide excision repair (NER) in protecting mammalian cells against the genotoxic effects of monofunctional alkylating agents. The direct acting agent BNU was found to be mutagenic in normal and XPA mouse splenocytes after a single i.p. treatment in vivo. After 25 and 35 mg/kg BNU, but not after 75 mg/ kg, 2- to 3-fold more hprt mutants were detected in splenocytes from XPA mice than from normal mice. Using O6-alkylguanine-DNA alkyltransferase (AGT)-deficient hamster cells, it was found that NER-deficient CHO UV5 cells carrying a mutation in the ERCC-2 gene were 40% more mutable towards lesions induced by BNU when compared with parental NER-proficient CHO AA8 cells. UV5 cells were 1.4-fold more sensitive to the cytotoxic effects of BNU compared with AA8 cells. To investigate whether this increased sensitivity of NER-deficient cells is modulated by AGT activity, cell survival studies were performed in human and mouse primary fibroblasts as well. BNU was 2.7-fold more toxic for mouse XPA fibroblasts compared with normal mouse fibroblasts. Comparable results were found for human fibroblasts. Taken together these data indicate that the role of NER in protecting rodent cells against the mutagenic and cytotoxic effects of the alkylating agent BNU depends on AGT.

  15. Cyclosporin A inhibits nucleotide excision repair via downregulation of the xeroderma pigmentosum group A and G proteins, which is mediated by calcineurin inhibition.

    Science.gov (United States)

    Kuschal, Christiane; Thoms, Kai-Martin; Boeckmann, Lars; Laspe, Petra; Apel, Antje; Schön, Michael P; Emmert, Steffen

    2011-10-01

    Cyclosporin A (CsA) inhibits nucleotide excision repair (NER) in human cells, a process that contributes to the skin cancer proneness in organ transplant patients. We investigated the mechanisms of CsA-induced NER reduction by assessing all xeroderma pigmentosum (XP) genes (XPA-XPG). Western blot analyses revealed that XPA and XPG protein expression was reduced in normal human GM00637 fibroblasts exposed to 0.1 and 0.5 μm CsA. Interestingly, the CsA treatment reduced XPG, but not XPA, mRNA expression. Calcineurin knockdown in GM00637 fibroblasts using RNAi led to similar results suggesting that calcineurin-dependent signalling is involved in XPA and XPG protein regulation. CsA-induced reduction in NER could be complemented by the overexpression of either XPA or XPG protein. Likewise, XPA-deficient fibroblasts with stable overexpression of XPA (XP2OS-pCAH19WS) did not show the inhibitory effect of CsA on NER. In contrast, XPC-deficient fibroblasts overexpressing XPC showed CsA-reduced NER. Our data indicate that the CsA-induced inhibition of NER is a result of downregulation of XPA and XPG protein in a calcineurin-dependent manner. © 2011 John Wiley & Sons A/S.

  16. Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

    Science.gov (United States)

    Robu, Mihaela; Shah, Rashmi G; Purohit, Nupur K; Zhou, Pengbo; Naegeli, Hanspeter; Shah, Girish M

    2017-08-15

    Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

  17. UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Kelley N. Newton

    2012-01-01

    Full Text Available UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structures in vitro and is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair.

  18. Isolation of the functional human excision repair gene ERCC5 by intercosmid recombination

    International Nuclear Information System (INIS)

    Mudgett, J.S.; MacInnes, M.A.

    1990-01-01

    The complete human nucleotide exicision repair gene ERCC5 was isolated as a functional gene on overlapping cosmids. ERCC5 corrects the excision repair deficiency of Chinese hamster ovary cell line UV135, of complementation group 5. Cosmids that contained human sequences were obtained from a UV-resistant cell line derived from UV135 cells transformed with human genomic DNA. Individually, none of the cosmids complemented the UV135 repair defect; cosmid groups were formed to represent putative human genomic regions, and specific pairs of cosmids that effectively transformed UV135 cells to UV resistance were identified. Analysis of transformants derived from the active cosmid pairs showed that the functional 32-kbp ERCC5 gene was reconstructed by homologous intercosmid recombination. The cloned human sequences exhibited 100% concordance with the locus designated genetically as ERCC5 located on human chromosome 13q. Cosmid-transformed UV135 host cells repaired cytotoxic damage to levels about 70% of normal and repaired UV-irradiated shuttle vector DNA to levels about 82% of normal

  19. Substitution of Active Site Tyrosines with Tryptophan Alters the Free Energy for Nucleotide Flipping by Human Alkyladenine DNA Glycosylase†

    Science.gov (United States)

    Hendershot, Jenna M.; Wolfe, Abigail E.; O'Brien, Patrick J.

    2011-01-01

    Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N6-ethenoadenine (εA), within 4-fold of that of the wildtype enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and εA fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the εA fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the εA fluorescence. Although these mutations do not have large effects on the rate constant for excision of εA, there are dramatic effects on the rate constants for nucleotide flipping that result in 40 to 100-fold decreases in the flipping equilibrium relative to wildtype. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in

  20. The possible role of human milk nucleotides as sleep inducers.

    Science.gov (United States)

    Sánchez, Cristina L; Cubero, Javier; Sánchez, Javier; Chanclón, Belén; Rivero, Montserrat; Rodríguez, Ana B; Barriga, Carmen

    2009-02-01

    Breast-milk contains a potent mixture of diverse components, such as the non-protein nitrogen fraction which includes nucleotides, whose variation in levels is evident throughout lactation. In addition, these substances play an important role in sleep homeostasis. In the present study, human milk samples were analyzed using a capillary electrophoresis system. The rhythmicity of each nucleotide was studied by cosinor analysis. It was found that the nucleotides 5'AMP, 5'GMP, 5'CMP, and 5'IMP have significant (P inducing the 'hypnotic' action of breast-milk at night in the infant.

  1. Important role for Mycobacterium tuberculosis UvrD1 in pathogenesis and persistence apart from its function in nucleotide excision repair.

    Science.gov (United States)

    Houghton, Joanna; Townsend, Carolin; Williams, Alan R; Rodgers, Angela; Rand, Lucinda; Walker, K Barry; Böttger, Erik C; Springer, Burkhard; Davis, Elaine O

    2012-06-01

    Mycobacterium tuberculosis survives and replicates in macrophages, where it is exposed to reactive oxygen and nitrogen species that damage DNA. In this study, we investigated the roles of UvrA and UvrD1, thought to be parts of the nucleotide excision repair pathway of M. tuberculosis. Strains in which uvrD1 was inactivated either alone or in conjunction with uvrA were constructed. Inactivation of uvrD1 resulted in a small colony phenotype, although growth in liquid culture was not significantly affected. The sensitivity of the mutant strains to UV irradiation and to mitomycin C highlighted the importance of the targeted genes for nucleotide excision repair. The mutant strains all exhibited heightened susceptibility to representatives of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). The uvrD1 and the uvrA uvrD1 mutants showed decreased intracellular multiplication following infection of macrophages. Most importantly, the uvrA uvrD1 mutant was markedly attenuated following infection of mice by either the aerosol or the intravenous route.

  2. Reconstitution of nucleotide excision nuclease with UvrA and UvrB proteins from Escherichia coli and UvrC protein from Bacillus subtilis

    International Nuclear Information System (INIS)

    Lin, J.J.; Sancar, A.

    1990-01-01

    Recently, an open reading frame which has a deduced amino acid sequence that shows 38% homology to Escherichia coli UvrC protein was found upstream of the aspartokinase II gene (ask) in Bacillus subtilis. We found that plasmids containing this open reading frame complement the uvrC mutations in E. coli. We joined the open reading frame to a tac promoter to amplify the gene product in E. coli and purified the protein to near homogeneity. The apparent molecular weight of the gene product is 69,000, which is consistent with the calculated molecular weight of 69,378 fro the deduced gene product of the open reading frame. The purified gene product causes the nicking of DNA at the 8th phosphodiester bond 5' and the 5th phosphodiester bond 3' to a thymine dimer when mixed with E. coli UvrA and UvrB proteins and a DNA substrate containing a uniquely located thymine dimer. We conclude that the gene product of the open reading frame is the B. subtilis UvrC protein. Our results suggest that the B. subtilis nucleotide excision repair system is quite similar to that of E. coli. Furthermore, complementation of the UvrA and UvrB proteins from a Gram-negative bacterium with the UvrC protein of Gram-positive B. subtilis indicates a significant evolutionary conservation of the nucleotide excision repair system

  3. Molecular cloning and biological characterization of the human excision repair gene ERCC-3

    International Nuclear Information System (INIS)

    Weeda, G.; van Ham, R.C.; Masurel, R.; Westerveld, A.; Odijk, H.; de Wit, J.; Bootsma, D.; van der Eb, A.J.; Hoeijmakers, J.H.

    1990-01-01

    In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion

  4. Terahertz pulsed imaging of freshly excised human colonic tissues

    Energy Technology Data Exchange (ETDEWEB)

    Reid, Caroline B; Gibson, Adam P [Department of Medical Physics and Bioengineering, University College London, London, WC1E 6BT (United Kingdom); Fitzgerald, Anthony; Wallace, Vincent P [School of Physics, University of Western Australia, Crawley 6009 (Australia); Reese, George; Tekkis, Paris [Division of Surgery, Chelsea and Westminster Campus, Imperial College London, London (United Kingdom); Goldin, Robert [Centre for Pathology, Imperial College London, St Mary' s Campus, London (United Kingdom); O' Kelly, P S [TeraView Ltd, Platinum Building, St John' s Innovation Park, Cowley Road, Cambridge, CB4 0WS (United Kingdom); Pickwell-MacPherson, Emma, E-mail: c.reid@medphys.ucl.ac.uk [Department of Electronic Engineering, Chinese University of Hong Kong, Shatin, NT (Hong Kong)

    2011-07-21

    We present the results from a feasibility study which measures properties in the terahertz frequency range of excised cancerous, dysplastic and healthy colonic tissues from 30 patients. We compare their absorption and refractive index spectra to identify trends which may enable different tissue types to be distinguished. In addition, we present statistical models based on variations between up to 17 parameters calculated from the reflected time and frequency domain signals of all the measured tissues. These models produce a sensitivity of 82% and a specificity of 77% in distinguishing between healthy and all diseased tissues and a sensitivity of 89% and a specificity of 71% in distinguishing between dysplastic and healthy tissues. The contrast between the tissue types was supported by histological staining studies which showed an increased vascularity in regions of increased terahertz absorption.

  5. Terahertz pulsed imaging of freshly excised human colonic tissues

    International Nuclear Information System (INIS)

    Reid, Caroline B; Gibson, Adam P; Fitzgerald, Anthony; Wallace, Vincent P; Reese, George; Tekkis, Paris; Goldin, Robert; O'Kelly, P S; Pickwell-MacPherson, Emma

    2011-01-01

    We present the results from a feasibility study which measures properties in the terahertz frequency range of excised cancerous, dysplastic and healthy colonic tissues from 30 patients. We compare their absorption and refractive index spectra to identify trends which may enable different tissue types to be distinguished. In addition, we present statistical models based on variations between up to 17 parameters calculated from the reflected time and frequency domain signals of all the measured tissues. These models produce a sensitivity of 82% and a specificity of 77% in distinguishing between healthy and all diseased tissues and a sensitivity of 89% and a specificity of 71% in distinguishing between dysplastic and healthy tissues. The contrast between the tissue types was supported by histological staining studies which showed an increased vascularity in regions of increased terahertz absorption.

  6. Statistical properties of nucleotides in human chromosomes 21 and 22

    International Nuclear Information System (INIS)

    Zhang Linxi; Sun Tingting

    2005-01-01

    In this paper the statistical properties of nucleotides in human chromosomes 21 and 22 are investigated. The n-tuple Zipf analysis with n = 3, 4, 5, 6, and 7 is used in our investigation. It is found that the most common n-tuples are those which consist only of adenine (A) and thymine (T), and the rarest n-tuples are those in which GC or CG pattern appears twice. With the n-tuples become more and more frequent, the double GC or CG pattern becomes a single GC or CG pattern. The percentage of four nucleotides in the rarest ten and the most common ten n-tuples are also considered in human chromosomes 21 and 22, and different behaviors are found in the percentage of four nucleotides. Frequency of appearance of n-tuple f(r) as a function of rank r is also examined. We find the n-tuple Zipf plot shows a power-law behavior for r n-1 and a rapid decrease for r > 4 n-1 . In order to explore the interior statistical properties of human chromosomes 21 and 22 in detail, we divide the chromosome sequence into some moving windows and we discuss the percentage of ξη (ξ, η = A, C, G, T) pair in those moving windows. In some particular regions, there are some obvious changes in the percentage of ξη pair, and there maybe exist functional differences. The normalized number of repeats N 0 (l) can be described by a power law: N 0 (l) ∼ l -μ . The distance distributions P 0 (S) between two nucleotides in human chromosomes 21 and 22 are also discussed. A two-order polynomial fit exists in those distance distributions: log P 0 (S) = a + bS + cS 2 , and it is quite different from the random sequence

  7. Single-nucleotide polymorphisms in base excision repair, nucleotide excision repair, and double strand break genes as markers for response to radiotherapy in patients with Stage I to II head-and-neck cancer

    International Nuclear Information System (INIS)

    Carles, Joan; Monzo, Mariano; Amat, Marta; Jansa, Sonia; Artells, Rosa; Navarro, Alfons; Foro, Palmira; Alameda, Francesc; Gayete, Angel; Gel, Bernat; Miguel, Maribel; Albanell, Joan; Fabregat, Xavier

    2006-01-01

    Purpose: Polymorphisms in DNA repair genes can influence response to radiotherapy. We analyzed single-nucleotide polymorphisms (SNP) in nine DNA repair genes in 108 patients with head-and-neck cancer (HNSCC) who had received radiotherapy only. Methods and Materials: From May 1993 to December 2004, patients with Stage I and II histopathologically confirmed HNSCC underwent radiotherapy. DNA was obtained from paraffin-embedded tissue, and SNP analysis was performed using a real-time polymerase chain reaction allelic discrimination TaqMan assay with minor modifications. Results: Patients were 101 men (93.5%) and 7 (6.5%) women, with a median age of 64 years (range, 40 to 89 years). Of the patients, 76 (70.4%) patients were Stage I and 32 (29.6%) were Stage II. The XPF/ERCC1 SNP at codon 259 and XPG/ERCC5 at codon 46 emerged as significant predictors of progression (p 0.00005 and 0.049, respectively) and survival (p = 0.0089 and 0.0066, respectively). Similarly, when variant alleles of XPF/ERCC1, XPG/ERCC5 and XPA were examined in combination, a greater number of variant alleles was associated with shorter time to progression (p = 0.0003) and survival (p 0.0002). Conclusions: Genetic polymorphisms in XPF/ERCC1, XPG/ERCC5, and XPA may significantly influence response to radiotherapy; large studies are warranted to confirm their role in HNSCC

  8. Frequency of intrachromosomal homologous recombination induced by UV radiation in normally repairing and excision repair-deficient human cells

    International Nuclear Information System (INIS)

    Tsujimura, T.; Maher, V.M.; McCormick, J.J.; Godwin, A.R.; Liskay, R.M.

    1990-01-01

    To investigate the role of DNA damage and nucleotide excision repair in intrachromosomal homologous recombination, a plasmid containing duplicated copies of the gene coding for hygromycin resistance was introduced into the genome of a repair-proficient human cell line, KMST-6, and two repair-deficient lines, XP2OS(SV) from xeroderma pigmentosum complementation group A and XP2YO(SV) from complementation group F. Neither hygromycin-resistance gene codes for a functional enzyme because each contains an insertion/deletion mutation at a unique site, but recombination between the two defective genes can yield hygromycin-resistant cells. The rates of spontaneous recombination in normal and xeroderma pigmentosum cell strains containing the recombination substrate were found to be similar. The frequency of UV-induced recombination was determined for three of these cell strains. At low doses, the group A cell strain and the group F cell strain showed a significant increase in frequency of recombinants. The repair-proficient cell strain required 10-to 20-fold higher doses of UV to exhibit comparable increases in frequency of recombinants. These results suggest that unexcised DNA damage, rather than the excision repair process per se, stimulates such recombination

  9. Prediction of Human Pharmacokinetic Profile After Transdermal Drug Application Using Excised Human Skin.

    Science.gov (United States)

    Yamamoto, Syunsuke; Karashima, Masatoshi; Arai, Yuta; Tohyama, Kimio; Amano, Nobuyuki

    2017-09-01

    Although several mathematical models have been reported for the estimation of human plasma concentration profiles of drug substances after dermal application, the successful cases that can predict human pharmacokinetic profiles are limited. Therefore, the aim of this study is to investigate the prediction of human plasma concentrations after dermal application using in vitro permeation parameters obtained from excised human skin. The in vitro skin permeability of 7 marketed drug products was evaluated. The plasma concentration-time profiles of the drug substances in humans after their dermal application were simulated using compartment models and the clinical pharmacokinetic parameters. The transdermal process was simulated using the in vitro skin permeation rate and lag time assuming a zero-order absorption. These simulated plasma concentration profiles were compared with the clinical data. The result revealed that the steady-state plasma concentration of diclofenac and the maximum concentrations of nicotine, bisoprolol, rivastigmine, and lidocaine after topical application were within 2-fold of the clinical data. Furthermore, the simulated concentration profiles of bisoprolol, nicotine, and rivastigmine reproduced the decrease in absorption due to drug depletion from the formulation. In conclusion, this simple compartment model using in vitro human skin permeation parameters as zero-order absorption predicted the human plasma concentrations accurately. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  10. Site-specific analysis of UV-induced cyclobutane pyrimidine dimers in nucleotide excision repair-proficient and -deficient hamster cells: Lack of correlation with mutational spectra

    International Nuclear Information System (INIS)

    Vreeswijk, Maaike P.G.; Meijers, Caro M.; Giphart-Gassler, Micheline; Vrieling, Harry; Zeeland, Albert A. van; Mullenders, Leon H.F.; Loenen, Wil A.M.

    2009-01-01

    Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C > T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

  11. Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein

    International Nuclear Information System (INIS)

    Rodriguez, K.; Talamantez, J.; Huang, W.; Reed, S.H.; Wang, Z.; Chen, L.; Feaver, W.J.; Friedberg, E.C.; Tomkinson, A.E.

    1998-01-01

    The nucleotide excision repair (NER) pathway of eukaryotes involves approximately 30 polypeptides. Reconstitution of this pathway with purified components is consistent with the sequential assembly of NER proteins at the DNA lesion. However, recent studies have suggested that NER proteins may be pre-assembled in a high molecular weight complex in the absence of DNA damage. To examine this model further, we have constructed a histidine-tagged version of the yeast DNA damage recognition protein Rad14. Affinity purification of this protein from yeast nuclear extracts resulted in the co-purification of Rad1, Rad7, Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of these proteins bound to the affinity resin in the absence of recombinant Rad14. Furthermore, many of the co-purifying proteins were present in approximately equimolar amounts. Co-elution of these proteins was also observed when the nuclear extract was fractionated by gel filtration, indicating that the NER proteins were associated in a complex with a molecular mass of >1000 kDa prior to affinity chromatography. The affinity purified NER complex catalyzed the incision of UV-irradiated DNA in an ATP-dependent reaction. We conclude that active high molecular weight complexes of NER proteins exist in undamaged yeast cells

  12. Decreased transcription-coupled nucleotide excision repair capacity is associated with increased p53- and MLH1-independent apoptosis in response to cisplatin

    International Nuclear Information System (INIS)

    Stubbert, Lawton J; Smith, Jennifer M; McKay, Bruce C

    2010-01-01

    One of the most commonly used classes of anti-cancer drugs presently in clinical practice is the platinum-based drugs, including cisplatin. The efficacy of cisplatin therapy is often limited by the emergence of resistant tumours following treatment. Cisplatin resistance is multi-factorial but can be associated with increased DNA repair capacity, mutations in p53 or loss of DNA mismatch repair capacity. RNA interference (RNAi) was used to reduce the transcription-coupled nucleotide excision repair (TC-NER) capacity of several prostate and colorectal carcinoma cell lines with specific defects in p53 and/or DNA mismatch repair. The effect of small inhibitory RNAs designed to target the CSB (Cockayne syndrome group B) transcript on TC-NER and the sensitivity of cells to cisplatin-induced apoptosis was determined. These prostate and colon cancer cell lines were initially TC-NER proficient and RNAi against CSB significantly reduced their DNA repair capacity. Decreased TC-NER capacity was associated with an increase in the sensitivity of tumour cells to cisplatin-induced apoptosis, even in p53 null and DNA mismatch repair-deficient cell lines. The present work indicates that CSB and TC-NER play a prominent role in determining the sensitivity of tumour cells to cisplatin even in the absence of p53 and DNA mismatch repair. These results further suggest that CSB represents a potential target for cancer therapy that may be important to overcome resistance to cisplatin in the clinic

  13. Gamma-ray excision repair in normal and diseased human cells

    International Nuclear Information System (INIS)

    Cerutti, P.A.; Remsen, J.F.

    1976-01-01

    Radiation products of the 5,6-dihydroxy-dihydrothymine type (t') are efficiently removed from the DNA during postirradiation incubation of bacterial and mammalian cells. In this chapter we describe the t'-excision system contained in normal human cells, in human carcinoma HeLa S-3 cells, and in skin fibroblasts from xeroderma pigmentosum (XP) and Fanconi's anemia (FA) patients. The latter diseases are characterized among other symptoms by a genetically increased susceptibility for the development of cancer

  14. Differing levels of excision repair in human fetal dermis and brain cells

    International Nuclear Information System (INIS)

    Gibson, R.E.; D'Ambrosio, S.M.; Ohio State Univ., Columbus

    1982-01-01

    The levels of DNA excision repair, as measured by unscheduled DNA synthesis (UDS) and the UV-endonuclease sensitive site assay, were compared in cells derived from human fetal brain and dermal tissues. The level of UDS induced following ultraviolet (UV) irradiation was found to be lower (approx. 60%) in the fetal brain cells than in fetal dermal cells. It was determined, using the UV-endonuclease sensitive site assay to confirm the UDS observation, that 50% of the dimers induced by UV in fetal dermal cells were repaired in 8 h. while only 15% were removed in the fetal brain cells during the same period of time. Even after 24 h. only 44% of the dimers induced by UV in the fetal brain cells were repaired, while 65% were removed in the dermal cells. These data suggest that cultured human fetal brain cells exhibit lower levels of excision repair compared to cultured human fetal dermal cells. (author)

  15. On pitch jumps between chest and falsetto registers in voice : Data from living and excised human larynges

    NARCIS (Netherlands)

    Svec, JG; Schutte, HK; Miller, DG

    The paper offers a new concept of studying abrupt chest-falsetto register transitions Clumps) based on the theory of nonlinear dynamics. The jumps were studied in an excised human larynx and in three living subjects tone female and two male). Data from the excised larynx revealed that a small and

  16. Inter-individual variation in nucleotide excision repair pathway is modulated by non-synonymous polymorphisms in ERCC4 and MBD4 genes

    Energy Technology Data Exchange (ETDEWEB)

    Allione, Alessandra, E-mail: alessandra.allione@hugef-torino.org [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Guarrera, Simonetta; Russo, Alessia [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Ricceri, Fulvio [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Department of Medical Sciences, University of Turin, Via Santena 19, 10126 Turin (Italy); Purohit, Rituraj [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Bioinformatics Division, School of Bio Sciences and Technology, Vellore Institute of Technology University, Vellore 632014, Tamil Nadu (India); Pagnani, Andrea; Rosa, Fabio; Polidoro, Silvia; Voglino, Floriana [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Matullo, Giuseppe [Human Genetics Foundation (HuGeF), Via Nizza 52, 10126 Turin (Italy); Department of Medical Sciences, University of Turin, Via Santena 19, 10126 Turin (Italy)

    2013-11-15

    Highlights: • We reported a large inter-individual variability of NER capacity. • ERCC4 rs1800124 and MBD4 rs10342 nsSNP variants were associated with DNA repair capacity. • DNA–protein interaction analyses showed alteration of binding for ERCC4 and MBD4 variants. • A new possible cross-talk between NER and BER pathways has been reported. - Abstract: Inter-individual differences in DNA repair capacity (DRC) may lead to genome instability and, consequently, modulate individual cancer risk. Among the different DNA repair pathways, nucleotide excision repair (NER) is one of the most versatile, as it can eliminate a wide range of helix-distorting DNA lesions caused by ultraviolet light irradiation and chemical mutagens. We performed a genotype–phenotype correlation study in 122 healthy subjects in order to assess if any associations exist between phenotypic profiles of NER and DNA repair gene single nucleotide polymorphisms (SNPs). Individuals were genotyped for 768 SNPs with a custom Illumina Golden Gate Assay, and peripheral blood mononuclear cells (PBMCs) of the same subjects were tested for a NER comet assay to measure DRC after challenging cells by benzo(a)pyrene diolepoxide (BPDE). We observed a large inter-individual variability of NER capacity, with women showing a statistically significant lower DRC (mean ± SD: 6.68 ± 4.76; p = 0.004) than men (mean ± SD: 8.89 ± 5.20). Moreover, DRC was significantly lower in individuals carrying a variant allele for the ERCC4 rs1800124 non-synonymous SNP (nsSNP) (p = 0.006) and significantly higher in subjects with the variant allele of MBD4 rs2005618 SNP (p = 0.008), in linkage disequilibrium (r{sup 2} = 0.908) with rs10342 nsSNP. Traditional in silico docking approaches on protein–DNA and protein–protein interaction showed that Gly875 variant in ERCC4 (rs1800124) decreases the DNA–protein interaction and that Ser273 and Thr273 variants in MBD4 (rs10342) indicate complete loss of protein

  17. Decreased transcription-coupled nucleotide excision repair capacity is associated with increased p53- and MLH1-independent apoptosis in response to cisplatin

    Directory of Open Access Journals (Sweden)

    Smith Jennifer M

    2010-05-01

    Full Text Available Abstract Background One of the most commonly used classes of anti-cancer drugs presently in clinical practice is the platinum-based drugs, including cisplatin. The efficacy of cisplatin therapy is often limited by the emergence of resistant tumours following treatment. Cisplatin resistance is multi-factorial but can be associated with increased DNA repair capacity, mutations in p53 or loss of DNA mismatch repair capacity. Methods RNA interference (RNAi was used to reduce the transcription-coupled nucleotide excision repair (TC-NER capacity of several prostate and colorectal carcinoma cell lines with specific defects in p53 and/or DNA mismatch repair. The effect of small inhibitory RNAs designed to target the CSB (Cockayne syndrome group B transcript on TC-NER and the sensitivity of cells to cisplatin-induced apoptosis was determined. Results These prostate and colon cancer cell lines were initially TC-NER proficient and RNAi against CSB significantly reduced their DNA repair capacity. Decreased TC-NER capacity was associated with an increase in the sensitivity of tumour cells to cisplatin-induced apoptosis, even in p53 null and DNA mismatch repair-deficient cell lines. Conclusion The present work indicates that CSB and TC-NER play a prominent role in determining the sensitivity of tumour cells to cisplatin even in the absence of p53 and DNA mismatch repair. These results further suggest that CSB represents a potential target for cancer therapy that may be important to overcome resistance to cisplatin in the clinic.

  18. Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group A protein on interaction with DNA intermediates of nucleotide excision repair.

    Science.gov (United States)

    Maltseva, E A; Krasikova, Y S; Naegeli, H; Lavrik, O I; Rechkunova, N I

    2014-06-01

    Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the interaction of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E > K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.

  19. Modulation of DNA polymerase beta-dependent base excision repair in cultured human cells after low dose exposure to arsenite

    International Nuclear Information System (INIS)

    Sykora, Peter; Snow, Elizabeth T.

    2008-01-01

    Base excision repair (BER) is crucial for development and for the repair of endogenous DNA damage. However, unlike nucleotide excision repair, the regulation of BER is not well understood. Arsenic, a well-established human carcinogen, is known to produce oxidative DNA damage, which is repaired primarily by BER, whilst high doses of arsenic can also inhibit DNA repair. However, the mechanism of repair inhibition by arsenic and the steps inhibited are not well defined. To address this question we have investigated the regulation of DNA polymerase β (Pol β) and AP endonuclease (APE1), in response to low, physiologically relevant doses of arsenic. GM847 lung fibroblasts and HaCaT keratinocytes were exposed to sodium arsenite, As(III), and mRNA, protein levels and BER activity were assessed. Both Pol β and APE1 mRNA exhibited significant dose-dependant down regulation at doses of As(III) above 1 μM. However, at lower doses Pol β mRNA and protein levels, and consequently, BER activity were significantly increased. In contrast, APE1 protein levels were only marginally increased by low doses of As(III) and there was no correlation between APE1 and overall BER activity. Enzyme supplementation of nuclear extracts confirmed that Pol β was rate limiting. These changes in BER correlated with overall protection against sunlight UV-induced toxicity at low doses of As(III) and produced synergistic toxicity at high doses. The results provide evidence that changes in BER due to low doses of arsenic could contribute to a non-linear, threshold dose response for arsenic carcinogenesis

  20. Nucleotide Excision Repair Is Not Induced in Human Embryonic Lung Fibroblasts Treated with Environmental Pollutants

    Czech Academy of Sciences Publication Activity Database

    Rössner Jr., P.; Mrhálková, A.; Uhlířová, Kateřina; Špátová, M.; Rössnerová, A.; Líbalová, H.; Schmuczerová, J.; Milcová, A.; Topinka, J.; Šrám, R.

    2013-01-01

    Roč. 8, č. 7 (2013), e69197 E-ISSN 1932-6203 Institutional support: RVO:60077344 Keywords : environmental pollutants * DNA * expression of DNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.534, year: 2013

  1. Nucleotide Excision Repair Is Not Induced in Human Embryonic Lung Fibroblasts Treated with Environmental Pollutants

    Czech Academy of Sciences Publication Activity Database

    Rössner ml., Pavel; Mrhálková, Andrea; Uhlířová, Kateřina; Špátová, Milada; Rössnerová, Andrea; Líbalová, Helena; Schmuczerová, Jana; Milcová, Alena; Topinka, Jan; Šrám, Radim

    2013-01-01

    Roč. 8, č. 7 (2013), e69197 E-ISSN 1932-6203 R&D Projects: GA ČR GAP503/11/0084 Institutional support: RVO:68378041 Keywords : environmental pollutants * DNA * expression of DNA Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.534, year: 2013

  2. Palindromic nucleotide analysis in human T cell receptor rearrangements.

    Directory of Open Access Journals (Sweden)

    Santosh K Srivastava

    Full Text Available Diversity of T cell receptor (TCR genes is primarily generated by nucleotide insertions upon rearrangement from their germ line-encoded V, D and J segments. Nucleotide insertions at V-D and D-J junctions are random, but some small subsets of these insertions are exceptional, in that one to three base pairs inversely repeat the sequence of the germline DNA. These short complementary palindromic sequences are called P nucleotides. We apply the ImmunoSeq deep-sequencing assay to the third complementarity determining region (CDR3 of the β chain of T cell receptors, and use the resulting data to study P nucleotides in the repertoire of naïve and memory CD8(+ and CD4(+ T cells. We estimate P nucleotide distributions in a cross section of healthy adults and different T cell subtypes. We show that P nucleotide frequency in all T cell subtypes ranges from 1% to 2%, and that the distribution is highly biased with respect to the coding end of the gene segment. Classification of observed palindromic sequences into P nucleotides using a maximum conditional probability model shows that single base P nucleotides are very rare in VDJ recombination; P nucleotides are primarily two bases long. To explore the role of P nucleotides in thymic selection, we compare P nucleotides in productive and non-productive sequences of CD8(+ naïve T cells. The naïve CD8(+ T cell clones with P nucleotides are more highly expanded.

  3. Nucleotide sequence of the human N-myc gene

    International Nuclear Information System (INIS)

    Stanton, L.W.; Schwab, M.; Bishop, J.M.

    1986-01-01

    Human neuroblastomas frequently display amplification and augmented expression of a gene known as N-myc because of its similarity to the protooncogene c-myc. It has therefore been proposed that N-myc is itself a protooncogene, and subsequent tests have shown that N-myc and c-myc have similar biological activities in cell culture. The authors have now detailed the kinship between N-myc and c-myc by determining the nucleotide sequence of human N-myc and deducing the amino acid sequence of the protein encoded by the gene. The topography of N-myc is strikingly similar to that of c-myc: both genes contain three exons of similar lengths; the coding elements of both genes are located in the second and third exons; and both genes have unusually long 5' untranslated regions in their mRNAs, with features that raise the possibility that expression of the genes may be subject to similar controls of translation. The resemblance between the proteins encoded by N-myc and c-myc sustains previous suspicions that the genes encode related functions

  4. Cryopreserved Ultra-Thick Human Amniotic Membrane for Conjunctival Surface Reconstruction After Excision of Conjunctival Tumors.

    Science.gov (United States)

    Tanaka, Thais S; Demirci, Hakan

    2016-04-01

    Cryopreserved ultra-thick human amniotic membrane (AM) is used for glaucoma surgery. We evaluated the use of cryopreserved ultra-thick human AM for conjunctival surface reconstruction after excision of a conjunctival tumor. We retrospectively reviewed the medical records of 28 patients who underwent conjunctival surface reconstruction with cryopreserved ultra-thick human AM after excision of the tumor. The AM was secured to the surrounding conjunctiva and underlying sclera with interrupted 8-0 Vicryl sutures. Clinical data regarding demographics, diagnosis, size and location of conjunctival tumors, patient outcome, and complications were gathered. Of 28 patients, 6 (21.4%) had malignant melanoma, 4 (14.3%) had squamous cell carcinoma, 6 (21.4%) had conjunctival intraepithelial neoplasia, 1 (3.6%) had sebaceous carcinoma, 1 (3.6%) had mucoepidermoid carcinoma, 1 (3.6%) had conjunctival intraepithelial dysplasia, 5 (17.9%) had pterygium, 2 (7.1%) had compound nevus, 1 (3.6%) had a large epithelial inclusion cyst, and 1 (3.6%) patient had a granuloma. The mean area of graft size was 156 ± 120 mm2. Postoperatively, the graft was well tolerated with no failure, discomfort, or dehiscence. During the 17-month mean follow-up, symblepharon, which was clinically nonsignificant, developed in 3 (11%) patients and partial stem cell deficiency was noted in 5 (18%) patients. Cryopreserved ultra-thick human AM is a well-tolerated, effective graft material that is easy to handle. It is a viable alternative for conjunctival surface reconstruction after excision of a conjunctival tumor.

  5. Excision without excision

    International Nuclear Information System (INIS)

    Brown, David; Sarbach, Olivier; Schnetter, Erik; Diener, Peter; Tiglio, Manuel; Hawke, Ian; Pollney, Denis

    2007-01-01

    to turducken (turduckens, turduckening, turduckened, turduckened) [math.]: To stuff a black hole. We analyze and apply an alternative to black hole excision based on smoothing the interior of black holes with arbitrary initial data, and solving the vacuum Einstein evolution equations everywhere. By deriving the constraint propagation system for our hyperbolic formulation of the BSSN evolution system we rigorously prove that the constraints propagate causally and so any constraint violations introduced inside the black holes cannot affect the exterior spacetime. We present evolutions of Cook-Pfeiffer binary black hole initial configurations showing that these techniques appear to work robustly for generic data. We also present evidence from spherically symmetric evolutions that for the gauge conditions used the same stationary end-state is approached irrespective of the choice of initial data and smoothing procedure

  6. Surgical excision for recurrent herpes simplex virus 2 (HSV-2) anogenital infection in a patient with human immunodeficiency virus (HIV).

    Science.gov (United States)

    Arinze, Folasade; Shaver, Aaron; Raffanti, Stephen

    2017-10-01

    Recurrent anogenital herpes simplex virus infections are common in patients with human immunodeficiency virus (HIV), of whom approximately 5% develop resistance to acyclovir. We present a case of a 49-year-old man with HIV who had an 8-year history of recurrent left inguinal herpes simplex virus type 2 ulcerations. He initially responded to oral acyclovir, but developed resistance to acyclovir and eventually foscarnet. The lesion progressed to a large hypertrophic mass that required surgical excision, which led to resolution without recurrences. Our case highlights the importance of surgical excision as a treatment option in refractory herpes simplex virus anogenital infections.

  7. A human beta cell line with drug inducible excision of immortalizing transgenes

    Science.gov (United States)

    Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

    2015-01-01

    Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

  8. Excision and crosslink repair of DNA and sister chromatid exchanges in cultured human fibroblasts with different repair capacities

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, Y; Kano, Y; Paul, P; Goto, K; Yamamoto, K [Kobe Univ. (Japan). School of Medicine

    1981-01-01

    Xeroderma pigmentosum (XP) groups A to G lacked the initial stage of ultraviolet (UV) excision repair in the order of A = G > C > D > E asymptotically equals F, while the XP variant was weakly defective in the later repair steps. Killing sensitivities were in the orders of A >= G > D > C > E asymptotically equals F asymptotically equals variant > normal to UV, A = G > D > F > C = E > variant > normal to 4-nitroquinoline-1-oxide (4NQO), and A > C > D = E = F = variant > G = normal to decarbamoyl mitomycin-C(DCMC). The induced sister chromatid exchange (SCE) frequency was unrelated to the extent of repair deficiency. The SCE induction rate was consistently 3 - 6 fold higher by these UV-like mutagens in XP group A cells than in normal cells. However, repair-proficient Cockayne's syndrome (CS) cells showed a higher SCE induction by UV, which was normalized by NAD/sup +/, suggesting that chromatin lesions as well as DNA damage contribute to SCE. Two-step crosslink repair involves a first rapid half-excision and a second slow nucleotide-excision repair. Fanconi's anemia (FA) cells had an impaired first half-excision and were supersensitive to MC, but not to UV and DCMC. The SCE frequency induced by MC (1 hr) was higher in FA cells than in normal cells despite their normal response to DCMC, and vice versa in XP cells. FA cells lacked the first rapid decline and showed higher remaining SCEs. Thus, part of the crosslink seems to lead to SCE formation. Caffeine synergistically elevated UV-induced SCEs, but not UV induced mutations in V79 cells, implying that SCE may not necessarily involve mutation.

  9. Excision and crosslink repair of DNA and sister chromatid exchanges in cultured human fibroblasts with different repair capacities

    International Nuclear Information System (INIS)

    Fujiwara, Yoshisada; Kano, Yoshio; Paul, P.; Goto, Kaoru; Yamamoto, Kazuo

    1981-01-01

    Xeroderma pigmentosum (XP) groups A to G lacked the initial stage of ultraviolet (UV) excision repair in the order of A = G > C > D > E asymptotically equals F, while the XP variant was weakly defective in the later repair steps. Killing sensitivities were in the orders of A >= G > D > C > E asymptotically equals F asymptotically equals variant > normal to UV, A = G > D > F > C = E > variant > normal to 4-nitroquinoline-1-oxide (4NQO), and A > C > D = E = F = variant > G = normal to decarbamoyl mitomycin-C(DCMC). The induced sister chromatid exchange (SCE) frequency was unrelated to the extent of repair deficiency. The SCE induction rate was consistently 3 - 6 fold higher by these UV-like mutagens in XP group A cells than in normal cells. However, repair-proficient Cockayne's syndrome (CS) cells showed a higher SCE induction by UV, which was normalized by NAD + , suggesting that chromatin lesions as well as DNA damage contribute to SCE. Two-step crosslink repair involves a first rapid half-excision and a second slow nucleotide-excision repair. Fanconi's anemia (FA) cells had an impaired first half-excision and were supersensitive to MC, but not to UV and DCMC. The SCE frequency induced by MC (1 hr) was higher in FA cells than in normal cells despite their normal response to DCMC, and vice versa in XP cells. FA cells lacked the first rapid decline and showed higher remaining SCEs. Thus, part of the crosslink seems to lead to SCE formation. Caffeine synergistically elevated UV-induced SCEs, but not UV induced mutations in V79 cells, implying that SCE may not necessarily involve mutation. (J.P.N.)

  10. Excision and crosslink repair of DNA and sister chromatid exchanges in cultured human fibroblasts with different repair capacities

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, Y.; Kano, Y.; Paul, P.; Goto, K.; Yamamoto, K. (Kobe Univ. (Japan). School of Medicine)

    1981-01-01

    Xeroderma pigmentosum (XP) groups A to G lacked the initial stage of ultraviolet (UV) excision repair in the order of A = G > C > D > E asymptotically equals F, while the XP variant was weakly defective in the later repair steps. Killing sensitivities were in the orders of A >= G > D > C > E asymptotically equals F asymptotically equals variant > normal to UV, A = G > D > F > C = E > variant > normal to 4-nitroquinoline-1-oxide (4NQO), and A > C > D = E = F = variant > G = normal to decarbamoyl mitomycin-C(DCMC). The induced sister chromatid exchange (SCE) frequency was unrelated to the extent of repair deficiency. The SCE induction rate was consistently 3 - 6 fold higher by these UV-like mutagens in XP group A cells than in normal cells. However, repair-proficient Cockayne's syndrome (CS) cells showed a higher SCE induction by UV, which was normalized by NAD/sup +/, suggesting that chromatin lesions as well as DNA damage contribute to SCE. Two-step crosslink repair involves a first rapid half-excision and a second slow nucleotide-excision repair. Fanconi's anemia (FA) cells had an impaired first half-excision and were supersensitive to MC, but not to UV and DCMC. The SCE frequency induced by MC (1 hr) was higher in FA cells than in normal cells despite their normal response to DCMC, and vice versa in XP cells. FA cells lacked the first rapid decline and showed higher remaining SCEs. Thus, part of the crosslink seems to lead to SCE formation. Caffeine synergistically elevated UV-induced SCEs, but not UV induced mutations in V79 cells, implying that SCE may not necessarily involve mutation.

  11. Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway.

    Science.gov (United States)

    Mu, Hong; Geacintov, Nicholas E; Min, Jung-Hyun; Zhang, Yingkai; Broyde, Suse

    2017-06-19

    The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N 2 -dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the "pre-flipped" base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [ Mu , H. , ( 2015 ) Biochemistry , 54 ( 34 ), 5263 - 7 ]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER.

  12. Polymerization by DNA polymerase eta is blocked by cis-diamminedichloroplatinum(II) 1,3-d(GpTpG) cross-link: implications for cytotoxic effects in nucleotide excision repair-negative tumor cells.

    Science.gov (United States)

    Chijiwa, Shotaro; Masutani, Chikahide; Hanaoka, Fumio; Iwai, Shigenori; Kuraoka, Isao

    2010-03-01

    cis-Diamminedichloroplatinum(II) (cisplatin) forms DNA adducts that interfere with replication and transcription. The most common adducts formed in vivo are 1,2-intrastrand d(GpG) cross-links (Pt-GG) and d(ApG) cross-links (Pt-AG), with minor amounts of 1,3-d(GpNpG) cross-links (Pt-GNG), interstrand cross-links and monoadducts. Although the relative contribution of these different adducts to toxicity is not known, literature implicates that Pt-GG and Pt-AG adducts block replication. Thus, nucleotide excision repair (NER), by which platinum adducts are excised, and translesion DNA synthesis (TLS), which permits adduct bypass, are thought to be associated with cisplatin resistance. Recent studies have reported that the clinical benefit from platinum-based chemotherapy is high if tumor cells express low levels of NER factors. To investigate the role of platinum-DNA adducts in mediating tumor cell survival by TLS, we examined whether 1,3-intrastrand d(GpTpG) platinum cross-links (Pt-GTG), which probably exist in NER-negative tumor cells but not in NER-positive tumor cells, are bypassed by the translesion DNA polymerase eta (pol eta), which is known to bypass Pt-GG. We show that pol eta can incorporate the correct deoxycytidine triphosphate opposite the first 3'-cross-linked G of Pt-GTG but cannot insert any nucleotides opposite the second intact T or the third 5'-cross-linked G of the adducts, thereby suggesting that TLS does not facilitate replication past Pt-GTG adducts. Thus, our findings implicate Pt-GNG adducts as mediating the cytotoxicity of platinum-DNA adducts in NER-negative tumors in vivo.

  13. Kinetics of thymine dimer excision in ultraviolet-irradiated human cells

    International Nuclear Information System (INIS)

    Ehmann, U.K.; Cook, K.H.; Friedberg, E.C.

    1978-01-01

    We have investigated the kinetics of the loss of thymine dimers from the acid-insoluble fraction of several ultraviolet (uv)-irradiated cultured human cell lines. Our results show that uv fluences between 10 and 40 J/m 2 produce an average of 21 to 85 x 10 5 thymine dimers per cell and an eventual maximal loss per cell of 12 to 20 x 10 5 thymine dimers. The time for half-maximal loss of dimers ranged from 12 to 22 h after uv irradiation. In contrast, the time for half-maximal repair synthesis of DNA measured by autoradiography was 4.5 h. This figure agrees well with reported half-maximal repair synthesis times, which range from 0.5 to 3.6 h based on our analysis. The discrepancy in the kinetics of the loss of thymine dimers from DNA and repair synthesis is discussed in terms of possible molecular mechanisms of thymine dimer excision in vivo and in terms of possible experimental artifacts

  14. Human renin 5'-flanking DNA to nucleotide-2750.

    Science.gov (United States)

    Smith, D L; Jeyapalan, S; Lang, J A; Guo, X H; Sigmund, C D; Morris, B J

    1995-01-01

    Renin is one of the most important factors in blood pressure and electrolyte regulation in mammals and the renin locus has been implicated in hypertension. To assist studies of promoter control we therefore determined the 5'-flanking sequence of the human gene (REN) to residue -2750 relative to the transcription start site (+1). Sites of homology to consensus sequences for binding of trans-acting factors involved in transcriptional control of other genes were identified, and functionality for two of these (a CRE and Pit-1 site) have so far been demonstrated.

  15. Nucleotide sequence of a human tRNA gene heterocluster

    International Nuclear Information System (INIS)

    Chang, Y.N.; Pirtle, I.L.; Pirtle, R.M.

    1986-01-01

    Leucine tRNA from bovine liver was used as a hybridization probe to screen a human gene library harbored in Charon-4A of bacteriophage lambda. The human DNA inserts from plaque-pure clones were characterized by restriction endonuclease mapping and Southern hybridization techniques, using both [3'- 32 P]-labeled bovine liver leucine tRNA and total tRNA as hybridization probes. An 8-kb Hind III fragment of one of these γ-clones was subcloned into the Hind III site of pBR322. Subsequent fine restriction mapping and DNA sequence analysis of this plasmid DNA indicated the presence of four tRNA genes within the 8-kb DNA fragment. A leucine tRNA gene with an anticodon of AAG and a proline tRNA gene with an anticodon of AGG are in a 1.6-kb subfragment. A threonine tRNA gene with an anticodon of UGU and an as yet unidentified tRNA gene are located in a 1.1-kb subfragment. These two different subfragments are separated by 2.8 kb. The coding regions of the three sequenced genes contain characteristic internal split promoter sequences and do not have intervening sequences. The 3'-flanking region of these three genes have typical RNA polymerase III termination sites of at least four consecutive T residues

  16. Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ping; Guliaev, Anton B.; Hang, Bo

    2006-02-28

    Cadmium (Cd{sup 2+}), nickel (Ni{sup 2+}) and cobalt (Co{sup 2+}) are human and/or animal carcinogens. Zinc (Zn{sup 2+}) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (var epsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1000 {micro}M, both Cd{sup 2+} and Zn{sup 2+} showed metal-dependent inhibition of the MPG catalytic activity. Ni{sup 2+} also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co{sup 2+} and Mg{sup 2+} did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the var epsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd{sup 2+}, Zn{sup 2+}, and Ni{sup 2+} at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn{sup 2+} showed that the MPG active site has a potential binding site for Zn{sup 2+}, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.

  17. Understanding specificity in metabolic pathways-Structural biology of human nucleotide metabolism

    International Nuclear Information System (INIS)

    Welin, Martin; Nordlund, Paer

    2010-01-01

    Interactions are the foundation of life at the molecular level. In the plethora of activities in the cell, the evolution of enzyme specificity requires the balancing of appropriate substrate affinity with a negative selection, in order to minimize interactions with other potential substrates in the cell. To understand the structural basis for enzyme specificity, the comparison of structural and biochemical data between enzymes within pathways using similar substrates and effectors is valuable. Nucleotide metabolism is one of the largest metabolic pathways in the human cell and is of outstanding therapeutic importance since it activates and catabolises nucleoside based anti-proliferative drugs and serves as a direct target for anti-proliferative drugs. In recent years the structural coverage of the enzymes involved in human nucleotide metabolism has been dramatically improved and is approaching completion. An important factor has been the contribution from the Structural Genomics Consortium (SGC) at Karolinska Institutet, which recently has solved 33 novel structures of enzymes and enzyme domains in human nucleotide metabolism pathways and homologs thereof. In this review we will discuss some of the principles for substrate specificity of enzymes in human nucleotide metabolism illustrated by a selected set of enzyme families where a detailed understanding of the structural determinants for specificity is now emerging.

  18. On the biased nucleotide composition of the human coronavirus RNA genome

    NARCIS (Netherlands)

    Berkhout, Ben; van Hemert, Formijn

    2015-01-01

    We investigated the nucleotide composition of the RNA genome of the six human coronaviruses. Some general coronavirus characteristics were apparent (e.g. high U, low C count), but we also detected species-specific signatures. Most strikingly, the high U and low C proportions are quite variable and

  19. Zinc finger nuclease: a new approach for excising HIV-1 proviral DNA from infected human T cells.

    Science.gov (United States)

    Qu, Xiying; Wang, Pengfei; Ding, Donglin; Wang, Xiaohui; Zhang, Gongmin; Zhou, Xin; Liu, Lin; Zhu, Xiaoli; Zeng, Hanxian; Zhu, Huanzhang

    2014-09-01

    A major reason that Acquired Immune Deficiency Syndrome (AIDS) cannot be completely cured is the human immunodeficiency virus 1 (HIV-1) provirus integrated into the human genome. Though existing therapies can inhibit replication of HIV-1, they cannot eradicate it. A molecular therapy gains popularity due to its specifically targeting to HIV-1 infected cells and effectively removing the HIV-1, regardless of viral genes being active or dormant. Now, we propose a new method which can excellently delete the HIV provirus from the infected human T cell genome. First, we designed zinc-finger nucleases (ZFNs) that target a sequence within the long terminal repeat (LTR) U3 region that is highly conserved in whole clade. Then, we screened out one pair of ZFN and named it as ZFN-U3. We discovered that ZFN-U3 can exactly target and eliminate the full-length HIV-1 proviral DNA after the infected human cell lines treated with it, and the frequency of its excision was about 30 % without cytotoxicity. These results prove that ZFN-U3 can efficiently excise integrated HIV-1 from the human genome in infected cells. This method to delete full length HIV-1 in human genome can therefore provide a novel approach to cure HIV-infected individuals in the future.

  20. Significant contribution of the 3′→5′ exonuclease activity to the high fidelity of nucleotide incorporation catalyzed by human DNA polymerase ϵ

    Science.gov (United States)

    Zahurancik, Walter J.; Klein, Seth J.; Suo, Zucai

    2014-01-01

    Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess a faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3′→5′ exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to overall low polymerase error frequency (one error per 106–108 incorporations) and support faithful eukaryotic genome replication. Eukaryotic DNA polymerase ϵ (Polϵ) is one of three main replicative DNA polymerases for nuclear genomic replication and is responsible for leading strand synthesis. Here, we employed pre-steady-state kinetic methods and determined the overall fidelity of human Polϵ (hPolϵ) by measuring the individual contributions of its polymerase and 3′→5′ exonuclease activities. The polymerase activity of hPolϵ has a high base substitution fidelity (10−4–10−7) resulting from large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides. The 3′→5′ exonuclease activity of hPolϵ further enhances polymerization fidelity by an unprecedented 3.5 × 102 to 1.2 × 104-fold. The resulting overall fidelity of hPolϵ (10−6–10−11) justifies hPolϵ to be a primary enzyme to replicate human nuclear genome (0.1–1.0 error per round). Consistently, somatic mutations in hPolϵ, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation. PMID:25414327

  1. Nucleotide excision repair genes are expressed at low levels and are not detectably inducible in Caenorhabditis elegans somatic tissues, but their function is required for normal adult life after UVC exposure

    International Nuclear Information System (INIS)

    Boyd, Windy A.; Crocker, Tracey L.; Rodriguez, Ana M.; Leung, Maxwell C.K.; Wade Lehmann, D.; Freedman, Jonathan H.; Van Houten, Ben; Meyer, Joel N.

    2010-01-01

    We performed experiments to characterize the inducibility of nucleotide excision repair (NER) in Caenorhabditis elegans, and to examine global gene expression in NER-deficient and -proficient strains as well as germline vs. somatic tissues, with and without genotoxic stress. We also carried out experiments to elucidate the importance of NER in the adult life of C. elegans under genotoxin-stressed and control conditions. Adult lifespan was not detectably different between wild-type and NER-deficient xpa-1 nematodes under control conditions. However, exposure to 6 J/m 2 /day of ultraviolet C radiation (UVC) decreased lifespan in xpa-1 nematodes more than a dose of 100 J/m 2 /day in wild-type. Similar differential sensitivities were observed for adult size and feeding. Remarkably, global gene expression was nearly identical in young adult wild-type and xpa-1 nematodes, both in control conditions and 3 h after exposure to 50 J/m 2 UVC. Neither NER genes nor repair activity were detectably inducible in young adults that lacked germ cells and developing embryos (glp-1 strain). However, expression levels of dozens of NER and other DNA damage response genes were much (5-30-fold) lower in adults lacking germ cells and developing embryos, suggesting that somatic and post-mitotic cells have a much lower DNA repair ability. Finally, we describe a refinement of our DNA damage assay that allows damage measurement in single nematodes.

  2. Nucleotide excision repair genes are expressed at low levels and are not detectably inducible in Caenorhabditis elegans somatic tissues, but their function is required for normal adult life after UVC exposure

    Energy Technology Data Exchange (ETDEWEB)

    Boyd, Windy A. [Biomolecular Screening Branch, National Toxicology Program, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC (United States); Crocker, Tracey L. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Rodriguez, Ana M. [Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC (United States); Leung, Maxwell C.K. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Wade Lehmann, D. [Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC (United States); Freedman, Jonathan H. [Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC (United States); Van Houten, Ben [Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC (United States); Meyer, Joel N., E-mail: joel.meyer@duke.edu [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States)

    2010-01-05

    We performed experiments to characterize the inducibility of nucleotide excision repair (NER) in Caenorhabditis elegans, and to examine global gene expression in NER-deficient and -proficient strains as well as germline vs. somatic tissues, with and without genotoxic stress. We also carried out experiments to elucidate the importance of NER in the adult life of C. elegans under genotoxin-stressed and control conditions. Adult lifespan was not detectably different between wild-type and NER-deficient xpa-1 nematodes under control conditions. However, exposure to 6 J/m{sup 2}/day of ultraviolet C radiation (UVC) decreased lifespan in xpa-1 nematodes more than a dose of 100 J/m{sup 2}/day in wild-type. Similar differential sensitivities were observed for adult size and feeding. Remarkably, global gene expression was nearly identical in young adult wild-type and xpa-1 nematodes, both in control conditions and 3 h after exposure to 50 J/m{sup 2} UVC. Neither NER genes nor repair activity were detectably inducible in young adults that lacked germ cells and developing embryos (glp-1 strain). However, expression levels of dozens of NER and other DNA damage response genes were much (5-30-fold) lower in adults lacking germ cells and developing embryos, suggesting that somatic and post-mitotic cells have a much lower DNA repair ability. Finally, we describe a refinement of our DNA damage assay that allows damage measurement in single nematodes.

  3. Metabolic modulation of mammalian DNA excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Schrader, T.J.

    1988-01-01

    First, ultraviolet light (UVL)- and dimethylsulfate (DMS)-induced excision repair was examined in quiescent and lectin-stimulated bovine lymphocytes. Upon mitogenic stimulation, UVL-induced repair increased by a factor of 2 to 3, and reached this maximum 2 days before the onset of DNA replication. However, DMS-induced repair increased sevenfold in parallel with DNA replication. Repair patch sizes were smaller for DMS-induced damage reflecting patches of 7 nucleotides in quiescent lymphocytes compared to 20 nucleotides induced by UVL. The patch size increased during lymphocyte stimulation until one day prior to the peak of DNA replication when patch sizes of 45 and 35 nucleotides were produced in response to UVL- and DMS-induced damage, respectively. At the peak of DNA replication, the patch sizes were equal for both damaging agents at 34 nucleotides. In the second study, a small amount of repair replication was observed in undamaged quiescent and concanavalin A-stimulated bovine lymphocytes as well as in human T98G glioblastoma cells. Repair incorporation doubled in the presence of hydroxyurea. Thirdly, the enhanced repair replication induced by the poly (ADP-ribose) polymerase inhibitor, 3-aminobenzamide, (3-AB), could not be correlated either with an increased rate of repair in the presence of 3-AB or with the use of hydroxyurea in the repair protocol. Finally, treatment of unstimulated lymphocytes with hyperthermia was accompanied by decreased repair replication while the repair patches remained constant at 20 nucleotides.

  4. The nucleotide sequence of human transition protein 1 cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Luerssen, H; Hoyer-Fender, S; Engel, W [Universitaet Goettingen (West Germany)

    1988-08-11

    The authors have screened a human testis cDNA library with an oligonucleotide of 81 mer prepared according to a part of the published nucleotide sequence of the rat transition protein TP 1. They have isolated a cDNA clone with the length of 441 bp containing the coding region of 162 bp for human transition protein 1. There is about 84% homology in the coding region of the sequence compared to rat. The human cDNA-clone encodes a polypeptide of 54 amino acids of which 7 are different to that of rat.

  5. A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

    Science.gov (United States)

    Fu, Yuan; Lin, Hongyu; Wisitpitthaya, Somsinee; Blessing, William A; Aye, Yimon

    2014-11-24

    Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Inducible nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers in the cell cycle of the budding yeast Saccharomyces cerevisiae: evidence that inducible NER is confined to the G1 phase of the mitotic cell cycle

    International Nuclear Information System (INIS)

    Scott, A.D.; Waters, R.

    1997-01-01

    We previously reported on an inducible component of nucleotide excision repair in Saccharomyces cerevisiae that is controlled by the RAD16 gene. Here we describe a study of this event at the MAT alpha and HML alpha mating-type loci and on the transcribed (TS) and nontranscribed (NTS) strands of the RAD16 gene. Events were examined at various stages of the mitotic cycle in cells synchronised by centrifugal elutriation. Repair of cyclobutane pyrimidine dimers (CPDs) following a single UV dose does not vary significantly in different stages of the mitotic cell cycle. CPDs are removed more rapidly from the transcriptionally active MAT alpha locus than from the silent HML alpha locus, and the TS of RAD16 is repaired faster than the NTS in all stages of the cycle following a single UV irradiation. Enhanced excision of CPDs at MAT alpha and HML alpha can be induced only in the G1 and early S stages of the cell cycle. Here prior irradiation of cells with 25 J/m 2 enhances the removal of CPDs following a second UV dose of 70 J/m 2 . The level of enhancement of repair does not differ significantly between MAT alpha and HML alpha in G1. Enhanced removal of CPDs is absent when cells receive the inducing dose in late S or G2/M. Repair of CPDs in both strands of RAD16 is similarly enhanced only if cells receive the initial irradiation in G1 and early S. The level of enhanced removal of CPDs is not significantly different in the TS and NTS of RAD16 either in asynchronous cells or in cells preirradiated in G1 and early S. It has been shown by others that UV-induced expression of RAD16 remains at high levels if cells are held in G1 by treatment with alpha factor. Therefore the increase in RAD16 transcript levels in G1 may be responsible for the ability to enhance NER solely in this stage of the cell cycle

  7. Molecular cloning and complete nucleotide sequence of a human ventricular myosin light chain 1

    Energy Technology Data Exchange (ETDEWEB)

    Hoffmann, E; Shi, Q W; Floroff, M; Mickle, D A.G.; Wu, T W; Olley, P M; Jackowski, G

    1988-03-25

    Human ventricular plasmid library was constructed. The library was screened with the oligonucleotide probe (17-mer) corresponding to a conserve region of myosin light chain 1 near the carboxy terminal. Full length cDNA recombinant plasmid containing 1100 bp insert was isolated. RNA blot hybridization with this insert detected a message of approximately 1500 bp corresponding to the size of VLCl and mRNA. Complete nucleotide sequence of the coding region was determined in M13 subclones using dideoxy chain termination method. With the isolation of this clone (pCD HLVCl), the publication of the complete nucleotide sequence of HVLCl and the predicted secondary structure of this protein will aid in understanding of the biochemistry of myosin and its function in contraction, the evolution of myosin light genes and the genetic, developmental and physiological regulation of myosin genes.

  8. Effects of fenspiride on human bronchial cyclic nucleotide phosphodiesterase isoenzymes: functional and biochemical study.

    Science.gov (United States)

    Cortijo, J; Naline, E; Ortiz, J L; Berto, L; Girard, V; Malbezin, M; Advenier, C; Morcillo, E J

    1998-01-02

    We have investigated the role of human bronchial cyclic nucleotide phosphodiesterases in the effects of fenspiride, a drug endowed with bronchodilator and anti-inflammatory properties. Functional studies on human isolated bronchi showed that fenspiride (10(-6)-3 x 10(-3) M, 30 min) induced a shift to the left of the concentration-response curves for isoprenaline and sodium nitroprusside with -logEC50 values of 4.1+/-0.1 (n = 7) and 3.5+/-0.2 (n = 8), respectively. Biochemical studies were carried out on three human bronchi in which separation of cyclic nucleotide phosphodiesterase isoenzymes was performed by ion exchange chromatography followed by determination of phosphodiesterase activity with a radioisotopic method. Phosphodiesterase 4 (cyclic AMP-specific) and phosphodiesterase 5 (cyclic GMP-specific) were the major phosphodiesterase isoforms present in the human bronchial tissue. The presence of phosphodiesterase 1 (Ca2+/calmodulin-stimulated), phosphodiesterase 2 (cyclic GMP-stimulated) and, in two cases, phosphodiesterase 3 (cyclic GMP-inhibited) was also identified. Fenspiride inhibited phosphodiesterase 4 and phosphodiesterase 3 activities with -logIC50 values of 4.16+/-0.09 and 3.44+/-0.12, respectively. Phosphodiesterase 5 activity was also inhibited with a -logIC50 value of approximately 3.8. Fenspiride (fenspiride is an effective inhibitor of both cyclic AMP and cyclic GMP hydrolytic activity in human bronchial tissues and this action may contribute to its airway effects.

  9. Differential pathway control in nucleotide excision repair

    NARCIS (Netherlands)

    G.J.C. van Belle (Gijsbert)

    2015-01-01

    markdownabstractAbstract The stability and integrity of the genome is crucial for all cellular life on earth. This integrity is continuously challenged by internal and external genotoxic agents. These agents cause DNA damages which interfere with important cellular processes like replication of

  10. Base excision repair activities differ in human lung cancer cells and corresponding normal controls

    DEFF Research Database (Denmark)

    Karahalil, Bensu; Bohr, Vilhelm A; De Souza-Pinto, Nadja C

    2010-01-01

    Oxidative damage to DNA is thought to play a role in carcinogenesis by causing mutations, and indeed accumulation of oxidized DNA bases has been observed in samples obtained from tumors but not from surrounding tissue within the same patient. Base excision repair (BER) is the main pathway...... for the repair of oxidized modifications both in nuclear and mitochondrial DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non......-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three...

  11. Identification of genes and proteins involved in excision repair of human cells

    International Nuclear Information System (INIS)

    Hoeijmakers, J.H.J.; Westerveld, A.; Van Duin, M.; Vermeulen, W.; Odijk, H.; De Wit, J.; Bootsma, D.

    1986-01-01

    The autosomal, recessive disorder xeroderma pigmentosum (XP) is characterized by extreme sensitivity of the skin to sun exposure and prediposition to skin cancer. The basic defect in most XP patients is thought to reside in an inefficient removal of UV-induced lesions in the DNA by excision repair. The biochemical complexity of this process is amply illustrated by the fact that so far nine complementary groups within this syndrome have been identified. Despite extensive research, none of these genes or proteins involved have been isolated. Using a microinjection assay system the authors identified components in crude cell extracts that transiently correct the defect in (injected) fibroblasts of all excision-deficient XP complementation groups, as indicated by temporary restoration of UV-induced unscheduled DNA synthesis. This correction is complementation group specific, since it is only found when extracts from complementing XP cells are injected. After incubation of extracts with proteinase K the XP-A and KP-G correcting activities were lost, indicating that the complementation is due to proteins. The XP-A correcting protein was found to precipitate between 30 and 60% ammonium sulfate saturation. Furthermore this protein binds to DEAE-cellulose and to (UV-irradiated) double-strand (ds) DNA attached to cellulose. The latter affinity chromatography step allows a considerable purification, since less than 1% of the proteins applied to such columns is retained. It has to be established whether the XP-A correcting proteins binds by itself or via other proteins to the UV-irradiated DNA and whether it also binds to nonirradiated (ds or ss) DNA. Similar experiments with the XP-G correcting protein are in progress

  12. Recovery from inhibition by UV-irradiation of ornithine decarboxylase induction in human cells: implication of excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Hur, E.; Prager, A. (Nuclear Research Centre-Negev, Beer-Sheva (Israel)); Buonaguro, F. (Argonne National Lab., IL (USA))

    1982-05-01

    Exposure of stationary-phase human breast carcinoma (T-47D) cells to far-UV light (254nm) inhibited the appearance of induced ornithine decarboxylase (ODC) activity. The fluence response curve had a shoulder (Dsub(q)=2Jm/sup -2/) followed by an exponential decline (D/sub 0/=4.2Jm/sup -2/). The cells could recover from this inhibition when the stimulus of induction of ODC was delayed for 20-24h after irradiation. Hydroxyurea (HU) when present at 3mM during the recovery period eliminated completely the ability of the cells to recover. This effect of HU on ODC induction was partially reversed by 50..mu..M of the four deoxyribonucleosides required for DNA synthesis. Neither HU nor the deoxyribonucleosides by themselves affected ODC induction in unirradiated cells. Since HU inhibited the recovery from potentially lethal UV damage and is a known inhibitor of excision repair, it is suggested that recovery from UV-induced inhibition of ODC induction depends on excision-repair of DNA damage. This interpretation is strongly supported by the finding that specific photolysis of 5-bromodeoxyuridine, incorporated into DNA during the recovery period, inhibited recovery of ODC induction from inhibition by UV light.

  13. In vivo excision of pyrimidine dimers is mediated by a DNA N-glycosylase in Micrococcus luteus but not in human fibroblasts

    International Nuclear Information System (INIS)

    La Belle, M.; Linn, S.

    1982-01-01

    It has been previously shown that Micrococcus luteus possesses a pyrimidine dimer-specific endonuclease which in vitro, functions as both an endonuclease and DNA-glycosylase. To determine if these combined activities function in vivo, the excision products of UV-irradiated M. luteus were isolated and examined. In addition, a procedure was devised to isolate and examine the excision products from UV-irradiated human fibroblasts to determine if an endonuclease/glycosylase activity functions in the excision of UV-induced pyrimidine dimers in human fibroblasts. It was shown that, in vivo, an endonuclease/glycosylase mechanism is utilized extensively in the repair of pyrimidine dimers by M. luteus, but that human fibroblasts do not appear to use this mechanism. (author)

  14. In vivo excision of pyrimidine dimers is mediated by a DNA N-glycosylase in Micrococcus luteus but not in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    La Belle, M; Linn, S [California Univ., Berkeley (USA). Dept. of Biochemistry

    1982-09-01

    It has been previously shown that Micrococcus luteus possesses a pyrimidine dimer-specific endonuclease which in vitro, functions as both an endonuclease and DNA-glycosylase. To determine if these combined activities function in vivo, the excision products of UV-irradiated M. luteus were isolated and examined. In addition, a procedure was devised to isolate and examine the excision products from UV-irradiated human fibroblasts to determine if an endonuclease/glycosylase activity functions in the excision of UV-induced pyrimidine dimers in human fibroblasts. It was shown that, in vivo, an endonuclease/glycosylase mechanism is utilized extensively in the repair of pyrimidine dimers by M. luteus, but that human fibroblasts do not appear to use this mechanism.

  15. Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells

    International Nuclear Information System (INIS)

    Intine, R.V.; Rainbow, A.J.

    1990-01-01

    A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in part at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair

  16. DREMECELS: A Curated Database for Base Excision and Mismatch Repair Mechanisms Associated Human Malignancies.

    Directory of Open Access Journals (Sweden)

    Ankita Shukla

    Full Text Available DNA repair mechanisms act as a warrior combating various damaging processes that ensue critical malignancies. DREMECELS was designed considering the malignancies with frequent alterations in DNA repair pathways, that is, colorectal and endometrial cancers, associated with Lynch syndrome (also known as HNPCC. Since lynch syndrome carries high risk (~40-60% for both cancers, therefore we decided to cover all three diseases in this portal. Although a large population is presently affected by these malignancies, many resources are available for various cancer types but no database archives information on the genes specifically for only these cancers and disorders. The database contains 156 genes and two repair mechanisms, base excision repair (BER and mismatch repair (MMR. Other parameters include some of the regulatory processes that have roles in these disease progressions due to incompetent repair mechanisms, specifically BER and MMR. However, our unique database mainly provides qualitative and quantitative information on these cancer types along with methylation, drug sensitivity, miRNAs, copy number variation (CNV and somatic mutations data. This database would serve the scientific community by providing integrated information on these disease types, thus sustaining diagnostic and therapeutic processes. This repository would serve as an excellent accompaniment for researchers and biomedical professionals and facilitate in understanding such critical diseases. DREMECELS is publicly available at http://www.bioinfoindia.org/dremecels.

  17. The Nucleotide Synthesis Enzyme CAD Inhibits NOD2 Antibacterial Function in Human Intestinal Epithelial Cells

    Science.gov (United States)

    Richmond, Amy L.; Kabi, Amrita; Homer, Craig R.; García, Noemí Marina; Nickerson, Kourtney P.; NesvizhskiI, Alexey I.; Sreekumar, Arun; Chinnaiyan, Arul M.; Nuñez, Gabriel; McDonald, Christine

    2013-01-01

    BACKGROUND & AIMS Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohn’s disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies. METHODS Carbamoyl phosphate synthetase/ aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors. RESULTS CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor κB and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD. CONCLUSIONS The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD. PMID:22387394

  18. Effects of an extract from the sea squirt Ecteinascidia turbinata on DNA synthesis and excision repair in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Dunn, W.C.; Carrier, W.L.; Regan, J.D.

    1982-01-01

    An aqueous ethanol extract from the marine tunicate species Ecteinascidia turbinata was studied to determine its effect on semiconservative DNA synthesis in human skin fibroblast cultures as measured by (/sup 3/H) thymidine uptake in acid-insoluble cell fractions. In addition, the effect of this extract on DNA excision repair in ultraviolet light (254 nm) irradiated fibroblasts was measured by the bromodeoxyuridine photolysis assay, thymine dimer chromatography, and DNA single-strand break analysis on alkaline sucrose gradients. Repair inhibition was accompanied by an accumulation of single-strand DNA breaks which was enhanced by the addtion of 2 mM hydroxyurea. These results are discussed with respect to a mechanism of action of the marine tunicate extract at the level of DNA polymerases and are contrasted with previously studied inhibitory mechanisms of arabinofuranosyl nucleosides.

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  20. The effect of ultraviolet light on the cyclic nucleotide system of human fibroblasts

    International Nuclear Information System (INIS)

    Fertel, R.H.; Tejwani, G.A.; Albrightson, C.R.; Hart, R.W.

    1981-01-01

    The concentrations of cyclic AMP and cyclic GMP in in human skin fibroblasts in culture were determined after exposing the cells to varying fluences of UV (254 nm) light. The cyclic nucleotide concentrations of cells irradiated in the log phase of growth were unchanged relative to controls. In contrast, there was a rise in the concentration of cyclic AMP in cells irradiated after they reached confluency. The increase in concentration was observed as early as 30 min after irradiation, reached a maximum of about 200% of control at 4 to 6 h after exposure, and returned to control values by 24 h after irradiation. The effect was proportional to a UV fluence from 5 to 20 J/m 2 , and was blocked by the addition of the UV absorbing agent para-aminobenzoic acid. In contrast, the results indicated that UV light had no effect on the concentration of cyclic GMP in human fibroblast cell cultures. Because of the importance of cyclic nucleotides in the regulation of cellular function, it is reasonable to hypothesize that changes in cyclic AMP induced by UV light may effect the extranuclear functions of irradiated cells. (author)

  1. The pyrimidine nucleotide carrier PNC1 and mitochondrial trafficking of thymidine phosphates in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Franzolin, Elisa; Miazzi, Cristina; Frangini, Miriam; Palumbo, Elisa; Rampazzo, Chiara [Department of Biology, University of Padova, Via Ugo Bassi 58B, I-35131 Padova (Italy); Bianchi, Vera, E-mail: vbianchi@bio.unipd.it [Department of Biology, University of Padova, Via Ugo Bassi 58B, I-35131 Padova (Italy)

    2012-10-15

    In cycling cells cytosolic de novo synthesis of deoxynucleotides is the main source of precursors for mitochondrial (mt) DNA synthesis. The transfer of deoxynucleotides across the inner mt membrane requires protein carriers. PNC1, a SLC25 family member, exchanges pyrimidine nucleoside triphosphates in liposomes and its downregulation decreases mtUTP concentration in cultured cells. By an isotope-flow protocol we confirmed transport of uridine nucleotides by PNC1 in intact cultured cells and investigated PNC1 involvement in the mt trafficking of thymidine phosphates. Key features of our approach were the manipulation of PNC1 expression by RNA interference or inducible overexpression, the employment of cells proficient or deficient for cytosolic thymidine kinase (TK1) to distinguish the direction of flow of thymidine nucleotides across the mt membrane during short pulses with [{sup 3}H]-thymidine, the determination of mtdTTP specific radioactivity to quantitate the rate of mtdTTP export to the cytoplasm. Downregulation of PNC1 in TK1{sup -} cells increased labeled dTTP in mitochondria due to a reduced rate of export. Overexpression of PNC1 in TK1{sup +} cells increased mtdTTP pool size and radioactivity, suggesting an involvement in the import of thymidine phosphates. Thus PNC1 is a component of the network regulating the mtdTTP pool in human cells. -- Highlights: Black-Right-Pointing-Pointer Thymidine phosphates exchange between mitochondria and cytosol in mammalian cells. Black-Right-Pointing-Pointer siRNA-downregulation of PNC1 delays mitochondrial dTTP export in TK1{sup -} cells. Black-Right-Pointing-Pointer PNC1 overexpression accumulates dTTP in mitochondria of TK1{sup +} cells. Black-Right-Pointing-Pointer PNC1 exchanges thymidine nucleotides across the mitochondrial inner membrane. Black-Right-Pointing-Pointer PNC1 participates in the regulation of the mtdTTP pool supporting mtDNA synthesis.

  2. The pyrimidine nucleotide carrier PNC1 and mitochondrial trafficking of thymidine phosphates in cultured human cells

    International Nuclear Information System (INIS)

    Franzolin, Elisa; Miazzi, Cristina; Frangini, Miriam; Palumbo, Elisa; Rampazzo, Chiara; Bianchi, Vera

    2012-01-01

    In cycling cells cytosolic de novo synthesis of deoxynucleotides is the main source of precursors for mitochondrial (mt) DNA synthesis. The transfer of deoxynucleotides across the inner mt membrane requires protein carriers. PNC1, a SLC25 family member, exchanges pyrimidine nucleoside triphosphates in liposomes and its downregulation decreases mtUTP concentration in cultured cells. By an isotope-flow protocol we confirmed transport of uridine nucleotides by PNC1 in intact cultured cells and investigated PNC1 involvement in the mt trafficking of thymidine phosphates. Key features of our approach were the manipulation of PNC1 expression by RNA interference or inducible overexpression, the employment of cells proficient or deficient for cytosolic thymidine kinase (TK1) to distinguish the direction of flow of thymidine nucleotides across the mt membrane during short pulses with [ 3 H]-thymidine, the determination of mtdTTP specific radioactivity to quantitate the rate of mtdTTP export to the cytoplasm. Downregulation of PNC1 in TK1 − cells increased labeled dTTP in mitochondria due to a reduced rate of export. Overexpression of PNC1 in TK1 + cells increased mtdTTP pool size and radioactivity, suggesting an involvement in the import of thymidine phosphates. Thus PNC1 is a component of the network regulating the mtdTTP pool in human cells. -- Highlights: ► Thymidine phosphates exchange between mitochondria and cytosol in mammalian cells. ► siRNA-downregulation of PNC1 delays mitochondrial dTTP export in TK1 − cells. ► PNC1 overexpression accumulates dTTP in mitochondria of TK1 + cells. ► PNC1 exchanges thymidine nucleotides across the mitochondrial inner membrane. ► PNC1 participates in the regulation of the mtdTTP pool supporting mtDNA synthesis.

  3. Few single nucleotide variations in exomes of human cord blood induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Rui-Jun Su

    Full Text Available The effect of the cellular reprogramming process per se on mutation load remains unclear. To address this issue, we performed whole exome sequencing analysis of induced pluripotent stem cells (iPSCs reprogrammed from human cord blood (CB CD34(+ cells. Cells from a single donor and improved lentiviral vectors for high-efficiency (2-14% reprogramming were used to examine the effects of three different combinations of reprogramming factors: OCT4 and SOX2 (OS, OS and ZSCAN4 (OSZ, OS and MYC and KLF4 (OSMK. Five clones from each group were subject to whole exome sequencing analysis. We identified 14, 11, and 9 single nucleotide variations (SNVs, in exomes, including untranslated regions (UTR, in the five clones of OSMK, OS, and OSZ iPSC lines. Only 8, 7, and 4 of these, respectively, were protein-coding mutations. An average of 1.3 coding mutations per CB iPSC line is remarkably lower than previous studies using fibroblasts and low-efficiency reprogramming approaches. These data demonstrate that point nucleotide mutations during cord blood reprogramming are negligible and that the inclusion of genome stabilizers like ZSCAN4 during reprogramming may further decrease reprogramming-associated mutations. Our findings provide evidence that CB is a superior source of cells for iPSC banking.

  4. Modification of nucleotide metabolism in relationship with differentiation and in response to irradiation in human tumour cells

    International Nuclear Information System (INIS)

    Wei, Shuang

    1998-01-01

    This research thesis reports the study of the metabolism of nucleotides in human tumour cells. The first part addresses the modifications of nucleotide (more specifically purine) metabolism in relationship with human melanoma cell proliferation and differentiation. The second part addresses the modifications of this metabolism in response to an irradiation in human colon tumour cells. For each part, the author proposes a bibliographic synthesis, and a presentation of studied cells and of methods used to grow cells, and respectively to proliferate and differentiate them or to irradiate them, and then discusses the obtained results [fr

  5. A Single Nucleotide Polymorphism in Human APOBEC3C Enhances Restriction of Lentiviruses.

    Directory of Open Access Journals (Sweden)

    Cristina J Wittkopp

    2016-10-01

    Full Text Available Humans express seven human APOBEC3 proteins, which can inhibit viruses and endogenous retroelements through cytidine deaminase activity. The seven paralogs differ in the potency of their antiviral effects, as well as in their antiviral targets. One APOBEC3, APOBEC3C, is exceptional as it has been found to only weakly block viruses and endogenous retroelements compared to other APOBEC3s. However, our positive selection analyses suggest that APOBEC3C has played a role in pathogen defense during primate evolution. Here, we describe a single nucleotide polymorphism in human APOBEC3C, a change from serine to isoleucine at position 188 (I188 that confers potent antiviral activity against HIV-1. The gain-of-function APOBEC3C SNP results in increased enzymatic activity and hypermutation of target sequences when tested in vitro, and correlates with increased dimerization of the protein. The I188 is widely distributed in human African populations, and is the ancestral primate allele, but is not found in chimpanzees or gorillas. Thus, while other hominids have lost activity of this antiviral gene, it has been maintained, or re-acquired, as a more active antiviral gene in a subset of humans. Taken together, our results suggest that APOBEC3C is in fact involved in protecting hosts from lentiviruses.

  6. Transient correction of excision repair defects in fibroblasts of 9 xeroderma pigmentosum complementation groups by microinjection of crude human cell extract.

    NARCIS (Netherlands)

    W. Vermeulen (Wim); P. Osseweijer; A.J.R. de Jonge; J.H.J. Hoeijmakers (Jan)

    1986-01-01

    textabstractCrude extracts from human cells were microinjected into the cytoplasm of cultured fibroblasts from 9 excision-deficient xeroderma pigmentosum (XP) complementation groups. The level of UV-induced unscheduled DNA synthesis (UDS) was measured to determine the effect of the extract on the

  7. Current trend of annotating single nucleotide variation in humans--A case study on SNVrap.

    Science.gov (United States)

    Li, Mulin Jun; Wang, Junwen

    2015-06-01

    As high throughput methods, such as whole genome genotyping arrays, whole exome sequencing (WES) and whole genome sequencing (WGS), have detected huge amounts of genetic variants associated with human diseases, function annotation of these variants is an indispensable step in understanding disease etiology. Large-scale functional genomics projects, such as The ENCODE Project and Roadmap Epigenomics Project, provide genome-wide profiling of functional elements across different human cell types and tissues. With the urgent demands for identification of disease-causal variants, comprehensive and easy-to-use annotation tool is highly in demand. Here we review and discuss current progress and trend of the variant annotation field. Furthermore, we introduce a comprehensive web portal for annotating human genetic variants. We use gene-based features and the latest functional genomics datasets to annotate single nucleotide variation (SNVs) in human, at whole genome scale. We further apply several function prediction algorithms to annotate SNVs that might affect different biological processes, including transcriptional gene regulation, alternative splicing, post-transcriptional regulation, translation and post-translational modifications. The SNVrap web portal is freely available at http://jjwanglab.org/snvrap. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. The role of DNA base excision repair in brain homeostasis and disease

    DEFF Research Database (Denmark)

    Akbari, Mansour; Morevati, Marya; Croteau, Deborah

    2015-01-01

    Chemical modification and spontaneous loss of nucleotide bases from DNA are estimated to occur at the rate of thousands per human cell per day. DNA base excision repair (BER) is a critical mechanism for repairing such lesions in nuclear and mitochondrial DNA. Defective expression or function of p...... energy homeostasis, mitochondrial function and cellular bioenergetics, with especially strong influence on neurological function. Further studies in this area could lead to novel approaches to prevent and treat human neurodegenerative disease....

  9. Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor

    International Nuclear Information System (INIS)

    Dewji, N.N.; Wenger, D.A.; O'Brien, J.S.

    1987-01-01

    Two cDNA clones encoding prepro-sphingolipid activator protein 1 (SAP-1) were isolated from a λ gt11 human hepatoma expression library using polyclonal antibodies. These had inserts of ≅ 2 kilobases (λ-S-1.2 and λ-S-1.3) and both were both homologous with a previously isolated clone (λ-S-1.1) for mature SAP-1. The authors report here the nucleotide sequence of the longer two EcoRI fragments of S-1.2 and S-1.3 that were not the same and the derived amino acid sequences of mature SAP-1 and its prepro form. The open reading frame encodes 19 amino acids, which are colinear with the amino-terminal sequence of mature SAP-1, and extends far beyond the predicted carboxyl terminus of mature SAP-1, indicating extensive carboxyl-terminal processing. The nucleotide sequence of cDNA encoding prepro-SAP-1 includes 1449 bases from the assigned initiation codon ATG at base-pair 472 to the stop codon TGA at base-pair 1921. The first 23 amino acids coded after the initiation ATG are characteristic of a signal peptide. The calculated molecular mass for a polypeptide encoded by 1449 bases is ≅ 53 kDa, in keeping with the reported value for pro-SAP-1. The data indicate that after removal of the signal peptide mature SAP-1 is generated by removing an additional 7 amino acids from the amino terminus and ≅ 373 amino acids from the carboxyl terminus. One potential glycosylation site was previously found in mature SAP-1. Three additional potential glycosylation sites are present in the processed carboxyl-terminal polypeptide, which they designate as P-2

  10. Ultraviolet-induced DNA excision repair in human B and T lymphocytes. II

    International Nuclear Information System (INIS)

    Yew, F.F.-H.; Johnson, R.T.

    1979-01-01

    Despite their great sensitivity to ultraviolet light purified human B and T lymphocytes are capable of complete repair provided that the ultraviolet dose does not exceed 0.5 Jm -2 . Their capacity to repair, as measured by the restoration of DNA supercoiling in preparations of nucleoids, and their survival are significantly increased in the presence of deoxyribonucleosides. Certain agents which inhibit semi-conservative DNA synthesis (hydroxyurea, 1-β-D-arabino-furanosylcytosine (arafCyt) either stop or delay the repair process in lymphocytes. The effect of hydroxyurea is eventually overcome spontaneously, but changes in the sedimentation behaviour of ultraviolet-irradiated nucleoids caused by arafCyt can only be neutralized by addition of deoxycytidine. The effective inhibition of repair by arafCyt permits the detection of extremely small amounts of ultraviolet damage and also the estimation of when repair is complete. (Auth.)

  11. A single-nucleotide polymorphism of human neuropeptide s gene originated from Europe shows decreased bioactivity.

    Directory of Open Access Journals (Sweden)

    Cheng Deng

    Full Text Available Using accumulating SNP (Single-Nucleotide Polymorphism data, we performed a genome-wide search for polypeptide hormone ligands showing changes in the mature regions to elucidate genotype/phenotype diversity among various human populations. Neuropeptide S (NPS, a brain peptide hormone highly conserved in vertebrates, has diverse physiological effects on anxiety, fear, hyperactivity, food intake, and sleeping time through its cognate receptor-NPSR. Here, we report a SNP rs4751440 (L(6-NPS causing non-synonymous substitution on the 6(th position (V to L of the NPS mature peptide region. L(6-NPS has a higher allele frequency in Europeans than other populations and probably originated from European ancestors ~25,000 yrs ago based on haplotype analysis and Approximate Bayesian Computation. Functional analyses indicate that L(6-NPS exhibits a significant lower bioactivity than the wild type NPS, with ~20-fold higher EC50 values in the stimulation of NPSR. Additional evolutionary and mutagenesis studies further demonstrate the importance of the valine residue in the 6(th position for NPS functions. Given the known physiological roles of NPS receptor in inflammatory bowel diseases, asthma pathogenesis, macrophage immune responses, and brain functions, our study provides the basis to elucidate NPS evolution and signaling diversity among human populations.

  12. Functional expression of human adenine nucleotide translocase 4 in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Takashi Hamazaki

    2011-04-01

    Full Text Available The adenine nucleotide translocase (ANT mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31 was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4 in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells.

  13. Electrical conductivity measurement of excised human metastatic liver tumours before and after thermal ablation.

    Science.gov (United States)

    Haemmerich, Dieter; Schutt, David J; Wright, Andrew W; Webster, John G; Mahvi, David M

    2009-05-01

    We measured the ex vivo electrical conductivity of eight human metastatic liver tumours and six normal liver tissue samples from six patients using the four electrode method over the frequency range 10 Hz to 1 MHz. In addition, in a single patient we measured the electrical conductivity before and after the thermal ablation of normal and tumour tissue. The average conductivity of tumour tissue was significantly higher than normal tissue over the entire frequency range (from 4.11 versus 0.75 mS cm(-1) at 10 Hz, to 5.33 versus 2.88 mS cm(-1) at 1 MHz). We found no significant correlation between tumour size and measured electrical conductivity. While before ablation tumour tissue had considerably higher conductivity than normal tissue, the two had similar conductivity throughout the frequency range after ablation. Tumour tissue conductivity changed by +25% and -7% at 10 Hz and 1 MHz after ablation (0.23-0.29 at 10 Hz, and 0.43-0.40 at 1 MHz), while normal tissue conductivity increased by +270% and +10% at 10 Hz and 1 MHz (0.09-0.32 at 10 Hz and 0.37-0.41 at 1 MHz). These data can potentially be used to differentiate tumour from normal tissue diagnostically.

  14. A Comprehensive Experiment for Molecular Biology: Determination of Single Nucleotide Polymorphism in Human REV3 Gene Using PCR-RFLP

    Science.gov (United States)

    Zhang, Xu; Shao, Meng; Gao, Lu; Zhao, Yuanyuan; Sun, Zixuan; Zhou, Liping; Yan, Yongmin; Shao, Qixiang; Xu, Wenrong; Qian, Hui

    2017-01-01

    Laboratory exercise is helpful for medical students to understand the basic principles of molecular biology and to learn about the practical applications of molecular biology. We have designed a lab course on molecular biology about the determination of single nucleotide polymorphism (SNP) in human REV3 gene, the product of which is a subunit of…

  15. Structural basis for the binding and incorporation of nucleotide analogs with L-stereochemistry by human DNA polymerase λ

    OpenAIRE

    Vyas, Rajan; Zahurancik, Walter J.; Suo, Zucai

    2014-01-01

    DNA polymerases are known to select against L-nucleotides, the enantiomers of natural D-nucleotides. However, the structural basis for D-stereoselectivity of a DNA polymerase has not been established, although two L-nucleoside analogs, lamivudine and emtricitabine, have been widely used as anti-HIV and anti-hepatitis B drugs. Here, we report ternary crystal structures of human DNA polymerase λ in complex with DNA and L-deoxycytidine 5′-triphosphate, or its analogs (the triphosphates of lamivu...

  16. The regulated secretory pathway and human disease: insights from gene variants and single nucleotide polymorphisms

    Directory of Open Access Journals (Sweden)

    Stephen eSalton

    2013-08-01

    Full Text Available The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs, where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs, with neuropsychiatric, endocrine and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A of the human brain-derived neurotrophic factor (BDNF gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired.

  17. Nucleotide, cytogenetic and expression impact of the human chromosome 8p23.1 inversion polymorphism.

    Science.gov (United States)

    Bosch, Nina; Morell, Marta; Ponsa, Immaculada; Mercader, Josep Maria; Armengol, Lluís; Estivill, Xavier

    2009-12-14

    The human chromosome 8p23.1 region contains a 3.8-4.5 Mb segment which can be found in different orientations (defined as genomic inversion) among individuals. The identification of single nucleotide polymorphisms (SNPs) tightly linked to the genomic orientation of a given region should be useful to indirectly evaluate the genotypes of large genomic orientations in the individuals. We have identified 16 SNPs, which are in linkage disequilibrium (LD) with the 8p23.1 inversion as detected by fluorescent in situ hybridization (FISH). The variability of the 8p23.1 orientation in 150 HapMap samples was predicted using this set of SNPs and was verified by FISH in a subset of samples. Four genes (NEIL2, MSRA, CTSB and BLK) were found differentially expressed (pinversion occurred. Moreover, an impact of 8p23.1 inversion on gene expression levels cannot be ruled out, since four genes from this region have statistically significant different expression levels depending on the inversion status. FISH results in lymphoblastoid cell lines suggest the presence of mosaicism regarding the 8p23.1 inversion.

  18. Guanine nucleotides stimulate hydrolysis of phosphatidyl inositol bis phosphate in human myelin membranes

    International Nuclear Information System (INIS)

    Boulias, C.; Moscarello, M.A.

    1989-01-01

    Phosphodiesterase activity was stimulated in myelin membranes in the presence of guanine nucleotide analogues. This activity was reduced in myelin membranes which had been adenosine diphosphate ribosylated in the presence of cholera toxin which ADP-ribosylated three proteins of Mr 46,000, 43,000 and 18,500. Aluminum fluoride treatment of myelin had the same stimulatory effects on phosphodiesterase activity as did the guanine nucleotides

  19. Dependence of u.v.-induced DNA excision repair on deoxyribonucleoside triphosphate concentrations in permeable human fibroblasts: a model for the inhibition of repair by hydroxyurea

    International Nuclear Information System (INIS)

    Hunting, D.J.; Dresler, S.L.

    1985-01-01

    We have tested the hypothesis that the inhibition by hydroxyurea of repair patch ligation and chromatin rearrangement during u.v.-induced DNA excision repair results from a reduction in cellular deoxyribonucleotide concentrations and not from a direct effect of hydroxyurea on the repair process. Using permeable human fibroblasts, we have shown that hydroxyurea has no direct effect on either repair synthesis or repair patch ligation. We also have shown that by reducing the deoxyribonucleoside triphosphate concentrations in the permeable cell reaction mixture, we can mimic the inhibition of repair patch ligation and chromatin rearrangement seen when u.v.-damaged intact confluent fibroblasts are treated with hydroxyurea. Our results are consistent with the concept that hydroxyurea inhibits DNA repair in intact cells by inhibiting deoxyribonucleotide synthesis through its effect on ribonucleotide reductase and, conversely, that continued deoxyribonucleotide synthesis is required for the excision repair of u.v.-induced DNA damage even in resting cells

  20. Low Prevalence of Oral and Nasal Human Papillomavirus in Employees Performing CO2-laser Evaporation of Genital Warts or Loop Electrode Excision Procedure of Cervical Dysplasia

    DEFF Research Database (Denmark)

    Kofoed, Kristian; Norrbom, Christina; Forslund, Ola

    2014-01-01

    Risk of human papillomavirus (HPV) transmission during laser vaporisation of genital warts or loop electrode excision procedure is controversial. An oral rinse, a nasal swabs, history of HPV related diseases and data on HPV exposure were collected from 287 employees at departments of dermato......, or loop electrode excision procedure compared with those who did not. HPV 6 or 11 were not detected in any samples. Hand warts after the age of 24 years was more common among dermatology than among non-dermatology personnel (18% vs. 8.0%, p = 0.03). Mucosal HPV types are infrequent in the oral and nasal...... cavity of health care personnel, however, employees at departments of dermato-venereology are at risk of acquiring hand warts....

  1. Evidence that novobiocin and nalidixic acid do not inhibit excision repair in u.v.-irradiated human skin fibroblasts at a pre-incision

    International Nuclear Information System (INIS)

    Keyse, S.M.; Tyrrell, R.M.

    1985-01-01

    The effects of novobiocin and nalidixic acid on the specific toxicity of aphidicolin towards u.v. irradiated arrested human skin fibroblasts have been determined. Contrary to the result expected if either drug were causing inhibition of excision repair at a pre-incision step the sector of toxicity due to a combined treatment of 300 μg ml -1 nalidixic acid and 1.0 μg ml -1 aphidicolin is unchanged when compared with that due to treatment with 1.0 μg ml -1 aphidicolin alone, while that for 150 μg ml -1 novobiocin + 1.0 μg ml -1 aphidicolin was slightly increased. In parallel measurements of the inhibition of u.v.-induced DNA repair synthesis in arrested fibroblasts by these drugs, 150 μg ml -1 novobiocin inhibited repair synthesis by approx.60% over the fluence range employed. Nalidixic acid (300 μg ml -1 ) caused no detectable inhibition of repair synthesis. It was concluded that the mode of action of novobiocin in the inhibition of DNA excision repair is not via the inhibition of a pre-incision step and the data do not support the hypothesis that a type II topoisomerase mediated change in DNA supercoiling is an essential early step in excision repair of u.v.-induced damage. (author)

  2. Evidence that novobiocin and nalidixic acid do not inhibit excision repair in u.v.-irradiated human skin fibroblasts at a pre-incision step

    International Nuclear Information System (INIS)

    Keyse, S.M.; Tyrrell, R.M.

    1985-01-01

    The effects of novobiocin and nalidixic acid on the specific toxicity of aphidicolin towards u.v. irradiated arrested (nondividing) human skin fibroblasts have been determined. Contrary to the result expected if either drug were causing inhibition of excision repair at a pre-incision step the sector of toxicity due to a combined treatment of 300 micrograms ml -1 nalidixic acid and 1.0 micrograms ml -1 aphidicolin is unchanged when compared with that due to treatment with 1.0 micrograms ml -1 aphidicolin alone, while that for 150 micrograms ml -1 novobiocin + 1.0 micrograms ml -1 aphidicolin was slightly increased. In parallel measurements of the inhibition of u.v.-induced DNA repair synthesis in arrested fibroblasts by these drugs, 150 micrograms ml -1 novobiocin inhibited repair synthesis by approximately 60% over the fluence range employed. Nalidixic acid at a concentration of 300 micrograms ml -1 caused no detectable inhibition of repair synthesis. The authors conclude that the mode of action of novobiocin in the inhibition of DNA excision repair is not via the inhibition of a pre-incision step and the data do not support the hypothesis that a type II topoisomerase mediated change in DNA supercoiling is an essential early step in excision repair of u.v.-induced damage

  3. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: Two guanine nucleotide-dependent activators of cholera toxin

    International Nuclear Information System (INIS)

    Bobak, D.A.; Nightingale, M.S.; Murtagh, J.J.; Price, S.R.; Moss, J.; Vaughan, M.

    1989-01-01

    ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A) + RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A) + RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFS also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs

  4. Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A

    International Nuclear Information System (INIS)

    Siebert, P.D.; Fukuda, M.

    1987-01-01

    The authors describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector λgt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH 2 -terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. They used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. They established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes

  5. Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Langie, S. A. S.; Collins, A. R.; Vodička, Pavel

    2014-01-01

    Roč. 116, č. 5 (2014) ISSN 1664-8021 R&D Projects: GA ČR(CZ) GAP304/12/1585 Institutional support: RVO:68378041 Keywords : base excision repair * nucleotide excision repair * human solid tissue Subject RIV: EB - Genetics ; Molecular Biology

  6. A nucleotide-analogue-induced gain of function corrects the error-prone nature of human DNA polymerase iota.

    Science.gov (United States)

    Ketkar, Amit; Zafar, Maroof K; Banerjee, Surajit; Marquez, Victor E; Egli, Martin; Eoff, Robert L

    2012-06-27

    Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2'-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2'-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle, which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base-stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase.

  7. A nucleotide analogue induced gain of function corrects the error-prone nature of human DNA polymerase iota

    Science.gov (United States)

    Ketkar, Amit; Zafar, Maroof K.; Banerjee, Surajit; Marquez, Victor E.; Egli, Martin; Eoff, Robert L

    2012-01-01

    Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2′-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2′-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle (χ), which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase. PMID:22632140

  8. Nucleotide Metabolism

    DEFF Research Database (Denmark)

    Martinussen, Jan; Willemoës, M.; Kilstrup, Mogens

    2011-01-01

    Metabolic pathways are connected through their utilization of nucleotides as supplier of energy, allosteric effectors, and their role in activation of intermediates. Therefore, any attempt to exploit a given living organism in a biotechnological process will have an impact on nucleotide metabolis...

  9. Precise detection of de novo single nucleotide variants in human genomes.

    Science.gov (United States)

    Gómez-Romero, Laura; Palacios-Flores, Kim; Reyes, José; García, Delfino; Boege, Margareta; Dávila, Guillermo; Flores, Margarita; Schatz, Michael C; Palacios, Rafael

    2018-05-07

    The precise determination of de novo genetic variants has enormous implications across different fields of biology and medicine, particularly personalized medicine. Currently, de novo variations are identified by mapping sample reads from a parent-offspring trio to a reference genome, allowing for a certain degree of differences. While widely used, this approach often introduces false-positive (FP) results due to misaligned reads and mischaracterized sequencing errors. In a previous study, we developed an alternative approach to accurately identify single nucleotide variants (SNVs) using only perfect matches. However, this approach could be applied only to haploid regions of the genome and was computationally intensive. In this study, we present a unique approach, coverage-based single nucleotide variant identification (COBASI), which allows the exploration of the entire genome using second-generation short sequence reads without extensive computing requirements. COBASI identifies SNVs using changes in coverage of exactly matching unique substrings, and is particularly suited for pinpointing de novo SNVs. Unlike other approaches that require population frequencies across hundreds of samples to filter out any methodological biases, COBASI can be applied to detect de novo SNVs within isolated families. We demonstrate this capability through extensive simulation studies and by studying a parent-offspring trio we sequenced using short reads. Experimental validation of all 58 candidate de novo SNVs and a selection of non-de novo SNVs found in the trio confirmed zero FP calls. COBASI is available as open source at https://github.com/Laura-Gomez/COBASI for any researcher to use. Copyright © 2018 the Author(s). Published by PNAS.

  10. Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

    LENUS (Irish Health Repository)

    Gegenbauer, Kristina

    2013-11-01

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.

  11. Strand bias in complementary single-nucleotide polymorphisms of transcribed human sequences: evidence for functional effects of synonymous polymorphisms

    Directory of Open Access Journals (Sweden)

    Majewski Jacek

    2006-08-01

    Full Text Available Abstract Background Complementary single-nucleotide polymorphisms (SNPs may not be distributed equally between two DNA strands if the strands are functionally distinct, such as in transcribed genes. In introns, an excess of A↔G over the complementary C↔T substitutions had previously been found and attributed to transcription-coupled repair (TCR, demonstrating the valuable functional clues that can be obtained by studying such asymmetry. Here we studied asymmetry of human synonymous SNPs (sSNPs in the fourfold degenerate (FFD sites as compared to intronic SNPs (iSNPs. Results The identities of the ancestral bases and the direction of mutations were inferred from human-chimpanzee genomic alignment. After correction for background nucleotide composition, excess of A→G over the complementary T→C polymorphisms, which was observed previously and can be explained by TCR, was confirmed in FFD SNPs and iSNPs. However, when SNPs were separately examined according to whether they mapped to a CpG dinucleotide or not, an excess of C→T over G→A polymorphisms was found in non-CpG site FFD SNPs but was absent from iSNPs and CpG site FFD SNPs. Conclusion The genome-wide discrepancy of human FFD SNPs provides novel evidence for widespread selective pressure due to functional effects of sSNPs. The similar asymmetry pattern of FFD SNPs and iSNPs that map to a CpG can be explained by transcription-coupled mechanisms, including TCR and transcription-coupled mutation. Because of the hypermutability of CpG sites, more CpG site FFD SNPs are relatively younger and have confronted less selection effect than non-CpG FFD SNPs, which can explain the asymmetric discrepancy of CpG site FFD SNPs vs. non-CpG site FFD SNPs.

  12. Human mitochondrial Hsp70 (mortalin): shedding light on ATPase activity, interaction with adenosine nucleotides, solution structure and domain organization.

    Science.gov (United States)

    Dores-Silva, Paulo R; Barbosa, Leandro R S; Ramos, Carlos H I; Borges, Júlio C

    2015-01-01

    The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with age-related diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor compound screenings.

  13. RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures.

    Science.gov (United States)

    Theriot, Corey A; Hegde, Muralidhar L; Hazra, Tapas K; Mitra, Sankar

    2010-06-04

    The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K(d) approximately 20 nM) via the common interacting interface (residues 312-349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CDelta78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CDelta78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome. Copyright 2010 Elsevier B.V. All rights reserved.

  14. Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin 5)

    International Nuclear Information System (INIS)

    Campbell, H.D.; Tucker, W.Q.J.; Hort, Y.; Martinson, M.E.; Mayo, G.; Clutterbuck, E.J.; Sanderson, C.J.; Young, I.G.

    1987-01-01

    The human eosinophil differentiation factor (EDF) gene was cloned from a genomic library in λ phage EMBL3A by using a murine EDF cDNA clone as a probe. The DNA sequence of a 3.2-kilobase BamHI fragment spanning the gene was determined. The gene contains three introns. The predicted amino acid sequence of 134 amino acids is identical with that recently reported for human interleukin 5 but shows no significant homology with other known hemopoietic growth regulators. The amino acid sequence shows strong homology (∼ 70% identity) with that of murine EDF. Recombinant human EDF, expressed from the human EDF gene after transfection into monkey COS cells, stimulated the production of eosinophils and eosinophil colonies from normal human bone marrow but had no effect on the production of neutrophils or mononuclear cells (monocytes and lymphoid cells). The apparent specificity of human EDF for the eosinophil lineage in myeloid hemopoiesis contrasts with the properties of human interleukin 3 and granulocyte/macrophage and granulocyte colony-stimulating factors but is directly analogous to the biological properties of murine EDF. Human EDF therefore represents a distinct hemopoietic growth factor that could play a central role in the regulation of eosinophilia

  15. Molecular cloning and nucleotide sequence of cDNA for human liver arginase

    International Nuclear Information System (INIS)

    Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

    1987-01-01

    Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in λ gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated λ hARG6 and λ hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying λ hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes

  16. The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

    Science.gov (United States)

    Sirena, Dominique; Ruzsics, Zsolt; Schaffner, Walter; Greber, Urs F; Hemmi, Silvio

    2005-12-20

    Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings.

  17. Pre-Steady State Kinetic Investigation of the Incorporation of Anti-Hepatitis B Nucleotide Analogs Catalyzed by Non-Canonical Human DNA Polymerases

    Science.gov (United States)

    Brown, Jessica A.; Pack, Lindsey R.; Fowler, Jason D.; Suo, Zucai

    2011-01-01

    Antiviral nucleoside analogs have been developed to inhibit the enzymatic activities of the hepatitis B virus (HBV) polymerase, thereby preventing the replication and production of HBV. However, the usage of these analogs can be limited by drug toxicity because the 5′-triphosphates of these nucleoside analogs (nucleotide analogs) are potential substrates for human DNA polymerases to incorporate into host DNA. Although they are poor substrates for human replicative DNA polymerases, it remains to be established whether these nucleotide analogs are substrates for the recently discovered human X- and Y-family DNA polymerases. Using pre-steady state kinetic techniques, we have measured the substrate specificity values for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active forms of the following anti-HBV nucleoside analogs approved for clinical use: adefovir, tenofovir, lamivudine, telbivudine, and entecavir. Compared to the incorporation of a natural nucleotide, most of the nucleotide analogs were incorporated less efficiently (2 to >122,000) by the six human DNA polymerases. In addition, the potential for entecavir and telbivudine, two drugs which possess a 3′-hydroxyl, to become embedded into human DNA was examined by primer extension and DNA ligation assays. These results suggested that telbivudine functions as a chain terminator while entecavir was efficiently extended by the six enzymes and was a substrate for human DNA ligase I. Our findings suggested that incorporation of anti-HBV nucleotide analogs catalyzed by human X- and Y-family polymerases may contribute to clinical toxicity. PMID:22132702

  18. The complete nucleotide sequence, genome organization, and origin of human adenovirus type 11

    International Nuclear Information System (INIS)

    Stone, Daniel; Furthmann, Anne; Sandig, Volker; Lieber, Andre

    2003-01-01

    The complete DNA sequence and transcription map of human adenovirus type 11 are reported here. This is the first published sequence for a subgenera B human adenovirus and demonstrates a genome organization highly similar to those of other human adenoviruses. All of the genes from the early, intermediate, and late regions are present in the expected locations of the genome for a human adenovirus. The genome size is 34,794 bp in length and has a GC content of 48.9%. Sequence alignment with genomes of groups A (Ad12), C (Ad5), D (Ad17), E (Simian adenovirus 25), and F (Ad40) revealed homologies of 64, 54, 68, 75, and 52%, respectively. Detailed genomic analysis demonstrated that Ads 11 and 35 are highly conserved in all areas except the hexon hypervariable regions and fiber. Similarly, comparison of Ad11 with subgroup E SAV25 revealed poor homology between fibers but high homology in proteins encoded by all other areas of the genome. We propose an evolutionary model in which functional viruses can be reconstituted following fiber substitution from one serotype to another. According to this model either the Ad11 genome is a derivative of Ad35, from which the fiber was substituted with Ad7, or the Ad35 genome is the product of a fiber substitution from Ad21 into the Ad11 genome. This model also provides a possible explanation for the origin of group E Ads, which are evolutionarily derived from a group C fiber substitution into a group B genome

  19. Gene expression, nucleotide composition and codon usage bias of genes associated with human Y chromosome.

    Science.gov (United States)

    Choudhury, Monisha Nath; Uddin, Arif; Chakraborty, Supriyo

    2017-06-01

    Analysis of codon usage pattern is important to understand the genetic and evolutionary characteristics of genomes. We have used bioinformatic approaches to analyze the codon usage bias (CUB) of the genes located in human Y chromosome. Codon bias index (CBI) indicated that the overall extent of codon usage bias was low. The relative synonymous codon usage (RSCU) analysis suggested that approximately half of the codons out of 59 synonymous codons were most frequently used, and possessed a T or G at the third codon position. The codon usage pattern was different in different genes as revealed from correspondence analysis (COA). A significant correlation between effective number of codons (ENC) and various GC contents suggests that both mutation pressure and natural selection affect the codon usage pattern of genes located in human Y chromosome. In addition, Y-linked genes have significant difference in GC contents at the second and third codon positions, expression level, and codon usage pattern of some codons like the SPANX genes in X chromosome.

  20. Activation of nucleotide oligomerization domain 2 (NOD2 by human cytomegalovirus initiates innate immune responses and restricts virus replication.

    Directory of Open Access Journals (Sweden)

    Arun Kapoor

    Full Text Available Nucleotide-binding oligomerization domain 2 (NOD2 is an important innate immune sensor of bacterial pathogens. Its induction results in activation of the classic NF-κB pathway and alternative pathways including type I IFN and autophagy. Although the importance of NOD2 in recognizing RNA viruses has recently been identified, its role in sensing DNA viruses has not been studied. We report that infection with human cytomegalovirus (HCMV results in significant induction of NOD2 expression, beginning as early as 2 hours post infection and increasing steadily 24 hours post infection and afterwards. Infection with human herpesvirus 1 and 2 does not induce NOD2 expression. While the HCMV-encoded glycoprotein B is not required for NOD2 induction, a replication competent virion is necessary. Lentivirus-based NOD2 knockdown in human foreskin fibroblasts (HFFs and U373 glioma cells leads to enhanced HCMV replication along with decreased levels of interferon beta (IFN-β and the pro-inflammatory cytokine, IL8. NOD2 induction in HCMV-infected cells activates downstream NF-κB and interferon pathways supported by reduced nuclear localization of NF-κB and pIRF3 in NOD2 knockdown HFFs. Stable overexpression of NOD2 in HFFs restricts HCMV replication in association with increased levels of IFN-β and IL8. Similarly, transient overexpression of NOD2 in U373 cells or its downstream kinase, RIPK2, results in decreased HCMV replication and enhanced cytokine responses. However, overexpression of a mutant NOD2, 3020insC, associated with severe Crohn's disease, results in enhanced HCMV replication and decreased levels of IFN-β in U373 cells. These results show for the first time that NOD2 plays a significant role in HCMV replication and may provide a model for studies of HCMV recognition by the host cell and HCMV colitis in Crohn's disease.

  1. TH-AB-209-12: Tissue Equivalent Phantom with Excised Human Tissue for Assessing Clinical Capabilities of Coherent Scatter Imaging Applications

    Energy Technology Data Exchange (ETDEWEB)

    Albanese, K; Morris, R; Spencer, J [Medical Physics Graduate Program, Duke University, Durham, NC (United States); Greenberg, J [Dept. of Electrical and Computer Engineering, Duke University, Durham, NC (United States); Kapadia, A [Carl E Ravin Advanced Imaging Laboratories, Durham, NC (United States)

    2016-06-15

    Purpose: Previously we reported the development of anthropomorphic tissue-equivalent scatter phantoms of the human breast. Here we present the first results from the scatter imaging of the tissue equivalent breast phantoms for breast cancer diagnosis. Methods: A breast phantom was designed to assess the capability of coded aperture coherent x-ray scatter imaging to classify different types of breast tissue (adipose, fibroglandular, tumor). The phantom geometry was obtained from a prone breast geometry scanned on a dedicated breast CT system. The phantom was 3D printed using the segmented DICOM breast CT data. The 3D breast phantom was filled with lard (as a surrogate for adipose tissue) and scanned in different geometries alongside excised human breast tissues (obtained from lumpectomy and mastectomy procedures). The raw data were reconstructed using a model-based reconstruction algorithm and yielded the location and form factor (i.e., momentum transfer (q) spectrum) of the materials that were imaged. The measured material form factors were then compared to the ground truth measurements acquired by x-ray diffraction (XRD) imaging. Results: Our scatter imaging system was able to define the location and composition of the various materials and tissues within the phantom. Cancerous breast tissue was detected and classified through automated spectral matching and an 86% correlation threshold. The total scan time for the sample was approximately 10 minutes and approaches workflow times for clinical use in intra-operative or other diagnostic tasks. Conclusion: This work demonstrates the first results from an anthropomorphic tissue equivalent scatter phantom to characterize a coherent scatter imaging system. The functionality of the system shows promise in applications such as intra-operative margin detection or virtual biopsy in the diagnosis of breast cancer. Future work includes using additional patient-derived tissues (e.g., human fat), and modeling additional organs

  2. DNA damage and nucleotide excision repair capacity in healthy individuals

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Naccarati, Alessio; Poláková, Veronika; Pardini, Barbara; Vodičková, Ludmila; Štětina, R.; Schmuczerová, Jana; Šmerhovský, Z.; Lipská, L.; Vodička, Pavel

    2011-01-01

    Roč. 25, č. 7 (2011), s. 511-517 ISSN 0893-6692 R&D Projects: GA ČR GAP304/10/1286; GA MŠk 7F10069 Grant - others:GA MŠk(CZ) GAUK124710 Institutional research plan: CEZ:AV0Z50390512 Keywords : BPDE-induced DNA repair capacity * comet assay * interindividual variability Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.709, year: 2011

  3. Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?

    Directory of Open Access Journals (Sweden)

    Yi-Chih Tsai

    2013-01-01

    Full Text Available A random screening of human blood samples from 24 individuals of nonsmoker was conducted to examine the correlation between the oxidative DNA damage level of lymphocytes and the antioxidant capacity of serum or the base excision repair (BER activity of lymphocytes. The oxidative DNA damage level was measured with comet assay containing Fpg/Endo III cleavage, and the BER activity was estimated with a modified comet assay including nuclear extract of lymphocytes for enzymatic cleavage. Antioxidant capacity was determined with trolox equivalent antioxidant capacity assay. We found that though the endogenous DNA oxidation levels varied among the individuals, each individual level appeared to be steady for at least 1 month. Our results indicate that the oxidative DNA damage level is insignificantly or weakly correlated with antioxidant capacity or BER activity, respectively. However, lymphocytes from carriers of Helicobacter pylori (HP or Hepatitis B virus (HBV tend to give higher levels of oxidative DNA damage (P<0.05. Though sera of this group of individuals show no particular tendency with reduced antioxidant capacity, the respective BER activities of lymphocytes are lower in average (P<0.05. Thus, reduction of repair activity may be associated with the genotoxic effect of HP or HBV infection.

  4. DNA excision repair in human cells treated with ultraviolet radiation and 7,12-dimethylbenz(a)anthracene 5,6-oxide

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, F.E.; Gentil, A.; Renstein, B.S.; Setlow, R.B.

    1980-01-01

    Excision repair was measured in normal human and xeroderma pigmentosum group C cells treated with 7,12-dimethylbenz(a)anthracene 5,6-oxide and with ultraviolet radiation by the techniques of unscheduled DNA synthesis, repair replication, a modification and bromodeoxyuridine photolysis and endonuclease-sensitive sites assay. Radiautography and repair replication showed that in normal cells the magnitude of repair after a saturation dose of the epoxide to be 0.1 to 0.2, that after a saturating ultraviolet dose, though survival data showed that both doses gave nearly similar killings. Repair was of the long-patch type and repair kinetics after the epoxide treatment were similar to ultraviolet. After a combined treatment with both agents, unscheduled synthesis in normal cells was more than additive. The data indicate that there are different rate-limiting steps in the removal of the ultraviolet and the epoxide damages, and that the residual repair activity in xeroderma pigmentosum cells is accomplished by different, not just fewer, enzymes than in normal cells.

  5. DNA Base Excision Repair (BER) and Cancer Gene Therapy: Use of the Human N-mythlpurien DNA Glycosylase (MPG) to Sensitize Breast Cancer Cells to Low Dose Chemotherapy

    National Research Council Canada - National Science Library

    Harvey, Tia

    2003-01-01

    The DNA Base Excision Repair (PER) pathway is responsible for the repair of alkylation and oxidative DNA damage resulting in protection against the deleterious effects of endogenous and exogenous agents encountered on a daily basis...

  6. A history of the DNA repair and mutagenesis field: The discovery of base excision repair.

    Science.gov (United States)

    Friedberg, Errol C

    2016-01-01

    This article reviews the early history of the discovery of an DNA repair pathway designated as base excision repair (BER), since in contrast to the enzyme-catalyzed removal of damaged bases from DNA as nucleotides [called nucleotide excision repair (NER)], BER involves the removal of damaged or inappropriate bases, such as the presence of uracil instead of thymine, from DNA as free bases. Copyright © 2015. Published by Elsevier B.V.

  7. Highlights from the Functional Single Nucleotide Polymorphisms Associated with Human Muscle Size and Strength or FAMuSS Study

    Directory of Open Access Journals (Sweden)

    Linda S. Pescatello

    2013-01-01

    Full Text Available The purpose of the Functional Single Nucleotide Polymorphisms Associated with Human Muscle Size and Strength study or FAMuSS was to identify genetic factors that dictated the response of health-related fitness phenotypes to resistance exercise training (RT. The phenotypes examined were baseline muscle strength and muscle, fat, and bone volume and their response to RT. FAMuSS participants were 1300 young (24 years, healthy men (42% and women (58% that were primarily of European-American descent. They were genotyped for ~500 polymorphisms and completed the Paffenbarger Physical Activity Questionnaire to assess energy expenditure and time spent in light, moderate, and vigorous intensity habitual physical activity and sitting. Subjects then performed a 12-week progressive, unilateral RT program of the nondominant arm with the dominant arm used as a comparison. Before and after RT, muscle strength was measured with the maximum voluntary contraction and one repetition maximum, while MRI measured muscle, fat, and bone volume. We will discuss the history of how FAMuSS originated, provide a brief overview of the FAMuSS methods, and summarize our major findings regarding genotype associations with muscle strength and size, body composition, cardiometabolic biomarkers, and physical activity.

  8. A comprehensive experiment for molecular biology: Determination of single nucleotide polymorphism in human REV3 gene using PCR-RFLP.

    Science.gov (United States)

    Zhang, Xu; Shao, Meng; Gao, Lu; Zhao, Yuanyuan; Sun, Zixuan; Zhou, Liping; Yan, Yongmin; Shao, Qixiang; Xu, Wenrong; Qian, Hui

    2017-07-08

    Laboratory exercise is helpful for medical students to understand the basic principles of molecular biology and to learn about the practical applications of molecular biology. We have designed a lab course on molecular biology about the determination of single nucleotide polymorphism (SNP) in human REV3 gene, the product of which is a subunit of DNA polymerase ζ and SNPs in this gene are associated with altered susceptibility to cancer. This newly designed experiment is composed of three parts, including genomic DNA extraction, gene amplification by PCR, and genotyping by RFLP. By combining these activities, the students are not only able to learn a series of biotechniques in molecular biology, but also acquire the ability to link the learned knowledge with practical applications. This comprehensive experiment will help the medical students improve the conceptual understanding of SNP and the technical understanding of SNP detection. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(4):299-304, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

  9. Genetic analysis of the cardiac methylome at single nucleotide resolution in a model of human cardiovascular disease.

    Directory of Open Access Journals (Sweden)

    Michelle D Johnson

    2014-12-01

    Full Text Available Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR, a model of cardiovascular disease, and the Brown Norway (BN control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB, a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will

  10. Nucleotide-oligomerizing domain-1 (NOD1) receptor activation induces pro-inflammatory responses and autophagy in human alveolar macrophages.

    Science.gov (United States)

    Juárez, Esmeralda; Carranza, Claudia; Hernández-Sánchez, Fernando; Loyola, Elva; Escobedo, Dante; León-Contreras, Juan Carlos; Hernández-Pando, Rogelio; Torres, Martha; Sada, Eduardo

    2014-09-25

    Nucleotide-binding oligomerizing domain-1 (NOD1) is a cytoplasmic receptor involved in recognizing bacterial peptidoglycan fragments that localize to the cytosol. NOD1 activation triggers inflammation, antimicrobial mechanisms and autophagy in both epithelial cells and murine macrophages. NOD1 mediates intracellular pathogen clearance in the lungs of mice; however, little is known about NOD1's role in human alveolar macrophages (AMs) or its involvement in Mycobacterium tuberculosis (Mtb) infection. AMs, monocytes (MNs), and monocyte-derived macrophages (MDMs) from healthy subjects were assayed for NOD1 expression. Cells were stimulated with the NOD1 ligand Tri-DAP and cytokine production and autophagy were assessed. Cells were infected with Mtb and treated with Tri-DAP post-infection. CFUs counting determined growth control, and autophagy protein recruitment to pathogen localization sites was analyzed by immunoelectron microscopy. NOD1 was expressed in AMs, MDMs and to a lesser extent MNs. Tri-DAP stimulation induced NOD1 up-regulation and a significant production of IL1β, IL6, IL8, and TNFα in AMs and MDMs; however, the level of NOD1-dependent response in MNs was limited. Autophagy activity determined by expression of proteins Atg9, LC3, IRGM and p62 degradation was induced in a NOD1-dependent manner in AMs and MDMs but not in MNs. Infected AMs could be activated by stimulation with Tri-DAP to control the intracellular growth of Mtb. In addition, recruitment of NOD1 and the autophagy proteins IRGM and LC3 to the Mtb localization site was observed in infected AMs after treatment with Tri-DAP. NOD1 is involved in AM and MDM innate responses, which include proinflammatory cytokines and autophagy, with potential implications in the killing of Mtb in humans.

  11. Identification of Critical Residues for the Tight Binding of Both Correct and Incorrect Nucleotides to Human DNA Polymerase λ

    Science.gov (United States)

    Brown, Jessica A.; Pack, Lindsey R.; Sherrer, Shanen M.; Kshetry, Ajay K.; Newmister, Sean A.; Fowler, Jason D.; Taylor, John-Stephen; Suo, Zucai

    2010-01-01

    DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β (Pol β). Pre-steady state kinetic studies have shown that the Pol λ•DNA complex binds both correct and incorrect nucleotides 130-fold tighter on average than the Pol β•DNA complex, although, the base substitution fidelity of both polymerases is 10−4 to 10−5. To better understand Pol λ’s tight nucleotide binding affinity, we created single- and double-substitution mutants of Pol λ to disrupt interactions between active site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding with each of the common structural moieties in the following order: triphosphate ≫ base > ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and template base led to a moderate increase in the Kd. The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template. PMID:20851705

  12. Nuclear translocation contributes to regulation of DNA excision repair activities

    DEFF Research Database (Denmark)

    Knudsen, Nina Østergaard; Andersen, Sofie Dabros; Lützen, Anne

    2009-01-01

    for regulation of nuclear import that is necessary for proper localization of the repair proteins. This review summarizes the current knowledge on nuclear import mechanisms of DNA excision repair proteins and provides a model that categorizes the import by different mechanisms, including classical nuclear import......DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA......, it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927-939; M. Christmann, M...

  13. Prediction of the damage-associated non-synonymous single nucleotide polymorphisms in the human MC1R gene.

    Science.gov (United States)

    Hepp, Diego; Gonçalves, Gislene Lopes; de Freitas, Thales Renato Ochotorena

    2015-01-01

    The melanocortin 1 receptor (MC1R) is involved in the control of melanogenesis. Polymorphisms in this gene have been associated with variation in skin and hair color and with elevated risk for the development of melanoma. Here we used 11 computational tools based on different approaches to predict the damage-associated non-synonymous single nucleotide polymorphisms (nsSNPs) in the coding region of the human MC1R gene. Among the 92 nsSNPs arranged according to the predictions 62% were classified as damaging in more than five tools. The classification was significantly correlated with the scores of two consensus programs. Alleles associated with the red hair color (RHC) phenotype and with the risk of melanoma were examined. The R variants D84E, R142H, R151C, I155T, R160W and D294H were classified as damaging by the majority of the tools while the r variants V60L, V92M and R163Q have been predicted as neutral in most of the programs The combination of the prediction tools results in 14 nsSNPs indicated as the most damaging mutations in MC1R (L48P, R67W, H70Y, P72L, S83P, R151H, S172I, L206P, T242I, G255R, P256S, C273Y, C289R and R306H); C273Y showed to be highly damaging in SIFT, Polyphen-2, MutPred, PANTHER and PROVEAN scores. The computational analysis proved capable of identifying the potentially damaging nsSNPs in MC1R, which are candidates for further laboratory studies of the functional and pharmacological significance of the alterations in the receptor and the phenotypic outcomes.

  14. Prediction of the damage-associated non-synonymous single nucleotide polymorphisms in the human MC1R gene.

    Directory of Open Access Journals (Sweden)

    Diego Hepp

    Full Text Available The melanocortin 1 receptor (MC1R is involved in the control of melanogenesis. Polymorphisms in this gene have been associated with variation in skin and hair color and with elevated risk for the development of melanoma. Here we used 11 computational tools based on different approaches to predict the damage-associated non-synonymous single nucleotide polymorphisms (nsSNPs in the coding region of the human MC1R gene. Among the 92 nsSNPs arranged according to the predictions 62% were classified as damaging in more than five tools. The classification was significantly correlated with the scores of two consensus programs. Alleles associated with the red hair color (RHC phenotype and with the risk of melanoma were examined. The R variants D84E, R142H, R151C, I155T, R160W and D294H were classified as damaging by the majority of the tools while the r variants V60L, V92M and R163Q have been predicted as neutral in most of the programs The combination of the prediction tools results in 14 nsSNPs indicated as the most damaging mutations in MC1R (L48P, R67W, H70Y, P72L, S83P, R151H, S172I, L206P, T242I, G255R, P256S, C273Y, C289R and R306H; C273Y showed to be highly damaging in SIFT, Polyphen-2, MutPred, PANTHER and PROVEAN scores. The computational analysis proved capable of identifying the potentially damaging nsSNPs in MC1R, which are candidates for further laboratory studies of the functional and pharmacological significance of the alterations in the receptor and the phenotypic outcomes.

  15. Genotyping of human parvovirus B19 in clinical samples from Brazil and Paraguay using heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing

    Directory of Open Access Journals (Sweden)

    Marcos César Lima de Mendonça

    2011-06-01

    Full Text Available Heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing were utilised to genotype human parvovirus B19 samples from Brazil and Paraguay. Ninety-seven serum samples were collected from individuals presenting with abortion or erythema infectiosum, arthropathies, severe anaemia and transient aplastic crisis; two additional skin samples were collected by biopsy. After the procedure, all clinical samples were classified as genotype 1.

  16. Arthroscopic excision of ganglion cysts.

    Science.gov (United States)

    Bontempo, Nicholas A; Weiss, Arnold-Peter C

    2014-02-01

    Arthroscopy is an advancing field in orthopedics, the applications of which have been expanding over time. Traditionally, excision of ganglion cysts has been done in an open fashion. However, more recently, studies show outcomes following arthroscopic excision to be as good as open excision. Cosmetically, the incisions are smaller and heal faster following arthroscopy. In addition, there is the suggested benefit that patients will regain function and return to work faster following arthroscopic excision. More prospective studies comparing open and arthroscopic excision of ganglion cysts need to be done in order to delineate if there is a true functional benefit. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Single Nucleotide Polymorphism

    DEFF Research Database (Denmark)

    Børsting, Claus; Pereira, Vania; Andersen, Jeppe Dyrberg

    2014-01-01

    Single nucleotide polymorphisms (SNPs) are the most frequent DNA sequence variations in the genome. They have been studied extensively in the last decade with various purposes in mind. In this chapter, we will discuss the advantages and disadvantages of using SNPs for human identification...... of SNPs. This will allow acquisition of more information from the sample materials and open up for new possibilities as well as new challenges....

  18. Mitochondrial base excision repair assays

    DEFF Research Database (Denmark)

    Maynard, Scott; de Souza-Pinto, Nadja C; Scheibye-Knudsen, Morten

    2010-01-01

    The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur....... Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA...... glycosylases, AP endonuclease, DNA polymerase (POLgamma in mitochondria) and DNA ligase. This article outlines procedures for measuring oxidative damage formation and BER in mitochondria, including isolation of mitochondria from tissues and cells, protocols for measuring BER enzyme activities, gene...

  19. The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters*

    Science.gov (United States)

    Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

    2014-01-01

    The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. PMID:25320081

  20. The human SLC25A33 and SLC25A36 genes of solute carrier family 25 encode two mitochondrial pyrimidine nucleotide transporters.

    Science.gov (United States)

    Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

    2014-11-28

    The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Purification, crystallization and preliminary X-ray crystallographic analysis of the ATPase domain of human TAP in nucleotide-free and ADP-, vanadate- and azide-complexed forms

    International Nuclear Information System (INIS)

    Meena, Sita R.; Gangwar, Shanti P.; Saxena, Ajay K.

    2012-01-01

    The ATPase domain of human TAP in nucleotide free, ADP, vanadate and azide complexed forms were purified and crystallized. The X-ray diffraction data sets were collected for all crystals in the resolution range of 2.8–3.0 Å. The human transporter associated with antigen processing (TAP) protein belongs to the ATP-binding cassette (ABC) transporter superfamily and is formed by the heterodimerization of TAP1 and TAP2 subunits. TAP selectively pumps cytosolic peptides into the lumen of the endoplasmic reticulum in an ATP-dependent manner. The catalytic cycle of the ATPase domain of TAP is not understood at the molecular level. The structures of catalytic intermediates of the ATPase domain of TAP will contribute to the understanding of the chemical mechanism of ATP hydrolysis. In order to understand this mechanism, the ATPase domain of human TAP1 (NBD1) was expressed and purified, crystallized in nucleotide-free and transition-state complex forms and X-ray crystallographic studies were performed. The NBD1 protein was crystallized (i) in the nucleotide-free apo form; (ii) in complex with ADP–Mg 2+ , mimicking the product-bound state; (iii) in complex with vanadate–ADP–Mg 2+ , mimicking the ATP-bound state; and (iv) in complex with azide–ADP–Mg 2+ , also mimicking the ATP-bound state. X-ray diffraction data sets were collected for apo and complexed NBD1 using an in-house X-ray diffraction facility at a wavelength of 1.5418 Å. The apo and complexed NBD1 crystals belonged to the primitive hexagonal space group P6 2 , with one monomer in the asymmetric unit. Here, the crystallization, data collection and preliminary crystallographic analysis of apo and complexed NBD1 are reported

  2. Annotate-it: a Swiss-knife approach to annotation, analysis and interpretation of single nucleotide variation in human disease.

    Science.gov (United States)

    Sifrim, Alejandro; Van Houdt, Jeroen Kj; Tranchevent, Leon-Charles; Nowakowska, Beata; Sakai, Ryo; Pavlopoulos, Georgios A; Devriendt, Koen; Vermeesch, Joris R; Moreau, Yves; Aerts, Jan

    2012-01-01

    The increasing size and complexity of exome/genome sequencing data requires new tools for clinical geneticists to discover disease-causing variants. Bottlenecks in identifying the causative variation include poor cross-sample querying, constantly changing functional annotation and not considering existing knowledge concerning the phenotype. We describe a methodology that facilitates exploration of patient sequencing data towards identification of causal variants under different genetic hypotheses. Annotate-it facilitates handling, analysis and interpretation of high-throughput single nucleotide variant data. We demonstrate our strategy using three case studies. Annotate-it is freely available and test data are accessible to all users at http://www.annotate-it.org.

  3. Bioinformatic Analysis of Deleterious Non-Synonymous Single Nucleotide Polymorphisms (nsSNPs in the Coding Regions of Human Prion Protein Gene (PRNP

    Directory of Open Access Journals (Sweden)

    Kourosh Bamdad

    2016-12-01

    Full Text Available Background & Objective: Single nucleotide polymorphisms are the cause of genetic variation to living organisms. Single nucleotide polymorphisms alter residues in the protein sequence. In this investigation, the relationship between prion protein gene polymorphisms and its relevance to pathogenicity was studied. Material & Method: Amino acid sequence of the main isoform from the human prion protein gene (PRNP was extracted from UniProt database and evaluated by FoldAmyloid and AmylPred servers. All non-synonymous single nucleotide polymorphisms (nsSNPs from SNP database (dbSNP were further analyzed by bioinformatics servers including SIFT, PolyPhen-2, I-Mutant-3.0, PANTHER, SNPs & GO, PHD-SNP, Meta-SNP, and MutPred to determine the most damaging nsSNPs. Results: The results of the first structure analyses by FoldAmyloid and AmylPerd servers implied that regions including 5-15, 174-178, 180-184, 211-217, and 240-252 were the most sensitive parts of the protein sequence to amyloidosis. Screening all nsSNPs of the main protein isoform using bioinformatic servers revealed that substitution of Aspartic acid with Valine at position 178 (ID code: rs11538766 was the most deleterious nsSNP in the protein structure. Conclusion:  Substitution of the Aspartic acid with Valine at position 178 (D178V was the most pathogenic mutation in the human prion protein gene. Analyses from the MutPred server also showed that beta-sheets’ increment in the secondary structure was the main reason behind the molecular mechanism of the prion protein aggregation.

  4. NUCLEOTIDES IN INFANT FEEDING

    Directory of Open Access Journals (Sweden)

    L.G. Mamonova

    2007-01-01

    Full Text Available The article reviews the application of nucleotides-metabolites, playing a key role in many biological processes, for the infant feeding. The researcher provides the date on the nucleotides in the women's milk according to the lactation stages. She also analyzes the foreign experience in feeding newborns with nucleotides-containing milk formulas. The article gives a comparison of nucleotides in the adapted formulas represented in the domestic market of the given products.Key words: children, feeding, nucleotides.

  5. Human inherited diseases with altered mechanisms for DNA repair and mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Cleaver, J.E.

    1977-01-01

    A variety of human diseases involving clinical symptoms of increased cancer risk, and disorders of the central nervous system, and of hematopoietic, immunological, ocular, and cutaneous tissues and embryological development have defects in biochemical pathways for excision repair of damaged DNA. Excision repair has multiple branches by which damaged nucleotides, bases, and cross-links are excised and requires cofactors that control the access of repair enzymes to damage in DNA in chromatin. Diseases in which repair defects are a consistent feature of their biochemistry include xeroderma pigmentosum, ataxia telangiectasia and Fanconi's anemia.

  6. Heteronuclear multidimensional NMR and homology modelling studies of the C-terminal nucleotide-binding domain of the human mitochondrial ABC transporter ABCB6

    Energy Technology Data Exchange (ETDEWEB)

    Kurashima-Ito, Kaori [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Ikeya, Teppei [National Institute of Advanced Industrial Science and Technology (AIST), (Japan); Senbongi, Hiroshi [Mitochondrial Diseases Group, MRC Dunn Human NutritionUnit (United Kingdom); Tochio, Hidehito [International Graduate School of Arts and Sciences, Supramolecular Biology, Yokohama City University, Molecular Biophysics Laboratory (Japan); Mikawa, Tsutomu [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Shibata, Takehiko [RIKEN, Shibata Distinguished Senior Scientist Laboratory (Japan); Ito, Yutaka [RIKEN, Cellular and Molecular Biology Laboratory (Japan)], E-mail: ito-yutaka@center.tmu.ac.jp

    2006-05-15

    Human ATP-binding cassette, sub-family B, member 6 (ABCB6) is a mitochondrial ABC transporter, and presumably contributes to iron homeostasis. Aimed at understanding the structural basis for the conformational changes accompanying the substrate-transportation cycle, we have studied the C-terminal nucleotide-binding domain of ABCB6 (ABCB6-C) in both the nucleotide-free and ADP-bound states by heteronuclear multidimensional NMR and homology modelling. A non-linear sampling scheme was utilised for indirectly acquired {sup 13}C and {sup 15}N dimensions of all 3D triple-resonance NMR experiments, in order to overcome the instability and the low solubility of ABCB6-C. The backbone resonances for approximately 25% of non-proline residues, which are mostly distributed around the functionally important loops and in the Helical domain, were not observed for nucleotide-free form of ABCB6-C. From the pH, temperature and magnetic field strength dependencies of the resonance intensities, we concluded that this incompleteness in the assignments is mainly due to the exchange between multiple conformations at an intermediate rate on the NMR timescale. These localised conformational dynamics remained in ADP-bound ABCB6-C except for the loops responsible for adenine base and {alpha}/{beta}-phosphate binding. These results revealed that the localised dynamic cooperativity, which was recently proposed for a prokaryotic ABC MJ1267, also exists in a higher eukaryotic ABC, and is presumably shared by all members of the ABC family. Since the Helical domain is the putative interface to the transmembrane domain, this cooperativity may explain the coupled functions between domains in the substrate-transportation cycle.

  7. Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

    Energy Technology Data Exchange (ETDEWEB)

    Atwell, Shane; Brouillette, Christie G.; Conners, Kris; Emtage, Spencer; Gheyi, Tarun; Guggino, William B.; Hendle, Jorg; Hunt, John F.; Lewis, Hal A.; Lu, Frances; Protasevich, Irina I.; Rodgers, Logan A.; Romero, Rich; Wasserman, Stephen R.; Weber, Patricia C.; Wetmore, Diana; Zhang, Feiyu F.; Zhao, Xun (Cystic); (UAB); (JHU); (Columbia); (Lilly)

    2010-04-26

    Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646({Delta}405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-{angstrom} resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The {Delta}F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  8. Glu504Lys Single Nucleotide Polymorphism of Aldehyde Dehydrogenase 2 Gene and the Risk of Human Diseases

    Directory of Open Access Journals (Sweden)

    Yan Zhao

    2015-01-01

    Full Text Available Aldehyde dehydrogenase (ALDH 2 is a mitochondrial enzyme that is known for its important role in oxidation and detoxification of ethanol metabolite acetaldehyde. ALDH2 also metabolizes other reactive aldehydes such as 4-hydroxy-2-nonenal and acrolein. The Glu504Lys single nucleotide polymorphism (SNP of ALDH2 gene, which is found in approximately 40% of the East Asian populations, causes defect in the enzyme activity of ALDH2, leading to alterations in acetaldehyde metabolism and alcohol-induced “flushing” syndrome. Evidence suggests that ALDH2 Glu504Lys SNP is a potential candidate genetic risk factor for a variety of chronic diseases such as cardiovascular disease, cancer, and late-onset Alzheimer’s disease. In addition, the association between ALDH2 Glu504Lys SNP and the development of these chronic diseases appears to be affected by the interaction between the SNP and lifestyle factors such as alcohol consumption as well as by the presence of other genetic variations.

  9. Nucleotide-binding oligomerization domain 2 (NOD2) activation induces apoptosis of human oral squamous cell carcinoma cells.

    Science.gov (United States)

    Yoon, Hyo-Eun; Ahn, Mee-Young; Kwon, Seong-Min; Kim, Dong-Jae; Lee, Jun; Yoon, Jung-Hoon

    2016-04-01

    Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells. NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death. The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis. Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. The role of nucleoside/nucleotide transport and metabolism in the uptake and retention of 3'-fluoro-3'-deoxythymidine in human B-lymphoblast cells

    International Nuclear Information System (INIS)

    Plotnik, David A.; McLaughlin, Lena J.; Chan, Jenny; Redmayne-Titley, Joshua N.; Schwartz, Jeffrey L.

    2011-01-01

    Introduction: Recent studies in the human adenocarcinoma cell line A549 have identified cell growth-dependent equilibrative nucleoside transporter-1 (hENT1) as a modifier of 3'-fluoro-3'-deoxythymidine (FLT) uptake and retention. In the present study, we used the ability to isolate human lymphoblastoid clones deficient in thymidine kinase 1 (TK1) to study how metabolism and nucleoside transport influence FLT uptake and retention. Methods: Transport and metabolism of FLT were measured in the human lymphoblastoid cell line TK6 and in eight clones isolated from TK6. Four clones were TK1-proficient, while four were TK1-deficient. Both influx and efflux of FLT were measured under conditions where concentrative and equilibrative transport could be distinguished. Results: Sodium-dependent concentrative FLT transport dominated over equilibrative transport mechanisms and while inhibition of hENT1 reduced FLT uptake, there were no correlations between clonal variations in hENT1 levels and FLT uptake. There was an absolute requirement of TK1 for concentration of FLT in TK6 cells. FLT uptake reached a peak after 60 min of incubation with FLT after which intracellular levels of FLT and FLT metabolites declined. Efflux was rapid and was associated with reductions in FLT and each of its metabolites. Both FLT and FLT-monophosphate were found in the efflux buffer. Conclusions: Initial rates of FLT uptake were a function of both concentrative and equilibrative transporters. TK1 activity was an absolute requirement for the accumulation of FLT. Retention was dependent on nucleoside/nucleotide efflux and retrograde metabolism of FLT nucleotides.

  11. Comparative anatomy of the human APRT gene and enzyme: nucleotide sequence divergence and conservation of a nonrandom CpG dinucleotide arrangement

    International Nuclear Information System (INIS)

    Broderick, T.P.; Schaff, D.A.; Bertino, A.M.; Dush, M.K.; Tischfield, J.A.; Stambrook, P.J.

    1987-01-01

    The functional human adenine phosphoribosyltransferase (APRT) gene is <2.6 kilobases in length and contains five exons. The amino acid sequences of APRTs have been highly conserved throughout evolution. The human enzyme is 82%, 90%, and 40% identical to the mouse, hamster, and Escherichia coli enzymes, respectively. The promoter region of the human APRT gene, like that of several other housekeeping genes, lacks TATA and CCAAT boxes but contains five GC boxes that are potential binding sites for the Sp1 transcription factor. The distal three, however, are dispensable for gene expression. Comparison between human and mouse APRT gene nucleotide sequences reveals a high degree of homology within protein coding regions but an absence of significant homology in 5' flanking, 3' untranslated, and intron sequences, except for similarly positioned GC boxes in the promoter region and a 26-base-pair region in intron 3. This 26-base-pair sequence is 92% identical with a similarly positioned sequence in the mouse gene and is also found in intron 3 of the hamster gene, suggesting that its retention may be a consequence of stringent selection. The positions of all introns have been precisely retained in the human and both rodent genes. Retention of an elevated CpG dinucleotide content, despite loss of sequence homology, suggests that there may be selection for CpG dinucleotides in these regions and that their maintenance may be important for APRT gene function

  12. Conserved XPB Core Structure and Motifs for DNA Unwinding:Implications for Pathway Selection of Transcription or ExcisionRepair

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Li; Arval, Andrew S.; Cooper, Priscilla K.; Iwai, Shigenori; Hanaoka, Fumio; Tainer, John A.

    2005-04-01

    The human xeroderma pigmentosum group B (XPB) helicase is essential for transcription, nucleotide excision repair, and TFIIH functional assembly. Here, we determined crystal structures of an Archaeoglobus fulgidus XPB homolog (AfXPB) that characterize two RecA-like XPB helicase domains and discover a DNA damage recognition domain (DRD), a unique RED motif, a flexible thumb motif (ThM), and implied conformational changes within a conserved functional core. RED motif mutations dramatically reduce helicase activity, and the DRD and ThM, which flank the RED motif, appear structurally as well as functionally analogous to the MutS mismatch recognition and DNA polymerase thumb domains. Substrate specificity is altered by DNA damage, such that AfXPB unwinds dsDNA with 3' extensions, but not blunt-ended dsDNA, unless it contains a lesion, as shown for CPD or (6-4) photoproducts. Together, these results provide an unexpected mechanism of DNA unwinding with Implications for XPB damage verification in nucleotide excision repair.

  13. Cell Line Controls for the Genotyping of a Spectrum of Human Single Nucleotide Polymorphisms in the Clinical Laboratory.

    Science.gov (United States)

    Kimbacher, Christine; Paar, Christian; Freystetter, Andrea; Berg, Joerg

    2018-05-01

    Genotyping for clinically important single nucleotide polymorphisms (SNPs) is performed by many clinical routine laboratories. To support testing, quality controls and reference materials are needed. Those may be derived from residual patient samples, left over samples of external quality assurance schemes, plasmid DNA or DNA from cell lines. DNAs from cell lines are commutable and available in large amounts. DNA from 38 cell lines were examined for suitability as controls in 11 SNP assays that are frequently used in a clinical routine laboratory: FV (1691G>A), FII (20210G>A), PAI-1 4G/5G polymorphism, MTHFR (677C>T, 1298A>C), HFE (H63D, S65C, C282Y), APOE (E2, E3, E4), LPH (-13910C>T), UGT1A1 (*28, *36, *37), TPMT (*2, *3A, *3B, *3C), VKORC1 (-1639G>A, 1173C>T), CYP2C9 (*2, *3, *5). Genotyping was performed by real-time PCR with melting curve analysis and confirmed by bi-directional sequencing. We find an almost complete spectrum of genotypic constellations within these 38 cell lines. About 12 cell lines appear sufficient as genotypic controls for the 11 SNP assays by covering almost all of the genotypes. However, hetero- and homozygous genotypes for FII and the alleles TPMT*2, UGT1A1*37 and CYP2C9*5 were not detected in any of the cell lines. DNA from most of the examined cell lines appear suitable as quality controls for these SNP assays in the laboratory routine, as to the implementation of those assays or to prepare samples for quality assurance schemes. Our study may serve as a pilot to further characterize these cell lines to arrive at the status of reference materials.

  14. Nucleotide sequences of cDNAs for human papillomavirus type 18 transcripts in HeLa cells

    International Nuclear Information System (INIS)

    Inagaki, Yutaka; Tsunokawa, Youko; Takebe, Naoko; Terada, Masaaki; Sugimura, Takashi; Nawa, Hiroyuki; Nakanishi, Shigetada

    1988-01-01

    HeLa cells expressed 3.4- and 1.6-kilobase (kb) transcripts of the integrated human papillomavirus (HPV) type 18 genome. Two types of cDNA clones representing each size of HPV type 18 transcript were isolated. Sequence analysis of these two types of cDNA clones revealed that the 3.4-kb transcript contained E6, E7, the 5' portion of E1, and human sequence and that the 1.6-kb transcript contained spliced and frameshifted E6 (E6 * ), E7, and human sequence. There was a common human sequence containing a poly(A) addition signal in the 3' end portions of both transcripts, indicating that they were transcribed from the HPV genome at the same integration site with different splicing. Furthermore, the 1.6-kb transcript contained both of the two viral TATA boxes upstream of E6, strongly indicating that a cellular promoter was used for its transcription

  15. A novel peptide-nucleotide dual vaccine of human telomerase reverse transcriptase induces a potent cytotoxic T-cell response in vivo

    International Nuclear Information System (INIS)

    Guo, Hong; Hao, Jia; Wu, Chao; Shi, Yun; Zhao, Xiao-yan; Fang, Dian-chun

    2007-01-01

    Human telomerase reverse transcriptase (hTERT) is highly expressed in over 85% of human cancers, which makes it a broadly applicable molecular target for cancer therapy. Several groups have demonstrated that hTERT can efficiently evoke specific cytotoxic T lymphocytes (CTL) responses for malignant tumors. In the present study, we developed a novel virus-like particulate peptide-nucleotide dual vaccine (PNDV) of hTERT, which was composed of a low-affinity epitope variant with encoding full-length gene in the same virus-size particulate. We verified the formation of PNDV by DNA retarding assay, DNase I protection assay and transmission electron microscopy, and confirmed its immunogenicity and transfection activities in mammalian cells. Furthermore, in vivo immunization of HLA-A2.1 transgenic mice generated efficient IFN-γ secretion and hTERT-specific CTLs which are known to cause selective cell death of telomerase positive gastrointestinal cancer cells. To our knowledge, this represents the first report on collocating a low-affinity epitope variant with a full-length hTERT gene for anti-cancer vaccine design. This novel strategy for vaccine design not only enables enhanced immunity to a universal tumor antigen, but also has the potential to generate CTLs effective in telomerase-positive tumor cells of diverse tissue origins. Therefore, our findings bear significant implications for immunotherapy of human cancers

  16. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    International Nuclear Information System (INIS)

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J.

    1988-01-01

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 10 6 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria

  17. A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation

    DEFF Research Database (Denmark)

    Rasmussen, Trine Nygaard; Plenge, Per; Bay, Tina

    2009-01-01

    The human serotonin transporter (hSERT) is responsible for reuptake of serotonin (5-HT) from the synaptic cleft and is target for antidepressant medicine. Differential hSERT activity caused by genetic polymorphisms is believed to affect the risk of developing depression and, moreover, to affect t...

  18. Genetic variability of human respiratory syncytial virus A strains circulating in Ontario: a novel genotype with a 72 nucleotide G gene duplication.

    Directory of Open Access Journals (Sweden)

    Alireza Eshaghi

    Full Text Available Human respiratory syncytial virus (HRSV is the main cause of acute lower respiratory infections in children under 2 years of age and causes repeated infections throughout life. We investigated the genetic variability of RSV-A circulating in Ontario during 2010-2011 winter season by sequencing and phylogenetic analysis of the G glycoprotein gene.Among the 201 consecutive RSV isolates studied, RSV-A (55.7% was more commonly observed than RSV-B (42.3%. 59.8% and 90.1% of RSV-A infections were among children ≤12 months and ≤5 years old, respectively. On phylogenetic analysis of the second hypervariable region of the 112 RSV-A strains, 110 (98.2% clustered within or adjacent to the NA1 genotype; two isolates were GA5 genotype. Eleven (10% NA1-related isolates clustered together phylogenetically as a novel RSV-A genotype, named ON1, containing a 72 nucleotide duplication in the C-terminal region of the attachment (G glycoprotein. The predicted polypeptide is lengthened by 24 amino acids and includes a23 amino acid duplication. Using RNA secondary structural software, a possible mechanism of duplication occurrence was derived. The 23 amino acid ON1 G gene duplication results in a repeat of 7 potential O-glycosylation sites including three O-linked sugar acceptors at residues 270, 275, and 283. Using Phylogenetic Analysis by Maximum Likelihood analysis, a total of 19 positively selected sites were observed among Ontario NA1 isolates; six were found to be codons which reverted to the previous state observed in the prototype RSV-A2 strain. The tendency of codon regression in the G-ectodomain may infer a decreased avidity of antibody to the current circulating strains. Further work is needed to document and further understand the emergence, virulence, pathogenicity and transmissibility of this novel RSV-A genotype with a72 nucleotide G gene duplication.

  19. Assay for identification of heterozygous single-nucleotide polymorphism (Ala67Thr in human poliovirus receptor gene

    Directory of Open Access Journals (Sweden)

    Shyam Sundar Nandi

    2016-01-01

    Results: A new SNP assay for detection of heterozygous Ala67Thr genotype was developed and validated by testing 150 DNA samples. Heterozygous CD155 was detected in 27.33 per cent (41/150 of DNA samples tested by both SNP detection assay and sequencing. Interpretation & conclusions: The SNP detection assay was successfully developed for identification of Ala67Thr polymorphism in human PVR/CD155 gene. The SNP assay will be useful for large scale screening of DNA samples.

  20. Synthesis and evaluation of translocator 18 kDa protein (TSPO) positron emission tomography (PET) radioligands with low binding sensitivity to human single nucleotide polymorphism rs6971.

    Science.gov (United States)

    Zanotti-Fregonara, Paolo; Zhang, Yi; Jenko, Kimberly J; Gladding, Robert L; Zoghbi, Sami S; Fujita, Masahiro; Sbardella, Gianluca; Castellano, Sabrina; Taliani, Sabrina; Martini, Claudia; Innis, Robert B; Da Settimo, Federico; Pike, Victor W

    2014-10-15

    The imaging of translocator 18 kDa protein (TSPO) in living human brain with radioligands by positron emission tomography (PET) has become an important means for the study of neuroinflammatory conditions occurring in several neuropsychiatric disorders. The widely used prototypical PET radioligand [(11)C](R)-PK 11195 ([(11)C](R)-1; [N-methyl-(11)C](R)-N-sec-butyl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide) gives a low PET signal and is difficult to quantify, whereas later generation radioligands have binding sensitivity to a human single nucleotide polymorphism (SNP) rs6971, which imposes limitations on their utility for comparative quantitative PET studies of normal and diseased subjects. Recently, azaisosteres of 1 have been developed with improved drug-like properties, including enhanced TSPO affinity accompanied by moderated lipophilicity. Here we selected three of these new ligands (7-9) for labeling with carbon-11 and for evaluation in monkey as candidate PET radioligands for imaging brain TSPO. Each radioligand was readily prepared by (11)C-methylation of an N-desmethyl precursor and was found to give a high proportion of TSPO-specific binding in monkey brain. One of these radioligands, [(11)C]7, the direct 4-azaisostere of 1, presents many radioligand properties that are superior to those reported for [(11)C]1, including higher affinity, lower lipophilicity, and stable quantifiable PET signal. Importantly, 7 was also found to show very low sensitivity to the human SNP rs6971 in vitro. Therefore, [(11)C]7 now warrants evaluation in human subjects with PET to assess its utility for imaging TSPO in human brain, irrespective of subject genotype.

  1. Modulation of DNA base excision repair during neuronal differentiation

    DEFF Research Database (Denmark)

    Sykora, Peter; Yang, Jenq-Lin; Ferrarelli, Leslie K

    2013-01-01

    DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because...

  2. Single-nucleotide variant in multiple copies of a deleted in azoospermia (DAZ) sequence - a human Y chromosome quantitative polymorphism.

    Science.gov (United States)

    Szmulewicz, Martin N; Ruiz, Luis M; Reategui, Erika P; Hussini, Saeed; Herrera, Rene J

    2002-01-01

    The evolution of the deleted in azoospermia (DAZ) gene family supports prevalent theories on the origin and development of sex chromosomes and sexual dimorphism. The ancestral DAZL gene in human chromosome 3 is known to be involved in germline development of both males and females. The available phylogenetic data suggest that some time after the divergence of the New World and Old World monkey lineages, the DAZL gene, which is found in all mammals, was copied to the Y chromosome of an ancestor to the Old World monkeys, but not New World monkeys. In modern man, the Y-linked DAZ gene complex is located on the distal part of the q arm. It is thought that after being copied to the Y chromosome, and after the divergence of the human and great ape lineages, the DAZ gene in the former underwent internal rearrangements. This included tandem duplications as well as a T > C transition altering an MboI restriction enzyme site in a duplicated sequence. In this study, we report on the ratios of MboI-/MboI+ variant sequences in individuals from seven worldwide human populations (Basque, Benin, Egypt, Formosa, Kungurtug, Oman and Rwanda) in the DAZ complex. The ratio of PCR MboI- and MboI+ amplicons can be used to characterize individuals and populations. Our results show a nonrandom distribution of MboI-/MboI+ sequence ratios in all populations examined, as well as significant differences in ratios between populations when compared pairwise. The multiple ratios imply that there have been more than one recent reorganization events at this locus. Considering the dynamic nature of this locus and its involvement in male fertility, we investigated the extent and distribution of this polymorphism. Copyright 2002 S. Karger AG, Basel

  3. Nucleotide sequence of a human cDNA encoding a ras-related protein (rap1B)

    Energy Technology Data Exchange (ETDEWEB)

    Pizon, V; Lerosey, I; Chardin, P; Tavitian, A [INSERM, Paris (France)

    1988-08-11

    The authors have previously characterized two human ras-related genes rap1 and rap2. Using the rap1 clone as probe they isolated and sequenced a new rap cDNA encoding the 184aa rap1B protein. The rap1B protein is 95% identical to rap1 and shares several properties with the ras protein suggesting that it could bind GTP/GDP and have a membrane location. As for rap1, the structural characteristics of rap1B suggest that the rap and ras proteins might interact on the same effector.

  4. The mitochondrial transcription factor A functions in mitochondrial base excision repair

    DEFF Research Database (Denmark)

    Canugovi, Chandrika; Maynard, Scott; Bayne, Anne-Cécile V

    2010-01-01

    Mitochondrial transcription factor A (TFAM) is an essential component of mitochondrial nucleoids. TFAM plays an important role in mitochondrial transcription and replication. TFAM has been previously reported to inhibit nucleotide excision repair (NER) in vitro but NER has not yet been detected i...

  5. Apoptotic function of human PMS2 compromised by the nonsynonymous single-nucleotide polymorphic variant R20Q.

    Science.gov (United States)

    Marinovic-Terzic, Ivana; Yoshioka-Yamashita, Atsuko; Shimodaira, Hideki; Avdievich, Elena; Hunton, Irina C; Kolodner, Richard D; Edelmann, Winfried; Wang, Jean Y J

    2008-09-16

    Mismatch repair (MMR) corrects replication errors during DNA synthesis. The mammalian MMR proteins also activate cell cycle checkpoints and apoptosis in response to persistent DNA damage. MMR-deficient cells are resistant to cisplatin, a DNA cross-linking agent used in chemotherapy, because of impaired activation of apoptotic pathways. It is shown that postmeiotic segregation 2 (PMS2), an MMR protein, is required for cisplatin-induced activation of p73, a member of the p53 family of transcription factors with proapoptotic activity. The human PMS2 is highly polymorphic, with at least 12 known nonsynonymous codon changes identified. We show here that the PMS2(R20Q) variant is defective in activating p73-dependent apoptotic response to cisplatin. When expressed in Pms2-deficient mouse fibroblasts, human PMS2(R20Q) but not PMS2 interfered with the apoptotic response to cisplatin. Correspondingly, PMS2 but not PMS2(R20Q) enhanced the cytotoxic effect of cisplatin measured by clonogenic survival. Because PMS2(R20Q) lacks proapoptotic activity, this polymorphic allele may modulate tumor responses to cisplatin among cancer patients.

  6. Effect of pregnancy-associated plasma protein-A (PAPP-A) single-nucleotide polymorphisms on the level and activity of PAPP-A and the hormone profile in fluid from normal human small antral follicles

    DEFF Research Database (Denmark)

    Bøtkjær, Jane Alrø; Borgbo, Tanni; Kløverpris, Søren

    2016-01-01

    Objective: To reveal a possible relationship between two single nucleotide polymorphisms (SNPs) in PAPP-A—1224 (rs7020782) and 327 (rs12375498)—and the level and activity of PAPP-A in follicular fluid (FF) of human small antral follicles, and to analyze the intrafollicular hormone levels. Design:...

  7. Nucleotide sequence of the gene coding for human factor VII, a vitamin K-dependent protein participating in blood coagulation

    International Nuclear Information System (INIS)

    O'Hara, P.J.; Grant, F.J.; Haldeman, B.A.; Gray, C.L.; Insley, M.Y.; Hagen, F.S.; Murray, M.J.

    1987-01-01

    Activated factor VII (factor VIIa) is a vitamin K-dependent plasma serine protease that participates in a cascade of reactions leading to the coagulation of blood. Two overlapping genomic clones containing sequences encoding human factor VII were isolated and characterized. The complete sequence of the gene was determined and found to span about 12.8 kilobases. The mRNA for factor VII as demonstrated by cDNA cloning is polyadenylylated at multiple sites but contains only one AAUAAA poly(A) signal sequence. The mRNA can undergo alternative splicing, forming one transcript containing eight segments as exons and another with an additional exon that encodes a larger prepro leader sequence. The latter transcript has no known counterpart in the other vitamin K-dependent proteins. The positions of the introns with respect to the amino acid sequence encoded by the eight essential exons of factor VII are the same as those present in factor IX, factor X, protein C, and the first three exons of prothrombin. These exons code for domains generally conserved among members of this gene family. The comparable introns in these genes, however, are dissimilar with respect to size and sequence, with the exception of intron C in factor VII and protein C. The gene for factor VII also contains five regions made up of tandem repeats of oligonucleotide monomer elements. More than a quarter of the intron sequences and more than a third of the 3' untranslated portion of the mRNA transcript consist of these minisatellite tandem repeats

  8. Effects of single nucleotide polymorphisms on human N-acetyltransferase 2 structure and dynamics by molecular dynamics simulation.

    Directory of Open Access Journals (Sweden)

    M Rajasekaran

    Full Text Available BACKGROUND: Arylamine N-acetyltransferase 2 (NAT2 is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear. METHODOLOGY/PRINCIPAL FINDINGS: We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential. CONCLUSIONS/SIGNIFICANCE: Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants

  9. Molecular Comparison and Evolutionary Analyses of VP1 Nucleotide Sequences of New African Human Enterovirus 71 Isolates Reveal a Wide Genetic Diversity

    Science.gov (United States)

    Nougairède, Antoine; Joffret, Marie-Line; Deshpande, Jagadish M.; Dubot-Pérès, Audrey; Héraud, Jean-Michel

    2014-01-01

    Most circulating strains of Human enterovirus 71 (EV-A71) have been classified primarily into three genogroups (A to C) on the basis of genetic divergence between the 1D gene, which encodes the VP1 capsid protein. The aim of the present study was to provide further insights into the diversity of the EV-A71 genogroups following the recent description of highly divergent isolates, in particular those from African countries, including Madagascar. We classified recent EV-A71 isolates by a large comparison of 3,346 VP1 nucleotidic sequences collected from GenBank. Analysis of genetic distances and phylogenetic investigations indicated that some recently-reported isolates did not fall into the genogroups A-C and clustered into three additional genogroups, including one Indian genogroup (genogroup D) and 2 African ones (E and F). Our Bayesian phylogenetic analysis provided consistent data showing that the genogroup D isolates share a recent common ancestor with the members of genogroup E, while the isolates of genogroup F evolved from a recent common ancestor shared with the members of the genogroup B. Our results reveal the wide diversity that exists among EV-A71 isolates and suggest that the number of circulating genogroups is probably underestimated, particularly in developing countries where EV-A71 epidemiology has been poorly studied. PMID:24598878

  10. Effects of cyclic-nucleotide derivatives on the growth of human colonic carcinoma xenografts and on cell production in the rat colonic crypt epithelium.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1981-08-01

    Previous studies have shown that various amine hormones are able to influence the growth rate of human colorectal carcinomas propagated as xenografts in immune-deprived mice, and it is now well known that the effects of many amine and other hormones are mediated by cyclic nucleotides, acting as second messengers within cells. In the present study the influence of various derivatives of cyclic adenosine monophosphate and cyclic guanosine monophosphate on the growth of two different lines of colorectal cancer growing in immune-deprived mice, and on the cell production rate in the colonic crypt epithelium of the rat, was assessed. Growth of each tumour line, as well as crypt-cell production, was suppressed by treatment wit N6O2' dibutyryl and N6 monobutyryl derivatives of cyclic adenosine monophosphate. Dibutyryl cyclic guanosine monophosphate, on the other hand, was found to promote the growth of Tumour HXK4 and to promote crypt cell production, but to have no significant effect on Tumour HXM2.

  11. The single nucleotide polymorphism Gly482Ser in the PGC-1α gene impairs exercise-induced slow-twitch muscle fibre transformation in humans.

    Directory of Open Access Journals (Sweden)

    Peter Steinbacher

    Full Text Available PGC-1α (peroxisome proliferator-activated receptor γ co-activator 1α is an important regulator of mitochondrial biogenesis and a master regulator of enzymes involved in oxidative phosphorylation. Recent evidence demonstrated that the Gly482Ser single nucleotide polymorphism (SNP in the PGC-1α gene affects insulin sensitivity, blood lipid metabolism and binding to myocyte enhancer factor 2 (MEF2. Individuals carrying this SNP were shown to have a reduced cardiorespiratory fitness and a higher risk to develop type 2 diabetes. Here, we investigated the responses of untrained men with the Gly482Ser SNP to a 10 week programme of endurance training (cycling, 3 x 60 min/week, heart rate at 70-90% VO2peak. Quantitative data from analysis of biopsies from vastus lateralis muscle revealed that the SNP group, in contrast to the control group, lacked a training-induced increase in content of slow contracting oxidative fibres. Capillary supply, mitochondrial density, mitochondrial enzyme activities and intramyocellular lipid content increased similarly in both groups. These results indicate that the impaired binding of MEF2 to PGC-1α in humans with this SNP impedes exercise-induced fast-to-slow muscle fibre transformation.

  12. The single nucleotide polymorphism Gly482Ser in the PGC-1α gene impairs exercise-induced slow-twitch muscle fibre transformation in humans.

    Science.gov (United States)

    Steinbacher, Peter; Feichtinger, René G; Kedenko, Lyudmyla; Kedenko, Igor; Reinhardt, Sandra; Schönauer, Anna-Lena; Leitner, Isabella; Sänger, Alexandra M; Stoiber, Walter; Kofler, Barbara; Förster, Holger; Paulweber, Bernhard; Ring-Dimitriou, Susanne

    2015-01-01

    PGC-1α (peroxisome proliferator-activated receptor γ co-activator 1α) is an important regulator of mitochondrial biogenesis and a master regulator of enzymes involved in oxidative phosphorylation. Recent evidence demonstrated that the Gly482Ser single nucleotide polymorphism (SNP) in the PGC-1α gene affects insulin sensitivity, blood lipid metabolism and binding to myocyte enhancer factor 2 (MEF2). Individuals carrying this SNP were shown to have a reduced cardiorespiratory fitness and a higher risk to develop type 2 diabetes. Here, we investigated the responses of untrained men with the Gly482Ser SNP to a 10 week programme of endurance training (cycling, 3 x 60 min/week, heart rate at 70-90% VO2peak). Quantitative data from analysis of biopsies from vastus lateralis muscle revealed that the SNP group, in contrast to the control group, lacked a training-induced increase in content of slow contracting oxidative fibres. Capillary supply, mitochondrial density, mitochondrial enzyme activities and intramyocellular lipid content increased similarly in both groups. These results indicate that the impaired binding of MEF2 to PGC-1α in humans with this SNP impedes exercise-induced fast-to-slow muscle fibre transformation.

  13. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    Directory of Open Access Journals (Sweden)

    Sanne H. Olesen

    2015-01-01

    Full Text Available The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2 did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM, while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

  14. Pan-pathway based interaction profiling of FDA-approved nucleoside and nucleobase analogs with enzymes of the human nucleotide metabolism.

    Science.gov (United States)

    Egeblad, Louise; Welin, Martin; Flodin, Susanne; Gräslund, Susanne; Wang, Liya; Balzarini, Jan; Eriksson, Staffan; Nordlund, Pär

    2012-01-01

    To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of "off target effects." However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔT(agg) around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design.

  15. Isolation and characterization of human glycophorin A cDNA clones by a synthetic oligonucleotide approach: nucleotide sequence and mRNA structure

    International Nuclear Information System (INIS)

    Siebert, P.D.; Fukuda, M.

    1986-01-01

    In an effort to understand the relationships among and the regulation of human glycophorins, the authors have isolated and characterized several glycophorin A-specific cDNA clones obtained from a human erythroleukemic K562 cell cDNA library. This was accomplished by using mixed synthetic oligonucleotides, corresponding to various regions of the known amino acid sequence, to prime the synthesis of the cDNA as well as to screen the cDNA library. They also used synthetic oligonucleotides to sequence the largest of the glycophorin cDNAs. The nucleotide sequence obtained suggests the presence of a potential leader peptide, consistent with the membrane localization of this glycoprotein. Examination of the structure of glycophorin mRNA by blot hybridization revealed the existence of several electrophoretically distinct mRNAs numbering three or four, depending on the size of the glycophorin cDNA used as a hybridization probe. The smaller cDNA hybridized to three mRNAs of approximately 2.8, 1.7, and 1.0 kilobases. In contrast, the larger cDNA hybridized to an additional mRNA of approximately 0.6 kilobases. Further examination of the relationships between these multiple mRNAs by blot hybridization was conducted with the use of exact-sequence oligonucleotide probes constructed from various regions of the cDNA representing portions of the amino acid sequence of glycophorin A with or without known homology with glycophorin B. In total, the results obtained are consistent with the hypothesis that the three larger mRNAs represent glycophorin A gene transcripts and that the smallest (0.6 kilobase) mRNA may be specific for glycophorin B

  16. Impact of the [delta]F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B.; Hunt, John F.; Emtage, Spencer (SG); (Columbia); (JHU)

    2010-07-19

    Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

  17. A single nucleotide polymorphism within the novel sex-linked testis-specific retrotransposed PGAM4 gene influences human male fertility.

    Directory of Open Access Journals (Sweden)

    Hidenobu Okuda

    Full Text Available The development of novel fertilization treatments, including in vitro fertilization and intracytoplasmic injection, has made pregnancy possible regardless of the level of activity of the spermatozoa; however, the etiology of male-factor infertility is poorly understood. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs as a cause of male infertility in an analysis of spermatogenesis-specific genes.We carried out the prevalence of SNPs in the coding region of phosphoglycerate mutase 4 (PGAM4 on the X chromosome by the direct sequencing of PCR-amplified DNA from male patients. Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility. PGAM4 is expressed in post-meiotic stages, including spermatids and spermatozoa in the testes, and the principal piece of the flagellum and acrosome in ejaculated spermatozoa. A case-control study revealed that 4.5% of infertile patients carry the G75C polymorphism, which causes an amino acid substitution in the encoded protein. Furthermore, an assay for enzymatic activity demonstrated that this polymorphism decreases the enzyme's activity both in vitro and in vivo.These results suggest that PGAM4, an X-linked retrogene, is a fundamental gene in human male reproduction and may escape meiotic sex chromosome inactivation. These findings provide fresh insight into elucidating the mechanisms of male infertility.

  18. Lumbar disc excision through fenestration

    Directory of Open Access Journals (Sweden)

    Sangwan S

    2006-01-01

    Full Text Available Background : Lumbar disc herniation often causes sciatica. Many different techniques have been advocated with the aim of least possible damage to other structures while dealing with prolapsed disc surgically in the properly selected and indicated cases. Methods : Twenty six patients with clinical symptoms and signs of prolapsed lumbar intervertebral disc having radiological correlation by MRI study were subjected to disc excision by interlaminar fenestration method. Results : The assessment at follow-up showed excellent results in 17 patients, good in 6 patients, fair in 2 patients and poor in 1 patient. The mean preoperative and postoperative Visual Analogue Scores were 9.34 ±0.84 and 2.19 ±0.84 on scale of 0-10 respectively. These were statistically significant (p value< 0.001, paired t test. No significant complications were recorded. Conclusion : Procedures of interlaminar fenestration and open disc excision under direct vision offers sufficient adequate exposure for lumbar disc excision with a smaller incision, lesser morbidity, shorter convalescence, early return to work and comparable overall results in the centers where recent laser and endoscopy facilities are not available.

  19. Safety and efficacy of a xenogeneic DNA vaccine encoding for human tyrosinase as adjunctive treatment for oral malignant melanoma in dogs following surgical excision of the primary tumor.

    Science.gov (United States)

    Grosenbaugh, Deborah A; Leard, A Timothy; Bergman, Philip J; Klein, Mary K; Meleo, Karri; Susaneck, Steven; Hess, Paul R; Jankowski, Monika K; Jones, Pamela D; Leibman, Nicole F; Johnson, Maribeth H; Kurzman, Ilene D; Wolchok, Jedd D

    2011-12-01

    To evaluate the safety and efficacy of a vaccine containing plasmid DNA with an insert encoding human tyrosinase (ie, huTyr vaccine) as adjunctive treatment for oral malignant melanoma (MM) in dogs. 111 dogs (58 prospectively enrolled in a multicenter clinical trial and 53 historical controls) with stage II or III oral MM (modified World Health Organization staging scale, I to IV) in which locoregional disease control was achieved. 58 dogs received an initial series of 4 injections of huTyr vaccine (102 μg of DNA/injection) administered transdermally by use of a needle-free IM vaccination device. Dogs were monitored for adverse reactions. Surviving dogs received booster injections at 6-month intervals thereafter. Survival time for vaccinates was compared with that of historical control dogs via Kaplan-Meier survival analysis for the outcome of death. Kaplan-Meier analysis of survival time until death attributable to MM was determined to be significantly improved for dogs that received the huTyr vaccine, compared with that of historical controls. However, median survival time could not be determined for vaccinates because dogs as adjunctive treatment for oral MM. Response to DNA vaccination in dogs with oral MM may be useful in development of plasmid DNA vaccination protocols for human patients with similar disease.

  20. Excision repair of bulky lesions in the DNA of mammalian cells

    International Nuclear Information System (INIS)

    Setlow, R.B.; Grist, E.

    1980-01-01

    The report examines the process of excision repair of pyrimidine dimers from uv-irradiated and chemically challenged human cells. It is shown by means of a sensitive endonuclease assay that the amount of excision observed depends upon the isotope used to label cells, and that XP heterozygotes are between normals and XPs

  1. Main: Nucleotide Analysis [KOME

    Lifescience Database Archive (English)

    Full Text Available Nucleotide Analysis Japonica genome blast search result Result of blastn search against jap...onica genome sequence kome_japonica_genome_blast_search_result.zip kome_japonica_genome_blast_search_result ...

  2. Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.

    Science.gov (United States)

    Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh

    2011-05-01

    Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Invasive Streptococcus mutans induces inflammatory cytokine production in human aortic endothelial cells via regulation of intracellular toll-like receptor 2 and nucleotide-binding oligomerization domain 2.

    Science.gov (United States)

    Nagata, E; Oho, T

    2017-04-01

    Streptococcus mutans, the primary etiologic agent of dental caries, can gain access to the bloodstream and has been associated with cardiovascular disease. However, the roles of S. mutans in inflammation in cardiovascular disease remain unclear. The aim of this study was to examine cytokine production induced by S. mutans in human aortic endothelial cells (HAECs) and to evaluate the participation of toll-like receptors (TLRs) and cytoplasmic nucleotide-binding oligomerization domain (NOD) -like receptors in HAECs. Cytokine production by HAECs was determined using enzyme-linked immunosorbent assays, and the expression of TLRs and NOD-like receptors was evaluated by real-time polymerase chain reaction, flow cytometry and immunocytochemistry. The involvement of TLR2 and NOD2 in cytokine production by invaded HAECs was examined using RNA interference. The invasion efficiencies of S. mutans strains were evaluated by means of antibiotic protection assays. Five of six strains of S. mutans of various serotypes induced interleukin-6, interleukin-8 and monocyte chemoattractant protein-1 production by HAECs. All S. mutans strains upregulated TLR2 and NOD2 mRNA levels in HAECs. Streptococcus mutans Xc upregulated the intracellular TLR2 and NOD2 protein levels in HAECs. Silencing of the TLR2 and NOD2 genes in HAECs invaded by S. mutans Xc led to a reduction in interleukin-6, interleukin-8 and monocyte chemoattractant protein-1 production. Cytokine production induced by invasive S. mutans via intracellular TLR2 and NOD2 in HAECs may be associated with inflammation in cardiovascular disease. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. In silico Analysis of the Functional and Structural Impacts of Non-synonymous Single Nucleotide Polymorphisms in the Human Paraxonase 1 Gene

    Directory of Open Access Journals (Sweden)

    Sudip Paul

    2015-09-01

    Full Text Available Computational approaches could help in identifying deleterious non-synonymous single nucleotide polymorphisms (nsSNPs in a disease related gene which is a difficult and laborious task through laboratory experiments. In the present study, we analyzed the impacts of nsSNPs on structure and function of Paraxonase 1 (PON1 using different bioinformatics tools. The human PON1 protein sequence and its corresponding gene's SNP information were collected from UniProt and dbSNP databases, respectively. We utilized SIFT, Polyphen, I-Mutant 2.0, MutPred, SNP and GO, PhD-SNP and PANTHER tools in order to examine the total 39 nsSNPs occurring in the PON1 coding region. We filtered the most pathological mutations by combining the scores of the aforementioned servers and found 8 SNPs (G344C, S302L, W281C, D279Y, H134R, F120S, L90P, C42R as deleterious and disease causing. The PDB structure of PON1 protein was obtained from RCSB Protein Data Bank (PDB ID: 1V04. The deleterious SNPs in native PON1 were introduced using Swiss-PDB Viewer package and changes in free energy were observed for six out of eight mutant structures. Two SNPs, S302L (substitution of serine to leucine at 302 position in amino acid sequence and L90P (substitution of leucine to proline at 90 position in amino acid sequence caused the highest energy increase amongst all. The findings implicate that these nsSNPs would be analyzed further in detail to enumerate their possible association with the protein deteriorating and disease causal potentialities.

  5. A child with xeroderma pigmentosum for excision of basal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Sridevi M Mulimani

    2013-01-01

    Full Text Available Xeroderma pigmentosum (XP is characterized by hypersensitivity to sunlight, ocular involvement, and progressive neurological complications. These manifestations are due to a cellular hypersensitivity to ultraviolet radiation leading to a defect in repair of DNA by the process of nucleotide excision repair. Basal cell carcinoma which is rare in children can occur with XP. Though the XP induced changes are predominately dermatologic, pose several challenges in anaesthetic management. Hence, we are reporting a 9-year-old child with XP scheduled for excision of basal cell carcinoma under general anaesthesia.

  6. Cyclic nucleotides and radioresistnace

    International Nuclear Information System (INIS)

    Kulinskij, V.I.; Mikheeva, G.A.; Zel'manovich, B.M.

    1982-01-01

    The addition of glucose to meat-peptone broth does not change the radiosensitizing effect (RSE) of cAMP at the logarithmic phase (LP) and the radioprotective effect (RPE) at the stationary phase (SP), but sensitization, characteristic of cGMP, disappears in SP and turns into RPE in LP. Introduction of glucose into the broth for 20 min eliminates all the effects of both cyclic nucleotides in the cya + strain while cya - mutant exhibits RSE. RSE of both cyclic nucleotides is only manifested on minimal media. These data brought confirmation of the dependence of the influence of cyclic media. These data brought confirmation of the dependence of the influence of cyclic nucleotides on radioresistance upon the metabolic status of the cell [ru

  7. Estimate at the nucleotide resolution level of genetic changes in the humans residing in the ecologically unfavourable regions of the Techa river

    International Nuclear Information System (INIS)

    Sojfer, V.N.; Petrova, N.V.; Timofeeva, O.A.; Filipenko, M.L.; Solov'eva, N.A.; Popovskij, A.V.; Vlasko, V.V.

    1998-01-01

    To study DNA at the nucleotide level of resolution in residents of settlements located along the Techa river, studies are performed by direct sequencing of gene sequences preliminary amplified and selected by means of analysis of changes of the conformation of DNA unifilament fragments (SSCP-method). Results are presented in details [ru

  8. Nucleotide sequences of the cDNAs encoding the V-regions of H- and L-chains of a human monoclonal antibody with broad reactivity to malignant tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Kishimoto, Toshimitsu; Okajima, Hideki; Okumoto, Takeki [Yoshitomi Pharmaceutical Industries, Ltd., Saitama (Japan); Taniguchi, Masaru [Chiba Univ. (Japan)

    1989-06-12

    The human monoclonal antibody secreted from 4G12 hybridoma cells has broad reactivity to malignant tumor cells, especially for lung squamous cell carcinomas, and recognizes a new tumor-associated and differentiation antigen. The antigen detected by 4G12 is a glycoprotein with MW 195,000 and MW 65,000 under nonreducing and reducing conditions, respectively. Screening of a 4G12 {lambda}gt10 cDNA library with constant region probes for human immunoglobulin yielded full length clones for H- and L-chains. Nucleotide sequences revealed that subtypes of the variable regions were V{sub HIII} and {lambda}{sub 1}, respectively.

  9. Feasibility study of transanal total mesorectal excision

    NARCIS (Netherlands)

    Velthuis, S.; Boezem, P.B. van den; Peet, D.L. van der; Cuesta, M.A.; Sietses, C.

    2013-01-01

    BACKGROUND: Laparoscopic resection of colorectal cancers is a safe alternative to open surgery. The conversion rate to open surgery remains fairly constant but is associated with increased morbidity. A new approach to the surgical excision of rectal cancer is transanal total mesorectal excision

  10. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara Elizabeth; Meier, Stuart Kurt; Gehring, Christoph A

    2016-01-01

    Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms

  11. Analysis of the relationship between single nucleotide polymorphism of the CD209, IL-10, IL-28 and CCR5 D32 genes with the human predisposition to developing tick-borne encephalitis

    Directory of Open Access Journals (Sweden)

    Piotr Czupryna

    2017-01-01

    Full Text Available Introduction: It is known that in the pathogenesis of tick-borne encephalitis (TBE various molecules play a significant role. The most prominent factors include IL-10, IL-28B, CD-209 and CCR5. It is reasonable to search for genetic predispositions to the development of various clinical forms of TBE related to the genetic variation of IL-10, IL-28B, CD-209 and CCR5. In this study we aimed to search for the relationship between single nucleotide polymorphism in the promoter region of the CD209, IL-10, IL-28 and 32 base pair deletion in CCR5 coding region (Δ 32 with the human predisposition to development of various clinical presentations of TBE. We tried to assess the relation between the presence of particular alleles and genotypes with laboratory and clinical parameters. Material/Methods 59 patients with TBE and 57 people, bitten by a tick who never developed TBE (Polish cohort, were included in the study. To assess the distribution of single nucleotide polymorphisms, TaqMan SNP genotyping assays were used for IL10: rs1800872 and rs1800896, for CD 209 rs4804803 and rs2287886, rs12979860 for IL 28B SNPs according to the manufacturer’s protocol using real-time PCR technology on the StepOne thermal cycler. Results Comparison between TBE patients and CG showed that in SNP rs2287886 CD 209 AG heterozygotes were more frequent in the TBE group, while homozygotes GG were more frequent in the CG group. Conclusions SNP rs2287886 CD 209 AG heterozygotes predispose humans to develop TBE. Single nucleotide polymorphism in the promoter region of the CD209, IL-10, IL-28 and CCR5 D32 genes does not correlate with the severity of TBE.

  12. The Gly16 allele of the G16R single nucleotide polymorphism in the β2-adrenergic receptor gene augments the glycemic response to adrenaline in humans

    DEFF Research Database (Denmark)

    Rokamp, Kim Z.; Staalsø, Jonatan M.; Zaar, Morten

    2017-01-01

    Cerebral non-oxidative carbohydrate consumption may be driven by a ß2-adrenergic mechanism. This study tested whether the 46G > A (G16R) single nucleotide polymorphism of the ß2-adrenergic receptor gene (ADRB2) influences the metabolic and cerebrovascular responses to administration of adrenaline....... Forty healthy Caucasian men were included from a group of genotyped individuals. Cardio- and cerebrovascular variables at baseline and during a 60-min adrenaline infusion (0.06 μg kg-1 min-1) were measured by Model flow, near-infrared spectroscopy and transcranial Doppler sonography. Blood samples were...... obtained from an artery and a retrograde catheter in the right internal jugular vein. The ADRB2 G16R variation had no effect on baseline arterial glucose, but during adrenaline infusion plasma glucose was up to 1.2 mM (CI95: 0.36-2.1, P

  13. Loss of Nucleotide Excision Repair as a Source of Genomic Instability in Breast Cancer

    National Research Council Canada - National Science Library

    Ford, James M

    2006-01-01

    .... Our objective is to study DNA repair activity in primary breast epithelial cells and cancer tissues from women at risk for or diagnosed with breast cancer to determine if NER activity can be reliably...

  14. Role of Rad23 and Dsk2 in Nucleotide Excision Repair and Spindle Pole Body Duplication

    Science.gov (United States)

    2007-03-01

    of Ddi1 in stabilizing Pds1–128, we overexpressed exogenous ddi1Δ184–285 in a pds1–128 DDI1 strain (Fig- ure 4C ) and asked whether this mutant could...the mutant forms after expression from the GAL1 promoter were all similar (Figure 4C ). This was also the case when tagged versions of DDI1 and...Axioplan2 Microscope, using an alpha-Plan-FLUAR 100X/1,45 oil objective, an Axiocam HRm camera and Axiovision soft- ware. The pB42AD-ddi1Δ184–285

  15. Nucleotide excision repair : a multi-step mechanism required to maintain genome integrity

    NARCIS (Netherlands)

    Moser, Jill

    2010-01-01

    DNA is continuously exposed to exogenous and genotoxic insults including ionizing and ultraviolet radiation as well as chemical agents. DNA damage can compromise the integrity of the genome and have potentially deleterious effects. Ultraviolet light (UV) can induce the formation of helix distorting

  16. Functional, genetic and epigenetic aspects of base and nucleotide excision repair in colorectal carcinomas

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Korenková, Vlasta; Collins, A. R.; Procházka, Pavel; Vodičková, Ludmila; Švec, Jiří; Lipská, L.; Levý, M.; Schneiderová, M.; Liška, V.; Holubec, L.; Kumar, R.; Souček, P.; Naccarati, Alessio; Vodička, Pavel

    2012-01-01

    Roč. 18, č. 21 (2012), s. 5878-5887 ISSN 1078-0432 R&D Projects: GA ČR GAP304/12/1585; GA ČR(CZ) GAP304/10/1286; GA MZd NT12025 Grant - others:UICC(XE) ICR/11/068/2011; EEA-research fund:(NO) B/CZ0046/40031 Institutional research plan: CEZ:AV0Z50390512 Institutional support: RVO:68378041 ; RVO:86652036 Keywords : DNA repair capacity * DNA repair gene expression * methylation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.837, year: 2012

  17. Differences in nucleotide excision repair capacity between newly diagnosed colorectal cancer patients and healthy controls

    Czech Academy of Sciences Publication Activity Database

    Slyšková, Jana; Naccarati, Alessio; Pardini, Barbara; Poláková, Veronika; Vodičková, Ludmila; Šmerhovský, Z.; Levý, M.; Lipská, L.; Liška, V.; Vodička, Pavel (ed.)

    2012-01-01

    Roč. 27, č. 2 (2012), s. 225-232 ISSN 0267-8357 R&D Projects: GA ČR GAP304/10/1286; GA MZd NS10230 Grant - others:EEA-research fund:(NO) A/CZ0046/2/0012 Institutional research plan: CEZ:AV0Z50390512 Keywords : biomarkers * DNA damage * DNA repair capacity Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.500, year: 2012

  18. Polymorphisms in miRNA binding sites of nucleotide excision repair genes and colorectal cancer risk

    Czech Academy of Sciences Publication Activity Database

    Naccarati, Alessio; Pardini, Barbara; Landi, S.; Landi, D.; Slyšková, Jana; Novotný, J.; Levý, M.; Poláková, Veronika; Lipská, L.; Vodička, Pavel

    2012-01-01

    Roč. 33, č. 7 (2012), s. 1346-1351 ISSN 0143-3334 R&D Projects: GA ČR GAP304/10/1286; GA ČR GP305/09/P194 Institutional research plan: CEZ:AV0Z50390703 Keywords : DNA repair * polymorphisms * miRNA binding sites Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.635, year: 2012

  19. Clinicopathologic Significance of Excision Repair Cross-Complementation 1 Expression in Patients Treated With Breast-Conserving Surgery and Radiation Therapy

    International Nuclear Information System (INIS)

    Goyal, Sharad; Parikh, Rahul R.; Green, Camille; Schiff, Devora B.S.; Moran, Meena S.; Yang Qifeng; Haffty, Bruce G.

    2010-01-01

    Purpose: The excision repair cross-complementation 1 (ERCC1) enzyme plays a rate-limiting role in the nucleotide excision repair pathway and is associated with resistance to platinum-based chemotherapy in cancers of the head and neck and the lung. The purpose of this study was to evaluate the clinicopathologic and prognostic significance of ERCC1 expression in a cohort of early-stage breast cancer patients treated with breast conservation therapy. Methods and Materials: Paraffin specimens from 504 women with early-stage breast cancer treated with breast conservation therapy were constructed into tissue microarrays. The array was stained for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) and ERCC1. This was then correlated with clinicopathologic factors and outcomes data. Results: ERCC-1 expression was evaluable in 366 cases (72%). In this group, 32% and 38% of patients received adjuvant chemotherapy and hormonal therapy, respectively. Increased ERCC-1 expression was found to be correlated with ER positivity (p 50 (p 50. To our knowledge, this is the first study investigating ERCC1 expression in patients receiving adjuvant radiation therapy for breast cancer.

  20. Tissue-Specific Methylation of Long Interspersed Nucleotide Element-1 of Homo Sapiens (L1Hs) During Human Embryogenesis and Roles in Neural Tube Defects.

    Science.gov (United States)

    Wang, L; Chang, S; Guan, J; Shangguan, S; Lu, X; Wang, Z; Wu, L; Zou, J; Zhao, H; Bao, Y; Qiu, Z; Niu, B; Zhang, T

    2015-01-01

    Epigenetic regulation of long interspersed nucleotide element-1 (LINE-1) retrotransposition events plays crucial roles during early development. Previously we showed that LINE-1 hypomethylation in neuronal tissues is associated with pathogenesis of neural tube defect (NTD). Herein, we further evaluated LINE-1 Homo sapiens (L1Hs) methylation in tissues derived from three germ layers of stillborn NTD fetuses, to define patterns of tissue specific methylation and site-specific hypomethylation at CpG sites within an L1Hs promoter region. Stable, tissue-specific L1Hs methylation patterns throughout three germ layer lineages of the fetus, placenta, and maternal peripheral blood were observed. Samples from maternal peripheral blood exhibited the highest level of L1Hs methylation (64.95%) and that from placenta showed the lowest (26.82%). Between samples from NTDs and controls, decrease in L1Hs methylation was only significant in NTD-affected brain tissue at 7.35%, especially in females (8.98%). L1Hs hypomethylation in NTDs was also associated with a significant increase in expression level of an L1Hs-encoded transcript in females (r = -0.846, p = 0.004). This could be due to genomic DNA instability and alternation in chromatins accessibility resulted from abnormal L1Hs hypomethylation, as showed in this study with HCT-15 cells treated with methylation inhibitor 5-Aza.

  1. [Efficacy of initial antiretroviral therapy based on lopinavir/ritonavir plus 2 nucleoside/nucleotide analogs in patients with human immunodeficiency virus type 1 infection].

    Science.gov (United States)

    Zamora, Laura; Gatell, José M

    2014-11-01

    Triple combination regimens consisting of lopinavir/ritonavir (LPV/r) plus 2 nucleoside/nucleotide analogs continue to be a valid option in initial antiretroviral therapy. Other protease inhibitors boosted with ritonavir (and in future with cobicistat) have been introduced, as well as other non-nucleoside analogs (rilpivirin) and 3 integrase inhibitors. None of the new regimens have shown superiority over LPV/r or comparisons are lacking. Therefore, regimens including LPV/r continue to be recommended as initial first-line or alternative strategies in most treatment guidelines. Dual combinations with LPV/r (plus raltegravir or lamivudine) are described in another article and can provide a similar response rate to triple combinations, better tolerance, and an improved cost-efficacy ratio, both for initial therapy and in simplification strategies. In contrast, LPV/r or darunavir/r monotherapy does not seem an acceptable option in treatment-naïve patients and is becoming increasingly less acceptable in simplification strategies. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

  2. G16R single nucleotide polymorphism but not haplotypes of the ß2-adrenergic receptor gene alters cardiac output in humans

    DEFF Research Database (Denmark)

    Rokamp, Kim Z; Staalsø, Jonatan M; Gartmann, Martin

    2013-01-01

    Variation in genes encoding the ß2-adrenergic receptor (ADRB2) and angiotensin-converting enzyme (ACE) may influence Q¿ (cardiac output). The 46G>A (G16R) SNP (single nucleotide polymorphism) has been associated with ß2-mediated vasodilation, but the effect of ADRB2 haplotypes on Q¿ has not been...... studied. Five SNPs within ADRB2 (46G>A, 79C>G, 491C>T, 523C>A and 1053G>C by a pairwise tagging principle) and the I/D (insertion/deletion) polymorphism in ACE were genotyped in 143 subjects. Cardiovascular variables were evaluated by the Model flow method at rest and during incremental cycling exercise...... V¿O2 (oxygen uptake) in G16G subjects, but the increase was 0.5 (0.0-0.9) l/min lower in Arg16 carriers (P=0.035). A similar effect size was observed for the Arg16 haplotypes ACCCG and ACCCC. No interaction was found between ADRB2 and ACE polymorphisms. During exercise, the increase in Q¿ was 0...

  3. The role of nucleoside/nucleotide transport and metabolism in the uptake and retention of 3'-fluoro-3'-deoxythymidine in human B-lymphoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Plotnik, David A.; McLaughlin, Lena J.; Chan, Jenny; Redmayne-Titley, Joshua N.; Schwartz, Jeffrey L., E-mail: jschwart@uw.edu

    2011-10-15

    Introduction: Recent studies in the human adenocarcinoma cell line A549 have identified cell growth-dependent equilibrative nucleoside transporter-1 (hENT1) as a modifier of 3'-fluoro-3'-deoxythymidine (FLT) uptake and retention. In the present study, we used the ability to isolate human lymphoblastoid clones deficient in thymidine kinase 1 (TK1) to study how metabolism and nucleoside transport influence FLT uptake and retention. Methods: Transport and metabolism of FLT were measured in the human lymphoblastoid cell line TK6 and in eight clones isolated from TK6. Four clones were TK1-proficient, while four were TK1-deficient. Both influx and efflux of FLT were measured under conditions where concentrative and equilibrative transport could be distinguished. Results: Sodium-dependent concentrative FLT transport dominated over equilibrative transport mechanisms and while inhibition of hENT1 reduced FLT uptake, there were no correlations between clonal variations in hENT1 levels and FLT uptake. There was an absolute requirement of TK1 for concentration of FLT in TK6 cells. FLT uptake reached a peak after 60 min of incubation with FLT after which intracellular levels of FLT and FLT metabolites declined. Efflux was rapid and was associated with reductions in FLT and each of its metabolites. Both FLT and FLT-monophosphate were found in the efflux buffer. Conclusions: Initial rates of FLT uptake were a function of both concentrative and equilibrative transporters. TK1 activity was an absolute requirement for the accumulation of FLT. Retention was dependent on nucleoside/nucleotide efflux and retrograde metabolism of FLT nucleotides.

  4. Ulnar nerve entrapment complicating radial head excision

    Directory of Open Access Journals (Sweden)

    Kevin Parfait Bienvenu Bouhelo-Pam

    Full Text Available Introduction: Several mechanisms are involved in ischemia or mechanical compression of ulnar nerve at the elbow. Presentation of case: We hereby present the case of a road accident victim, who received a radial head excision for an isolated fracture of the radial head and complicated by onset of cubital tunnel syndrome. This outcome could be the consequence of an iatrogenic valgus of the elbow due to excision of the radial head. Hitherto the surgical treatment of choice it is gradually been abandoned due to development of radial head implant arthroplasty. However, this management option is still being performed in some rural centers with low resources. Discussion: The radial head plays an important role in the stability of the elbow and his iatrogenic deformity can be complicated by cubital tunnel syndrome. Conclusion: An ulnar nerve release was performed with favorable outcome. Keywords: Cubital tunnel syndrome, Peripheral nerve palsy, Radial head excision, Elbow valgus

  5. Elliptical excisions: variations and the eccentric parallelogram.

    Science.gov (United States)

    Goldberg, Leonard H; Alam, Murad

    2004-02-01

    The elliptical (fusiform) excision is a basic tool of cutaneous surgery. To assess the design, functionality, ease of construction, and aesthetic outcomes of the ellipse. A systematic review of elliptical designs and their site-specific benefits and limitations. In particular, we consider the (1). context of prevailing relaxed skin tension lines and tissue laxity; and (2). removal of the smallest possible amount of tissue around the lesion and in the "dog-ears." Attention is focused on intuitive methods that can be reproducibly planned and executed. Elliptical variations are easily designed and can be adapted to many situations. The eccentric parallelogram excision is offered as a new technique that minimizes notching and focal tension in the center of an elliptical closure. Conclusion The elliptical (fusiform) excision is an efficient, elegant, and versatile technique that will remain a mainstay of the cutaneous surgical armamentarium.

  6. Modeling single nucleotide polymorphisms in the human AKR1C1 and AKR1C2 genes: implications for functional and genotyping analyses.

    Directory of Open Access Journals (Sweden)

    Jonathan W Arthur

    2010-12-01

    Full Text Available Enzymes encoded by the AKR1C1 and AKR1C2 genes are responsible for the metabolism of progesterone and 5α-dihydrotestosterone (DHT, respectively. The effect of amino acid substitutions, resulting from single nucleotide polymorphisms (SNPs in the AKR1C2 gene, on the enzyme kinetics of the AKR1C2 gene product were determined experimentally by Takashi et al. In this paper, we used homology modeling to predict and analyze the structure of AKR1C1 and AKR1C2 genetic variants. The experimental reduction in enzyme activity in the AKR1C2 variants F46Y and L172Q, as determined by Takahashi et al., is predicted to be due to increased instability in cofactor binding, caused by disruptions to the hydrogen bonds between NADP and AKR1C2, resulting from the insertion of polar residues into largely non-polar environments near the site of cofactor binding. Other AKR1C2 variants were shown to involve either conservative substitutions or changes taking place on the surface of the molecule and distant from the active site, confirming the experimental finding of Takahashi et al. that these variants do not result in any statistically significant reduction in enzyme activity. The AKR1C1 R258C variant is predicted to have no effect on enzyme activity for similar reasons. Thus, we provide further insight into the molecular mechanism of the enzyme kinetics of these proteins. Our data also highlight previously reported difficulties with online databases.

  7. Resistin in serum and gingival crevicular fluid as a marker of periodontal inflammation and its correlation with single-nucleotide polymorphism in human resistin gene at −420

    Directory of Open Access Journals (Sweden)

    Swati Pradeep Patel

    2013-01-01

    Full Text Available Aims: Resistin is an adipocytokine, which have been studied for its role in insulin resistance and recently in inflammation. The aim of the present study is to assess the concentration of resistin in serum and gingival crevicular fluid (GCF and to compare the levels between subjects with and without periodontitis and type 2 diabetes mellitus (T2DM and to further correlate the resistin levels with the single-nucleotide polymorphism (SNP at −420. Setting and Designs: A total of 96 subjects (48 males and 48 females were divided on the basis of gingival index (GI, probing pocket depth (PD, clinical attachment level (CAL and hemoglobin A 1c levels into healthy (group 1, n = 24, uncontrolled-diabetes related periodontitis (group 2, n = 24, controlled-diabetes related periodontitis (group 3, n = 24 and chronic periodontitis without T2DM (group 4, n = 24. Materials and Methods: The GCF and serum levels of resistin were quantified using the enzyme-linked immunosorbent assay and compared among the study groups. Further, the association of the resistin levels with periodontal inflammation and SNP at −420 was studied. Results and Conclusion: The resistin levels in GCF and serum from patients with periodontitis or diabetes mellitus related periodontitis (controlled or uncontrolled were higher than that of healthy subjects and correlated positively with GI. Further, subjects with GG genotype at −420 showed significantly higher GI, PD, CAL as compared with genotype group CC. Resistin was detected in all serum and GCF samples and was significantly higher in periodontitis. Further, GG genotype at −420 was associated significantly with periodontal inflammation and resistin levels.

  8. Stable Human Hepatoma Cell Lines for Efficient Regulated Expression of Nucleoside/Nucleotide Analog Resistant and Vaccine Escape Hepatitis B Virus Variants and Woolly Monkey Hepatitis B Virus.

    Directory of Open Access Journals (Sweden)

    Xin Cheng

    Full Text Available Hepatitis B virus (HBV causes acute and chronic hepatitis B (CHB. Due to its error-prone replication via reverse transcription, HBV can rapidly evolve variants that escape vaccination and/or become resistant to CHB treatment with nucleoside/nucleotide analogs (NAs. This is particularly problematic for the first generation NAs lamivudine and adefovir. Though now superseded by more potent NAs, both are still widely used. Furthermore, resistance against the older NAs can contribute to cross-resistance against more advanced NAs. For lack of feasible HBV infection systems, the biology of such variants is not well understood. From the recent discovery of Na+-taurocholate cotransporting polypeptide (NTCP as an HBV receptor new in vitro infection systems are emerging, yet access to the required large amounts of virions, in particular variants, remains a limiting factor. Stably HBV producing cell lines address both issues by allowing to study intracellular viral replication and as a permanent source of defined virions. Accordingly, we generated a panel of new tetracycline regulated TetOFF HepG2 hepatoma cell lines which produce six lamivudine and adefovir resistance-associated and two vaccine escape variants of HBV as well as the model virus woolly monkey HBV (WMHBV. The cell line-borne viruses reproduced the expected NA resistance profiles and all were equally sensitive against a non-NA drug. The new cell lines should be valuable to investigate under standardized conditions HBV resistance and cross-resistance. With titers of secreted virions reaching >3 x 10(7 viral genome equivalents per ml they should also facilitate exploitation of the new in vitro infection systems.

  9. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara Elizabeth

    2016-05-11

    Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

  10. Excised Abdominoplasty Material as a Systematic Plastic Surgical Training Model

    Directory of Open Access Journals (Sweden)

    M. Erol Demirseren

    2012-01-01

    Full Text Available Achieving a level of technical skill and confidence in surgical operations is the main goal of plastic surgical training. Operating rooms were accepted as the practical teaching venues of the traditional apprenticeship model. However, increased patient population, time, and ethical and legal considerations made preoperation room practical work a must for plastic surgical training. There are several plastic surgical teaching models and simulators which are very useful in preoperation room practical training and the evaluation of plastic surgery residents. The full thickness skin with its vascular network excised in abdominoplasty procedures is an easily obtainable real human tissue which could be used as a training model in plastic surgery.

  11. Exon sequence requirements for excision in vivo of the bacterial group II intron RmInt1

    Directory of Open Access Journals (Sweden)

    Toro Nicolás

    2011-05-01

    Full Text Available Abstract Background Group II intron splicing proceeds through two sequential transesterification reactions in which the 5' and 3'-exons are joined together and the lariat intron is released. The intron-encoded protein (IEP assists the splicing of the intron in vivo and remains bound to the excised intron lariat RNA in a ribonucleoprotein particle (RNP that promotes intron mobility. Exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain dI known as EBS1 and EBS2 and two stretches of sequence known as IBS1 and IBS2 on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from EBS1 [(δ-δ' interaction (subgroup IIA] or with a nucleotide [(EBS3-IBS3 interaction (subgroup IIB and IIC] located in the coordination-loop of dI. The δ nucleotide is involved in base pairing with another intron residue (δ' in subgroup IIB introns and this interaction facilitates base pairing between the 5' exon and the intron. Results In this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the group IIB intron RmInt1 in vivo. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo. Conclusions The excision in vivo of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.

  12. DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

    DEFF Research Database (Denmark)

    Osorio, Ana; Milne, Roger L; Kuchenbaecker, Karoline

    2014-01-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of th...

  13. DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

    NARCIS (Netherlands)

    A. Osorio (Ana); R.L. Milne (Roger); K.B. Kuchenbaecker (Karoline); T. Vaclová (Tereza); G. Pita (Guillermo); R. Alonso (Rosario); P. Peterlongo (Paolo); I. Blanco (Ignacio); M. de La Hoya (Miguel); M. Durán (Mercedes); O. Díez (Orland); T. Ramon Y Cajal; I. Konstantopoulou (I.); C. Martínez-Bouzas (Cristina); R. Andrés Conejero (Raquel); P. Soucy (Penny); L. McGuffog (Lesley); D. Barrowdale (Daniel); A. Lee (Andrew); B. Arver (Brita Wasteson); J. Rantala (Johanna); N. Loman (Niklas); H. Ehrencrona (Hans); O.I. Olopade (Olofunmilayo); M.S. Beattie (Mary); S.M. Domchek (Susan); K.L. Nathanson (Katherine); R. Rebbeck (Timothy); B.K. Arun (Banu); B.Y. Karlan (Beth); C.S. Walsh (Christine); K.J. Lester (Kathryn); E.M. John (Esther); A.S. Whittemore (Alice); M.B. Daly (Mary); M.C. Southey (Melissa); J.L. Hopper (John); M.-B. Terry (Mary-Beth); S.S. Buys (Saundra); R. Janavicius (Ramunas); C.M. Dorfling (Cecilia); E.J. van Rensburg (Elizabeth); L. Steele (Linda); S.L. Neuhausen (Susan); Y.C. Ding (Yuan); T.V.O. Hansen (Thomas); L. Jønson (Lars); B. Ejlertsen (Bent); A-M. Gerdes (Anne-Marie); J. Infante (Jon); B. Herráez (Belén); L.T. Moreno (Leticia Thais); J.N. Weitzel (Jeffrey); J. Herzog (Josef); K. Weeman (Kisa); S. Manoukian (Siranoush); B. Peissel (Bernard); D. Zaffaroni (D.); G. Scuvera (Giulietta); B. Bonnani (Bernardo); F. Mariette (F.); S. Volorio (Sara); A. Viel (Alessandra); L. Varesco (Liliana); L. Papi (Laura); L. Ottini (Laura); M.G. Tibiletti (Maria Grazia); P. Radice (Paolo); D. Yannoukakos (Drakoulis); J. Garber; S.D. Ellis (Steve); D. Frost (Debra); R. Platte (Radka); E. Fineberg (Elena); D.G. Evans (Gareth); F. Lalloo (Fiona); L. Izatt (Louise); R. Eeles (Rosalind); J.W. Adlard (Julian); R. Davidson (Rosemarie); T.J. Cole (Trevor); D. Eccles (Diana); J. Cook (Jackie); S.V. Hodgson (Shirley); C. Brewer (Carole); M. Tischkowitz (Marc); F. Douglas (Fiona); M.E. Porteous (Mary); L. Side (Lucy); L.J. Walker (Lisa); P.J. Morrison (Patrick); A. Donaldson (Alan); J. Kennedy (John); C. Foo (Claire); A.K. Godwin (Andrew); R.K. Schmutzler (Rita); B. Wapenschmidt (Barbara); K. Rhiem (Kerstin); C.W. Engel (Christoph); A. Meindl (Alfons); N. Ditsch (Nina); N. Arnold (Norbert); H. Plendl (Hansjoerg); D. Niederacher (Dieter); C. Sutter (Christian); S. Wang-Gohrke (Shan); D. Steinemann (Doris); S. Preisler-Adams (Sabine); K. Kast (Karin); R. Varon-Mateeva (Raymonda); P.A. Gehrig (Paola A.); D. Stoppa-Lyonnet (Dominique); O. Sinilnikova (Olga); S. Mazoyer (Sylvie); F. Damiola (Francesca); B. Poppe (Bruce); K. Claes (Kathleen); M. Piedmonte (Marion); K. Tucker (Kathryn); F.J. Backes (Floor); P.M. Rodríguez; W. Brewster (Wendy); K. Wakeley (Katie); T. Rutherford (Thomas); T. Caldes (Trinidad); H. Nevanlinna (Heli); K. Aittomäki (Kristiina); M.A. Rookus (Matti); T.A.M. van Os (Theo); L. van der Kolk (Lizet); J.L. de Lange (J.); E.J. Meijers-Heijboer (Hanne); A.H. van der Hout (Annemarie); C.J. van Asperen (Christi); E.B. Gómez García (Encarna); N. Hoogerbrugge (Nicoline); J.M. Collée (Margriet); C.H.M. van Deurzen (Carolien); R.B. van der Luijt (Rob); P. Devilee (Peter); E. Olah (Edith); C. Lazaro (Conxi); A. Teulé (A.); M. Menéndez (Mireia); A. Jakubowska (Anna); C. Cybulski (Cezary); J. Gronwald (Jacek); J. Lubinski (Jan); K. Durda (Katarzyna); K. Jaworska-Bieniek (Katarzyna); O.T. Johannson (Oskar); C. Maugard; M. Montagna (Marco); S. Tognazzo (Silvia); P.J. Teixeira; S. Healey (Sue); C. Olswold (Curtis); L. Guidugli (Lucia); N.M. Lindor (Noralane); S. Slager (Susan); C. Szabo (Csilla); J. Vijai (Joseph); M. Robson (Mark); N. Kauff (Noah); L. Zhang (Lingling); R. Rau-Murthy (Rohini); A. Fink-Retter (Anneliese); C.F. Singer (Christian); C. Rappaport (Christine); D. Geschwantler Kaulich (Daphne); G. Pfeiler (Georg); M.-K. Tea; A. Berger (Annemarie); C. Phelan (Catherine); M.H. Greene (Mark); P.L. Mai (Phuong); F. Lejbkowicz (Flavio); I.L. Andrulis (Irene); A.M. Mulligan (Anna Marie); G. Glendon (Gord); A.E. Toland (Amanda); S.E. Bojesen (Stig); I.S. Pedersen (Inge Sokilde); L. Sunde (Lone); M. Thomassen (Mads); T.A. Kruse (Torben); U.B. Jensen; E. Friedman (Eitan); Y. Laitman (Yael); S.P. Shimon (Shani Paluch); J. Simard (Jacques); D.F. Easton (Douglas); K. Offit (Kenneth); F.J. Couch (Fergus); G. Chenevix-Trench (Georgia); A.C. Antoniou (Antonis); J. Benítez (Javier)

    2014-01-01

    textabstractSingle Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between

  14. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers

    NARCIS (Netherlands)

    Osorio, A.; Milne, R.L.; Kuchenbaecker, K.; Vaclova, T.; Pita, G.; Alonso, R.; Peterlongo, P.; Blanco, I.; Hoya, M. de la; Duran, M.; Diez, O.; Ramon, Y.C.T.; Konstantopoulou, I.; Martinez-Bouzas, C.; Conejero, R. Andres; Soucy, P.; McGuffog, L.; Barrowdale, D.; Lee, A.; Swe, B.; Arver, B.; Rantala, J.; Loman, N.; Ehrencrona, H.; Olopade, O.I.; Beattie, M.S.; Domchek, S.M.; Nathanson, K.; Rebbeck, T.R.; Arun, B.K.; Karlan, B.Y.; Walsh, C.; Lester, J.; John, E.M.; Whittemore, A.S.; Daly, M.B.; Southey, M.; Hopper, J.; Terry, M.B.; Buys, S.S.; Janavicius, R.; Dorfling, C.M.; Rensburg, E.J. van; Steele, L.; Neuhausen, S.L.; Ding, Y.C.; Hansen, T.V.; Jonson, L.; Ejlertsen, B.; Gerdes, A.M.; Infante, M.; Herraez, B.; Moreno, L.T.; Weitzel, J.N.; Herzog, J.; Weeman, K.; Manoukian, S.; Peissel, B.; Zaffaroni, D.; Scuvera, G.; Bonanni, B.; Mariette, F.; Volorio, S.; Viel, A.; Varesco, L.; Papi, L.; Ottini, L.; Tibiletti, M.G.; Radice, P.; Yannoukakos, D.; Garber, J.; Ellis, S.; Frost, D.; Platte, R.; Fineberg, E.; Evans, G.; Lalloo, F.; Izatt, L.; Eeles, R.; Adlard, J.; Davidson, R.; Cole, T.; Eccles, D.; Cook, J; Hodgson, S.; Brewer, C.; Tischkowitz, M.; Douglas, F.; Porteous, M.; Side, L.; Walker, L.; Morrison, P.; Donaldson, A.; Kennedy, J.; Foo, C.; Godwin, A.K.; Schmutzler, R.K.; Wappenschmidt, B.; Rhiem, K.; Engel, C.; Hoogerbrugge-van der Linden, N.; et al.,

    2014-01-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the

  15. Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA.

    Directory of Open Access Journals (Sweden)

    Anna-Karin Berglund

    2017-02-01

    Full Text Available Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.

  16. Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA.

    Science.gov (United States)

    Berglund, Anna-Karin; Navarrete, Clara; Engqvist, Martin K M; Hoberg, Emily; Szilagyi, Zsolt; Taylor, Robert W; Gustafsson, Claes M; Falkenberg, Maria; Clausen, Anders R

    2017-02-01

    Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.

  17. Nucleotide sequence of a cDNA coding for the amino-terminal region of human prepro. alpha. 1(III) collagen

    Energy Technology Data Exchange (ETDEWEB)

    Toman, P D; Ricca, G A [Rorer Biotechnology, Inc., Springfield, VA (USA); de Crombrugghe, B [National Institutes of Health, Bethesda, MD (USA)

    1988-07-25

    Type III Collagen is synthesized in a variety of tissues as a precursor macromolecule containing a leader sequence, a N-propeptide, a N-telopeptide, the triple helical region, a C-telopeptide, and C-propeptide. To further characterize the human type III collagen precursor, a human placental cDNA library was constructed in gt11 using an oligonucleotide derived from a partial cDNA sequence corresponding to the carboxy-terminal part of the 1(III) collagen. A cDNA was identified which contains the leader sequence, the N-propeptide and N-telopeptide regions. The DNA sequence of these regions are presented here. The triple helical, C-telopeptide and C-propeptide amino acid sequence for human type III collagen has been determined previously. A comparison of the human amino acid sequence with mouse, chicken, and calf sequence shows 81%, 81%, and 92% similarity, respectively. At the DNA level, the sequence similarity between human and mouse or chicken type III collagen sequences in this area is 82% and 77%, respectively.

  18. Robotic Extramucosal Excision of Bladder Wall Leiomyoma

    Directory of Open Access Journals (Sweden)

    Khalid E. Al-Othman

    2014-01-01

    Full Text Available Introduction: Multiple case reports and reviews have been described in the literature for bladder wall leiomyoma resection via different approaches. The minimally invasive partial cystectomy remains the most widely accepted technique; however, case reports for enucleation of bladder wall leiomyoma have also been described. The purpose of this video is to demonstrate the robotic extramucosal excision of a bladder wall leiomyoma, without cystotomy, but with complete removal of the muscular layer. Materials and Methods: A 35-year old male present with lower urinary tract symptoms and imaging showed bladder wall mass with histopathology showed leiomyoma. The patient consented for mass excision with the possibility of a partial cystectomy. The patient was placed in the supine, 30-degree Trendelenburg position during the procedure. A total of 4 ports were inserted. A 3-arm da Vinci robotic surgical system was docked, and the arms were connected. Extramucosal excision was accomplished without cystotomy and muscle approximation was achieved by 2 0 Vicryle. Result: The operative time was 90 minutes, blood loss of approximately 50mL and the patient was discharged after 72 hours with no immediate complications and a 6 months follow-up showed no recurrence. Conclusion: Such a technique results in complete excision of the tumor, without cystotomy, and also maintains an intact mucosa. These steps, in addition to decreasing the risk of local recurrence, also shorten the period of postoperative catheterization and hospitalization.

  19. Base excision repair, aging and health span

    Czech Academy of Sciences Publication Activity Database

    Xu, G.; Herzig, M.; Rotrekl, Vladimír; Walter, Ch. A.

    2008-01-01

    Roč. 129, 7-8 (2008), s. 366-382 ISSN 0047-6374 Institutional research plan: CEZ:AV0Z50390512 Keywords : base excision repair * aging * DNA damage Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.915, year: 2008

  20. Uracil Excision for Assembly of Complex Pathways

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda; Nielsen, Morten Thrane; Kim, Se Hyeuk

    2015-01-01

    Despite decreasing prices on synthetic DNA constructs, higher-order assembly of PCR-generated DNA continues to be an important exercise in molecular and synthetic biology. Simplicity and robustness are attractive features met by the uracil excision DNA assembly method, which is one of the most in...

  1. Interaction between amiodarone and hepatitis-C virus nucleotide inhibitors in human induced pluripotent stem cell-derived cardiomyocytes and HEK-293 Cav1.2 over-expressing cells.

    Science.gov (United States)

    Lagrutta, Armando; Zeng, Haoyu; Imredy, John; Balasubramanian, Bharathi; Dech, Spencer; Lis, Edward; Wang, Jixin; Zhai, Jin; DeGeorge, Joseph; Sannajust, Frederick

    2016-10-01

    Several clinical cases of severe bradyarrhythmias have been reported upon co-administration of the Hepatitis-C NS5B Nucleotide Polymerase Inhibitor (HCV-NI) direct-acting antiviral agent, sofosbuvir (SOF), and the Class-III anti-arrhythmic amiodarone (AMIO). We model the cardiac drug-drug interaction (DDI) between AMIO and SOF, and between AMIO and a closely-related SOF analog, MNI-1 (Merck Nucleotide Inhibitor #1), in functional assays of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), to provide mechanistic insights into recently reported clinical cases. AMIO co-applied with SOF or MNI-1 increased beating rate or field potential (FP) rate and decreased impedance (IMP) and Ca(2+) transient amplitudes in hiPSC-CM syncytia. This action resembled that of Ca(2+) channel blockers (CCBs) in the model, but CCBs did not substitute for AMIO in the DDI. AMIO analog dronedarone (DRON) did not substitute for, but competed with AMIO in the DDI. Ryanodine and thapsigargin, decreasing intracellular Ca(2+) stores, and SEA-0400, a Na(+)/Ca(2+) exchanger-1 (NCX1) inhibitor, partially antagonized or suppressed DDI effects. Other agents affecting FP rate only exerted additive or subtractive effects, commensurate with their individual effects. We also describe an interaction between AMIO and MNI-1 on Cav1.2 ion channels in an over-expressing HEK-293 cell line. MNI-1 enhanced Cav1.2 channel inhibition by AMIO, but did not affect inhibition of Cav1.2 by DRON, verapamil, nifedipine, or diltiazem. Our data in hiPSC-CMs indicate that HCV-NI agents such as SOF and MNI-1 interact with key intracellular Ca(2+)-handling mechanisms. Additional study in a Cav1.2 HEK-293 cell-line suggests that HCV-NIs potentiate the inhibitory action of AMIO on L-type Ca(2+) channels. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Circumareolar Incision‑subdermal Tunneling Dissection for Excision ...

    African Journals Online (AJOL)

    2017 Nigerian Journal of Surgery | Published by Wolters Kluwer - Medknow. Excision of ... This is a report of excision of MF in multiple quadrants of the breast using a ... Agodirin, et al. .... the breast: The Ribeiro technique modified by Rezai.

  3. Interaction between amiodarone and hepatitis-C virus nucleotide inhibitors in human induced pluripotent stem cell-derived cardiomyocytes and HEK-293 Cav{sub 1.2} over-expressing cells

    Energy Technology Data Exchange (ETDEWEB)

    Lagrutta, Armando, E-mail: armando_lagrutta@merck.com; Zeng, Haoyu; Imredy, John; Balasubramanian, Bharathi; Dech, Spencer; Lis, Edward; Wang, Jixin; Zhai, Jin; DeGeorge, Joseph; Sannajust, Frederick

    2016-10-01

    Several clinical cases of severe bradyarrhythmias have been reported upon co-administration of the Hepatitis-C NS5B Nucleotide Polymerase Inhibitor (HCV-NI) direct-acting antiviral agent, sofosbuvir (SOF), and the Class-III anti-arrhythmic amiodarone (AMIO). We model the cardiac drug-drug interaction (DDI) between AMIO and SOF, and between AMIO and a closely-related SOF analog, MNI-1 (Merck Nucleotide Inhibitor #1), in functional assays of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), to provide mechanistic insights into recently reported clinical cases. AMIO co-applied with SOF or MNI-1 increased beating rate or field potential (FP) rate and decreased impedance (IMP) and Ca{sup 2+} transient amplitudes in hiPSC-CM syncytia. This action resembled that of Ca{sup 2+} channel blockers (CCBs) in the model, but CCBs did not substitute for AMIO in the DDI. AMIO analog dronedarone (DRON) did not substitute for, but competed with AMIO in the DDI. Ryanodine and thapsigargin, decreasing intracellular Ca{sup 2+} stores, and SEA-0400, a Na{sup +}/Ca{sup 2+} exchanger-1 (NCX1) inhibitor, partially antagonized or suppressed DDI effects. Other agents affecting FP rate only exerted additive or subtractive effects, commensurate with their individual effects. We also describe an interaction between AMIO and MNI-1 on Cav{sub 1.2} ion channels in an over-expressing HEK-293 cell line. MNI-1 enhanced Cav{sub 1.2} channel inhibition by AMIO, but did not affect inhibition of Cav{sub 1.2} by DRON, verapamil, nifedipine, or diltiazem. Our data in hiPSC-CMs indicate that HCV-NI agents such as SOF and MNI-1 interact with key intracellular Ca{sup 2+}-handling mechanisms. Additional study in a Cav{sub 1.2} HEK-293 cell-line suggests that HCV-NIs potentiate the inhibitory action of AMIO on L-type Ca{sup 2+} channels. - Highlights: • Adverse clinical interaction between amiodarone and HCV-NI drugs is captured by in vitro models. • Human iPSC-derived cardiomyocyte

  4. Antinociceptive effect of purine nucleotides.

    Science.gov (United States)

    Mello, C F; Begnini, J; De-La-Vega, D D; Lopes, F P; Schwartz, C C; Jimenez-Bernal, R E; Bellot, R G; Frussa-Filho, R

    1996-10-01

    The antinociceptive effect of purine nucleotides administered systematically (sc) was determined using the formalin and writhing tests in adult male albino mice. The mechanisms underlying nucleotide-induced antinociception were investigated by preinjecting the animals (sc) with specific antagonists for opioid (naloxone, 1 mg/kg), purinergic P1 (caffeine, 5, 10, of 30 mg/kg); theophylline, 10 mg/kg) or purinergic P2 receptors (suramin, 100 mg/kg; Coomassie blue, 30-300 mg/kg; quinidine, 10 mg/kg). Adenosine, adenosine monophosphate (AMP), diphosphate (ADP) and triphosphate (ATP) caused a reduction in the number of writhes and in the time of licking the formalin-injected paw. Naloxone had no effect on adenosine- or adenine nucleotide-induced antinociception. Caffeine (30 mg/kg) and theophylline (10 mg/kg) reversed the antinociceptive action of adenosine and adenine nucleotide derivatives in both tests. P2 antagonists did not reverse adenine nucleotide-induced antinociception. These results suggest that antinociceptive effect of adenine nucleotides is mediated by adenosine.

  5. Using Markov chains of nucleotide sequences as a possible precursor to predict functional roles of human genome: a case study on inactive chromatin regions.

    Science.gov (United States)

    Lee, K-E; Lee, E-J; Park, H-S

    2016-08-30

    Recent advances in computational epigenetics have provided new opportunities to evaluate n-gram probabilistic language models. In this paper, we describe a systematic genome-wide approach for predicting functional roles in inactive chromatin regions by using a sequence-based Markovian chromatin map of the human genome. We demonstrate that Markov chains of sequences can be used as a precursor to predict functional roles in heterochromatin regions and provide an example comparing two publicly available chromatin annotations of large-scale epigenomics projects: ENCODE project consortium and Roadmap Epigenomics consortium.

  6. Detection of single nucleotide polymorphisms in p53 mutation hotspots and expression of mutant p53 in human cell lines using an enzyme-linked electrochemical assay

    Czech Academy of Sciences Publication Activity Database

    Horáková Brázdilová, Petra; Šimková, Eva; Vychodilová, Zdenka; Brázdová, Marie; Fojta, Miroslav

    2009-01-01

    Roč. 21, č. 15 (2009), s. 1723-1729 ISSN 1040-0397 R&D Projects: GA ČR(CZ) GA203/07/1195; GA AV ČR(CZ) IAA400040901; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : enzyme-linked electrochemical assay * SNP typing * p53 mutation Subject RIV: AQ - Safety, Health Protection, Human - Machine Impact factor: 2.630, year: 2009

  7. Selective base excision repair of DNA damage by the non-base-flipping DNA glycosylase AlkC

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Rongxin; Mullins, Elwood A.; Shen, Xing; #8208; Xing; Lay, Kori T.; Yuen, Philip K.; David, Sheila S.; Rokas, Antonis; Eichman, Brandt F. (UCD); (Vanderbilt)

    2017-10-20

    DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

  8. Adenine nucleotide depletion from endothelial cells exposed to xanthine oxidase

    International Nuclear Information System (INIS)

    Aalto, T.K.; Raivio, K.O.

    1990-01-01

    Hypoxia causes breakdown of cellular nucleotides, accumulation of hypoxanthine (HX), and conversion of xanthine dehydrogenase into xanthine oxidase (XO). Upon reoxygenation, the HX-XO reaction generates free radicals, one potential mechanism of tissue damage. Because endothelial cells contain XO and are exposed to circulating HX, they are a likely target for damage. We studied the effect of XO and/or HX at physiologically relevant concentrations on nucleotide metabolism of cultured endothelial cells from human umbilical veins. Cells were labeled with [14C]adenine and incubated for up to 6 h with HX, XO, or both, in the absence or presence of serum. Adenine nucleotides from cell extracts and nucleotide breakdown products (HX, xanthine, and urate) from the medium were separated and counted. HX alone had no effect. XO (80 mU/ml) alone caused a 70% (no serum) or 40% (with serum) fall in adenine nucleotides and an equivalent increase of xanthine and urate. The combination of HX and XO caused a 90% (no serum) or 70% (with serum) decrease in nucleotides, decrease in energy charge, and detachment of cells from the culture plate. Nucleotide depletion was not accounted for by proteolytic activity in the XO preparation. Albumin was only half as effective as serum in preventing nucleotide loss. Thus exogenous XO, in the presence of endogenous HX, triggers adenine nucleotide catabolism, but endogenous XO activity is too low to influence nucleotide levels even at high exogenous HX concentrations. Serum limits the catabolic effect of XO and thus protects cells from free radical damage

  9. A human repair gene ERCC5 is involved in group G xeroderma pigmentosum

    International Nuclear Information System (INIS)

    Shiomi, Tadahiro

    1994-01-01

    In E. coli, ultraviolet-induced DNA damage is removed by the coordinated action of UVR A, B, C, and D proteins (1). In Saccharomyces cerevisiae, more than ten genes have been reported to be involved in excision repair (2). The nucleotide excision repair pathway has been extensively studied in these organisms. To facilitate studying nucleotide excision repair in mammalian cells. Ultraviolet-sensitive rodent cell mutants have been isolated and classified into 11 complementation groups (9,10). The human nucleotide excision repair genes which complement the defects of the mutants have been designated as the ERCC (excision repair cross-complementing) genes; a number is added to refer to the particular rodent complementation group that is corrected by the gene. Recently, several human DNA repair genes have been cloned using rodent cell lines sensitive to ultraviolet. These include ERCC2 (3), ERCC3 (4), and ERCC6 (5), which correspond to the defective genes in the ultraviolet-sensitive human disorders xeroderma pigmentosum (XP) group D (6) and group B (4), and Cockayne's syndrome (CS) group B (7), respectively. The human excision repair gene ERCC5 was cloned after DNA-mediated gene transfer of human HeLa cell genomic DNA into the ultraviolet-sensitive mouse mutant XL216, a member of rodent complementation group 5 (11,12) and the gene was mapped on human chromosome 13q32.3-q33.1 by the replication R-banding fluorescence in situ hybridization method (13). The ERCC5 cDNA encodes a predicted 133 kDa nuclear protein that shares some homology with product of the yeast DNA repair gene RAD 2. Transfection with mouse ERCC5 cDNA restored normal levels of ultraviolet-resistance to XL216 cells. Microinjection of ERCC5 cDNA specifically restored the defect of XP group G cells (XP-G) as measured by unscheduled DNA synthesis (UDS), and XP-G cells stably transformed with ERCC5 cDNA showed nearly normal ultraviolet resistance. (J.P.N.)

  10. An affinity pull-down approach to identify the plant cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara Elizabeth; Meier, Stuart Kurt

    2013-01-01

    Cyclic nucleotides (CNs) are intracellular second messengers that play an important role in mediating physiological responses to environmental and developmental signals, in species ranging from bacteria to humans. In response to these signals, CNs are synthesized by nucleotidyl cyclases and then act by binding to and altering the activity of downstream target proteins known as cyclic nucleotide-binding proteins (CNBPs). A number of CNBPs have been identified across kingdoms including transcription factors, protein kinases, phosphodiesterases, and channels, all of which harbor conserved CN-binding domains. In plants however, few CNBPs have been identified as homology searches fail to return plant sequences with significant matches to known CNBPs. Recently, affinity pull-down techniques have been successfully used to identify CNBPs in animals and have provided new insights into CN signaling. The application of these techniques to plants has not yet been extensively explored and offers an alternative approach toward the unbiased discovery of novel CNBP candidates in plants. Here, an affinity pull-down technique for the identification of the plant CN interactome is presented. In summary, the method involves an extraction of plant proteins which is incubated with a CN-bait, followed by a series of increasingly stringent elutions that eliminates proteins in a sequential manner according to their affinity to the bait. The eluted and bait-bound proteins are separated by one-dimensional gel electrophoresis, excised, and digested with trypsin after which the resultant peptides are identified by mass spectrometry - techniques that are commonplace in proteomics experiments. The discovery of plant CNBPs promises to provide valuable insight into the mechanism of CN signal transduction in plants. © Springer Science+Business Media New York 2013.

  11. An affinity pull-down approach to identify the plant cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara Elizabeth

    2013-09-03

    Cyclic nucleotides (CNs) are intracellular second messengers that play an important role in mediating physiological responses to environmental and developmental signals, in species ranging from bacteria to humans. In response to these signals, CNs are synthesized by nucleotidyl cyclases and then act by binding to and altering the activity of downstream target proteins known as cyclic nucleotide-binding proteins (CNBPs). A number of CNBPs have been identified across kingdoms including transcription factors, protein kinases, phosphodiesterases, and channels, all of which harbor conserved CN-binding domains. In plants however, few CNBPs have been identified as homology searches fail to return plant sequences with significant matches to known CNBPs. Recently, affinity pull-down techniques have been successfully used to identify CNBPs in animals and have provided new insights into CN signaling. The application of these techniques to plants has not yet been extensively explored and offers an alternative approach toward the unbiased discovery of novel CNBP candidates in plants. Here, an affinity pull-down technique for the identification of the plant CN interactome is presented. In summary, the method involves an extraction of plant proteins which is incubated with a CN-bait, followed by a series of increasingly stringent elutions that eliminates proteins in a sequential manner according to their affinity to the bait. The eluted and bait-bound proteins are separated by one-dimensional gel electrophoresis, excised, and digested with trypsin after which the resultant peptides are identified by mass spectrometry - techniques that are commonplace in proteomics experiments. The discovery of plant CNBPs promises to provide valuable insight into the mechanism of CN signal transduction in plants. © Springer Science+Business Media New York 2013.

  12. Genome-wide association study identifies single-nucleotide polymorphism in KCNB1 associated with left ventricular mass in humans: The HyperGEN Study

    Directory of Open Access Journals (Sweden)

    Kraemer Rachel

    2009-05-01

    Full Text Available Abstract Background We conducted a genome-wide association study (GWAS and validation study for left ventricular (LV mass in the Family Blood Pressure Program – HyperGEN population. LV mass is a sensitive predictor of cardiovascular mortality and morbidity in all genders, races, and ages. Polymorphisms of candidate genes in diverse pathways have been associated with LV mass. However, subsequent studies have often failed to replicate these associations. Genome-wide association studies have unprecedented power to identify potential genes with modest effects on left LV mass. We describe here a GWAS for LV mass in Caucasians using the Affymetrix GeneChip Human Mapping 100 k Set. Cases (N = 101 and controls (N = 101 were selected from extreme tails of the LV mass index distribution from 906 individuals in the HyperGEN study. Eleven of 12 promising (Q Results Despite the relatively small sample, we identified 12 promising SNPs in the GWAS. Eleven SNPs were successfully genotyped in the validation study of 704 Caucasians and 1467 African Americans; 5 SNPs on chromosomes 5, 12, and 20 were significantly (P ≤ 0.05 associated with LV mass after correction for multiple testing. One SNP (rs756529 is intragenic within KCNB1, which is dephosphorylated by calcineurin, a previously reported candidate gene for LV hypertrophy within this population. Conclusion These findings suggest KCNB1 may be involved in the development of LV hypertrophy in humans.

  13. Isolation and characterization of human glycophorin A cDNAs using a synthetic oligonucleotide approach: nucleotide sequence, mRNA structure and regulation by 12-O-tetradecanoylphorbol 13-acetate (TPA)

    International Nuclear Information System (INIS)

    Siebert, P.D.; Fukuda, M.

    1986-01-01

    The authors have previously shown that treatment of human erythroleukemic K562 cells with the tumor-promoting phorbol ester, TPA, results in a diminished expression of glycophorin A at the level of protein biosynthesis and in vitro mRNA translation activity. To further examine the structure, relationships and expression of human glycophorins they have successfully isolated and sequenced several glycophorin A specific cDNA clones derived from K562 cells, by making extensive use of mixed and exact synthetic oligonucleotides as primers and radioactively labeled probes. The nucleotide sequence obtained from the largest glycophorin A cDNA suggests the presence of a hydrophobic leader-like peptide of at least 19 amino acids. Northern gel analysis using both whole cDNA-plasmid and synthetic oligonucleotide probes revealed the existence of multiple mRNAs, three of which they believe to be glycophorin A-specific, whereas a fourth and smaller mRNA appears to be glycophorin B-specific. Furthermore, the abundance of all four glycophorin mRNAs were found to be extensively reduced following treatment of K562 cells with TPA suggesting coordinate regulation, possibly at the level of gene transcription

  14. 29 CFR 779.264 - Excise taxes separately stated.

    Science.gov (United States)

    2010-07-01

    ... AS APPLIED TO RETAILERS OF GOODS OR SERVICES Employment to Which the Act May Apply; Enterprise Coverage Excise Taxes § 779.264 Excise taxes separately stated. A tax is separately stated where it clearly... 29 Labor 3 2010-07-01 2010-07-01 false Excise taxes separately stated. 779.264 Section 779.264...

  15. 75 FR 9359 - Drawback of Internal Revenue Excise Tax

    Science.gov (United States)

    2010-03-02

    ... Drawback of Internal Revenue Excise Tax AGENCY: Customs and Border Protection, Department of Homeland... substitution drawback claim for internal revenue excise tax paid on imported merchandise in situations where no excise tax was paid upon the substituted merchandise or where the substituted merchandise is the subject...

  16. Symptomatic pancreatic heterotopia treated by local excision.

    Science.gov (United States)

    De Friend, D J; Saa-Gandi, F W; Humphrey, C S; Foster, D N

    1991-01-01

    Non-ulcer dyspepsia is a continuing problem and in many cases a precise cause is never identified. We present five patients with an allegedly uncommon condition--pancreatic heterotopia. They were managed by local excision of the tumour and after a mean (range) follow up of 42 (9-80) months all remain free of the original symptoms. Images Figure 1 Figure 2 PMID:2013433

  17. Relativistic hydrodynamic evolutions with black hole excision

    International Nuclear Information System (INIS)

    Duez, Matthew D.; Shapiro, Stuart L.; Yo, H.-J.

    2004-01-01

    We present a numerical code designed to study astrophysical phenomena involving dynamical spacetimes containing black holes in the presence of relativistic hydrodynamic matter. We present evolutions of the collapse of a fluid star from the onset of collapse to the settling of the resulting black hole to a final stationary state. In order to evolve stably after the black hole forms, we excise a region inside the hole before a singularity is encountered. This excision region is introduced after the appearance of an apparent horizon, but while a significant amount of matter remains outside the hole. We test our code by evolving accurately a vacuum Schwarzschild black hole, a relativistic Bondi accretion flow onto a black hole, Oppenheimer-Snyder dust collapse, and the collapse of nonrotating and rotating stars. These systems are tracked reliably for hundreds of M following excision, where M is the mass of the black hole. We perform these tests both in axisymmetry and in full 3+1 dimensions. We then apply our code to study the effect of the stellar spin parameter J/M 2 on the final outcome of gravitational collapse of rapidly rotating n=1 polytropes. We find that a black hole forms only if J/M 2 2 >1, the collapsing star forms a torus which fragments into nonaxisymmetric clumps, capable of generating appreciable 'splash' gravitational radiation

  18. Pisiform excision for pisotriquetral instability and arthritis.

    Science.gov (United States)

    Campion, Heather; Goad, Andrea; Rayan, Ghazi; Porembski, Margaret

    2014-07-01

    To evaluate wrist strength and kinematics after pisiform excision and preservation of its soft tissue confluence for pisotriquetral instability and arthritis. We evaluated 12 patients, (14 wrists) subjectively and objectively an average of 7.5 years after pisiform excision. Three additional patients were interviewed by phone. Subjective evaluation included inquiry about pain and satisfaction with the treatment. Objective testing included measuring wrist flexion and extension range of motion, grip strength, and static and dynamic flexion and ulnar deviation strengths of the operative hand compared with the nonsurgical normal hand. Four patients had concomitant ulnar nerve decompression at the wrist. All patients were satisfied with the outcome. Wrist flexion averaged 99% and wrist extension averaged 95% of the nonsurgical hand. Mean grip strength of the operative hand was 90% of the nonsurgical hand. Mean static flexion strength of the operative hand was 94% of the nonsurgical hand, whereas mean dynamic flexion strength was 113%. Mean static ulnar deviation strength of the operative hand was 87% of the nonsurgical hand. The mean dynamic ulnar deviation strength of the operative hand was 103% of the nonsurgical hand. Soft tissue confluence-preserving pisiform excision relieved pain and retained wrist motion and static and dynamic strength. Associated ulnar nerve compression was a confounding factor that may have affected outcomes. Therapeutic IV. Copyright © 2014 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.

  19. Transvaginal sling excision: tips and tricks.

    Science.gov (United States)

    Clifton, Marisa M; Goldman, Howard B

    2017-01-01

    Complications of synthetic midurethral sling surgery include bladder outlet obstruction, mesh extrusion, and vaginal pain. A treatment of these complications is transvaginal mesh removal. The objectives of this video are to present cases of complications after sling placement and describe techniques to help with successful sling removal. Three patients are presented in this video. One experienced urinary hesitancy and was found to have bladder outlet obstruction on urodynamic study. The second patient presented to the clinic with diminished force of stream and significant dyspareunia. The last patient presented with mesh extrusion. After discussion of management options, all three patients wished to pursue transvaginal sling excision. All patients had successful removal of a portion of their synthetic midurethral sling. This video presents techniques to aide with dissection, mesh excision and prevention of further mesh complications. These include using an individualized surgical technique based on patient presentation and surgeon expertise, planning surgical incisions based on where mesh can be identified or palpated, using a cystoscope sheath or urethral dilator to identify any bladder outlet obstruction, and using a knife blade to identify mesh from surrounding tissue. Sling excision can be successfully performed with careful surgical technique and dissection.

  20. Frequency and significance of the novel single nucleotide missense polymorphism Val109Asp in the human gene encoding omentin in Caucasian patients with type 2 diabetes mellitus or chronic inflammatory bowel diseases

    Directory of Open Access Journals (Sweden)

    Buechler Christa

    2007-02-01

    Full Text Available Background The omental adipose tissue is pathogenetically involved in both type 2 diabetes mellitus (T2D and chronic inflammatory bowel diseases (IBD such as Ulcerative colitis (UC and Crohn's Disease (CD. Thus, adipokines secreted from omental adipose tissue might play an important role in these diseases. Omentin represents a new adipokine expressed in and secreted by omental adipose tissue. Therefore, it was the aim to investigate the putative role of a newly described sequence missense variation in the human omentin gene. Methods The Val109Asp single nucleotide miss-sense polymorphism and the His86His polymorphism in exon-4 of the omentin gene were newly identified by random sequencing. Only the miss-sense polymorphism was investigated further. Genotyping was performed by restriction fragment length polymorphism (RFLP analysis of amplified DNA fragments. Three different cohorts of well-characterized individuals were included in the study. 114 patients suffering from T2D, 190 patients suffering from IBD (128 with CD and 62 with UC and 276 non-diabetic healthy controls without any history for IBD were analyzed. Results The following allelic frequencies were determined: controls: Val-allele: 0.26, Asp-allele: 0.74; T2D: Val-allele: 0.3, Asp-allele: 0.7; IBD: Val-allel: 0.31, Asp-allele: 0.69. UC and CD patients did not differ in regard to the allelic frequency. Similarly, controls, T2D patients and IBD patients did not show significant differences in genotype distribution among each other. Disease manifestation and pattern of infestation were not related to genotype subgroups, neither in CD nor in UC. Furthermore, there was no significant association between genotype subgroups and anthropometric or laboratory parameters in T2D patients. Conclusion Based on sequence comparisons and homology searches, the amino acid position 109 is conserved in the omentin gene of humans, mice and chimpanzee but is not completely conserved between other omentin

  1. Evaluation of human epidermal growth factor receptor 2 (HER2) single nucleotide polymorphisms (SNPs) in normal and breast tumor tissues and their link with breast cancer prognostic factors.

    Science.gov (United States)

    Furrer, Daniela; Lemieux, Julie; Côté, Marc-André; Provencher, Louise; Laflamme, Christian; Barabé, Frédéric; Jacob, Simon; Michaud, Annick; Diorio, Caroline

    2016-12-01

    Amplification of the human epidermal growth factor receptor 2 (HER2) gene is associated with worse prognosis and decreased overall survival in breast cancer patients. The HER2 gene contains several polymorphisms; two of the best-characterized HER2 polymorphisms are Ile655Val and Ala1170Pro. The aim of this study was to evaluate the association between these two HER2 polymorphisms in normal breast and breast cancer tissues and known breast cancer prognostic factors in a retrospective cohort study of 73 women with non-metastatic HER2-positive breast cancer. HER2 polymorphisms were assessed in breast cancer tissue and normal breast tissue using TaqMan assay. Ala1170Pro polymorphism in normal breast tissue was associated with age at diagnosis (p = 0.007), tumor size (p = 0.004) and lymphovascular invasion (p = 0.06). Similar significant associations in cancer tissues were observed. No association between the Ile655Val polymorphism and prognostic factors were observed. However, we found significant differences in the distribution of Ile655Val (p = 0.03) and Ala1170Pro (p = 0.01) genotypes between normal breast and breast tumor tissues. This study demonstrates that only the Ala1170Pro polymorphism is associated with prognostic factors in HER2-positive breast cancer patients. Moreover, our results suggest that both HER2 polymorphisms could play a significant role in carcinogenesis in non-metastatic HER2-positive breast cancer women. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Multidirectional Vector Excision Leads to Better Outcomes than Traditional Elliptical Excision of Facial Congenital Melanocytic Nevus

    Directory of Open Access Journals (Sweden)

    Seung Il Oh

    2013-09-01

    Full Text Available Background The elliptical excision is the standard method of removing benign skin lesions,such as congenital melanocytic nevi. This technique allows for primary closure, with little to nodog-ear deformity, but may sacrifice normal tissue adjacent to the lesion, resulting in scarswhich are unnecessarily long. This study was designed to compare the predicted results ofelliptical excision with those resulting from our excision technique.Methods Eighty-two patients with congenital melanocytic nevus on the face were prospectivelystudied. Each lesion was examined and an optimal ellipse was designed and marked onthe skin. After an incision on one side of the nevus margin, subcutaneous undermining wasperformed in the appropriate direction. The skin flap was pulled up and approximated alongseveral vectors to minimize the occurrence of dog-ear deformity.Results Overall, the final wound length was 21.1% shorter than that achieved by ellipticalexcision. Only 8.5% of the patients required dog-ear repair. There was no significant distortionof critical facial structures. All of the scars were deemed aesthetically acceptable based ontheir Patient and Observer Scar Assessment Scale scores.Conclusions When compared to elliptical excision, our technique appears to minimize dogeardeformity and decrease the final wound length. This technique should be considered analternative method for excision of facial nevi.

  3. Excision of thymine dimers from specifically incised DNA by extracts of xeroderma pigmentosum cells

    Energy Technology Data Exchange (ETDEWEB)

    Cook, K; Friedberg, E C; Slor, H; Cleaver, J E

    1975-07-17

    DNA repair defects as exhibited in fibroblasts from patients with xeroderma pigmentosa were studied. Five complementation groups for excision-repair defects were examined to test the hypothesis that a defective endonuclease or exonuclease may be the cause. No evidence was found to indicate that the enzyme activity functions in dimer excision. Since ultraviolet irradiated E. coli DNA incised with an endonuclease purified from phage-infected cells were used, it is possible that other factors may be involved in human UV endonuclease action. (JWP)

  4. Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release

    DEFF Research Database (Denmark)

    Leonardsen, L; DeClue, J E; Lybaek, H

    1996-01-01

    The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino ac...... and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive....

  5. Statistical properties and fractals of nucleotide clusters in DNA sequences

    International Nuclear Information System (INIS)

    Sun Tingting; Zhang Linxi; Chen Jin; Jiang Zhouting

    2004-01-01

    Statistical properties of nucleotide clusters in DNA sequences and their fractals are investigated in this paper. The average size of nucleotide clusters in non-coding sequence is larger than that in coding sequence. We investigate the cluster-size distribution P(S) for human chromosomes 21 and 22, and the results are different from previous works. The cluster-size distribution P(S 1 +S 2 ) with the total size of sequential Pu-cluster and Py-cluster S 1 +S 2 is studied. We observe that P(S 1 +S 2 ) follows an exponential decay both in coding and non-coding sequences. However, we get different results for human chromosomes 21 and 22. The probability distribution P(S 1 ,S 2 ) of nucleotide clusters with the size of sequential Pu-cluster and Py-cluster S 1 and S 2 respectively, is also examined. In the meantime, some of the linear correlations are obtained in the double logarithmic plots of the fluctuation F(l) versus nucleotide cluster distance l along the DNA chain. The power spectrums of nucleotide clusters are also discussed, and it is concluded that the curves are flat and hardly changed and the 1/3 frequency is neither observed in coding sequence nor in non-coding sequence. These investigations can provide some insights into the nucleotide clusters of DNA sequences

  6. Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

    Science.gov (United States)

    Wang, Sihua; Ding, Mingcui; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

    2017-09-01

    It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase ( hTERT ) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme Msp I for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency. © 2017 by the Association of Clinical Scientists, Inc.

  7. Value of histopathologic analysis of subcutis excisions by general practitioners

    Directory of Open Access Journals (Sweden)

    Verweij Wim

    2007-01-01

    Full Text Available Abstract Background Only around 60% of skin lesions excised by GPs are referred to a pathologist. Clinical diagnoses of skin excisions by GPs may not be very accurate. Subcutis excisions are rarely done by GPs, and there is hence little information in the literature on the histopathological yield of subcutis excisions by GPs with regard to malignancies. The aim of this study was to evaluate the yield of histopathological investigation of a relatively large group of subcutis excisions by GPs, with special emphasis on discrepancies between clinical and histopathological diagnoses of malignancy. Methods We investigated a series of 90 subcutis excisions, which was derived from a database of consecutive GP submissions from the years 1999–2000 where in the same time period 4595 skin excisions were performed by the same group of GPs. This underlines the apparent reluctance of GPs to perform subcutis excisions. Results The final diagnosis was benign in 88 cases (97.8% and malignant in 2 cases (2.2%. Seven cases had no clinical diagnosis, all of which were benign. Of the 83 clinically benign cases, 81 (97.6% were indeed benign and 2 (2.4% were malignant: one Merkel cell carcinoma and one dermatofibrosarcoma protuberans. The former was clinically thought to be a lipoma, and the latter a trichilemmal cyst. The dermatofibrosarcoma protuberans presented at the age of 27, and the Merkel cell carcinoma at the age of 60. Both were incompletely removed and required re-excision by a surgical oncologist. Conclusion Histopathological investigation of subcutis excisions by GPs yields unexpected and rare malignancies in about 2% of cases that may initially be excised inadequately. Based on these data, and because of the relatively rareness of these type of excisions, it could be argued that it may be worthwhile to have all subcutis excisions by GPs routinely investigated by histopathology.

  8. Role of DNA lesions and DNA repair in mutagenesis by carcinogens in diploid human fibroblasts

    International Nuclear Information System (INIS)

    Maher, V.M.; McCormick, J.J.

    1986-01-01

    The authors investigated the cytotoxicity, mutagenicity, and transforming activity of carcinogens and radiation in diploid human fibroblasts, using cells which differ in their DNA repair capacity. The results indicate that cell killing and induction of mutations are correlated with the number of specific lesions remaining unrepaired in the cells at a particular time posttreatment. DNA excision repair acts to eliminate potentially cytotoxic and mutagenic (and transforming) damage from DNA before these can be converted into permanent cellular effects. Normal human fibroblasts were derived from skin biopsies or circumcision material. Skin fibroblasts from xeroderma pigmentosum (XP) patients provided cells deficient in nucleotide excision repair of pyrimidine dimers or DNA adducts formed by bulky ring structures. Cytotoxicity was determined from loss of ability to form a colony. The genetic marker used was resistance to 6-thioguanine (TG). Transformation was measured by determining the frequency of anchorage-independent cells

  9. Alar base reduction: the boomerang-shaped excision.

    Science.gov (United States)

    Foda, Hossam M T

    2011-04-01

    A boomerang-shaped alar base excision is described to narrow the nasal base and correct the excessive alar flare. The boomerang excision combined the external alar wedge resection with an internal vestibular floor excision. The internal excision was inclined 30 to 45 degrees laterally to form the inner limb of the boomerang. The study included 46 patients presenting with wide nasal base and excessive alar flaring. All cases were followed for a mean period of 18 months (range, 8 to 36 months). The laterally oriented vestibular floor excision allowed for maximum preservation of the natural curvature of the alar rim where it meets the nostril floor and upon its closure resulted in a considerable medialization of alar lobule, which significantly reduced the amount of alar flare and the amount of external alar excision needed. This external alar excision measured, on average, 3.8 mm (range, 2 to 8 mm), which is significantly less than that needed when a standard vertical internal excision was used ( P boomerang alar base excision proved to be a safe and effective technique for narrowing the nasal base and elimination of the excessive flaring and resulted in a natural, well-proportioned nasal base with no obvious scarring. © Thieme Medical Publishers.

  10. Surgical excision of eroded mesh after prior abdominal sacrocolpopexy.

    Science.gov (United States)

    South, Mary M T; Foster, Raymond T; Webster, George D; Weidner, Alison C; Amundsen, Cindy L

    2007-12-01

    We previously described an endoscopic-assisted transvaginal mesh excision technique. This study compares surgical outcomes after transvaginal mesh excision vs endoscopic-assisted transvaginal mesh excision. In addition, we reviewed our postoperative outcomes with excision via laparotomy. This was an inclusive retrospective analysis of patients presenting to our institution from 1997 to 2006 for surgical management of vaginal erosion of permanent mesh after sacrocolpopexy. Three techniques were utilized: transvaginal, endoscopic-assisted transvaginal, and laparotomy. For the patients undergoing transvaginal excision, data recorded included number and type of excisions performed, number of prior excisions performed at outside facilities, intraoperative and postoperative complications (including blood transfusions, pelvic abscess, or bowel complications), use of postoperative antibiotics, persistent symptoms of vaginal bleeding and discharge at follow-up, and demographic characteristics. The intraoperative and postoperative complications and the postoperative symptoms were recorded for the laparotomy cases. Thirty-one patients underwent transvaginal mesh excision during this time period: 17 endoscopic-assisted transvaginal and 14 transvaginal without endoscope assistance. In addition, a total of 7 patients underwent abdominal excision via laparotomy. Comparison of the 2 vaginal methods revealed no difference in the demographics or success rate, with success defined as no symptoms at follow-up. Endoscopic-assisted transvaginal excision was successful in 7 of 17 patients and transvaginal without endoscopic assistance in 9 of 13 patients (1 patient excluded for lack of follow-up data) for a total vaginal success rate of 53.3%. No intraoperative and only minor postoperative complications occurred with either vaginal method. Three patients underwent 3 vaginal attempts to achieve complete symptom resolution. The average follow-up time for the entire vaginal group was 14

  11. DNA repair capacity and rate of excision repair in UV-irradiated mammalian cells

    International Nuclear Information System (INIS)

    Inoue, Masao; Takebe, Hiraku.

    1978-01-01

    Repair capacities of five mammalian cell strains were measured by colony-forming ability, HCR of UV-irradiated virus, UDS, pyrimidine dimer excision, and semi-conservative DNA replication. Colony-forming ability of UV-irradiated cells was high for human amnion FL cells and mouse L cells, slightly low for African green monkey CV-1 cells, and extremely low for xeroderma pigmentosum cells. HCR of UV-irradiated Herpes simplex virus was high in CV-1 cells, FL and normal human fibroblast cells, low in both XP and L cells. The amount of UDS was high in FL and normal human fibroblast cells, considerably low in CV-1 cells, and essentially no UDS was observed in XP cells. Rate of UDS after UV-irradiation was slower for CV-1 cells than FL and human fibroblast cells. Rate of the excision of thymine-containing dimers from the acid-insoluble fraction during post-irradiation incubation of the cells was rapid in FL and normal human cells and slow in CV-1 cells, and no excision took place in XP cells. Semi-conservative DNA synthesis was reduced after UV-irradiation in all cell lines, but subsequently recovered in FL, normal human and CV-1 cells. The onset of recovery was 4 h after UV-irradiation for FL and normal human cells, but about 6 h for CV-1 cells. The apparent intermediate repair of CV-1 cells except for HCR may be related to the slow rate of excision repair. ''Patch and cut'' model is more favorable than ''cut and patch'' model to elucidate these results. (auth.)

  12. Efficiency and Fidelity of Human DNA Polymerases λ and β during Gap-Filling DNA Synthesis

    Science.gov (United States)

    Brown, Jessica A.; Pack, Lindsey R.; Sanman, Laura E.; Suo, Zucai

    2010-01-01

    The base excision repair (BER) pathway coordinates the replacement of 1 to 10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1- to 10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5′-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER. PMID:20961817

  13. Trans-sphenoidal excision of craniopharyngiomas.

    Directory of Open Access Journals (Sweden)

    Nagpal R

    1991-04-01

    Full Text Available Craniopharyngiomas have been by and large excised by the transcranial route. Since 1982, 11 patients have been operated by the traus-sphensidal route in the department. The clinical features with which they were presented, diagnostic investigations, details of surgical procedures and follow-up analysis is being presented here. A retrospective study of radiological investigation was done to determine the features that help decide the choice of surgical approach to these lesions. Only lesions that were primarily intrasellar, cystic and those that expanded the sella could be treated by the trans-sphenoidal route. Associated suprasellar extensions could also be removed. Predominantly calcified or firm, fleshy tumours lent themselves poorly to removal by the trans-sphenoidal route.

  14. Piezosurgery for Excision of Large Osteoid Osteoma.

    Science.gov (United States)

    Gadre, Pushkar; Singh, Divya; Gadre, Kiran; Khan, Imran

    2016-10-01

    Osteoid osteoma, a rare benign osteoblastic tumor first described by Jaffe in 1935, is characterized as a small but painful lesion that mostly affects younger people. Usually benign and harmless, osteomas are removed for pain or esthetic reasons.Piezoelectric surgery is also increasingly being used effectively in major and minor osseous oral and maxillofacial surgeries, in delicate areas. It is used regularly for various procedures, including sinus lift procedures, bone graft harvesting, osteogenic distraction, ridge expansion, inferior alveolar nerve decompression and lateralization, cyst removal, dental extraction, and impacted tooth removal.The following report presents a patient of intraoral excision of a large osteoid osteoma from lingual aspect of mandibular lower border in the body region using piezoelectric surgery.

  15. Recovery of DNA synthesis after ultraviolet irradiation of xeroderma pigmentosum cells depends on excision repair and is blocked by caffeine

    International Nuclear Information System (INIS)

    Park, S.D.; Cleaver, J.E.

    1979-01-01

    Normal human and xeroderma pigmentosum (XP, excision-defective group A) cells (both SV40-transformed) pulse-labeled with [ 3 H] thymidine at various times after irradiation with ultraviolet light showed a decline and recovery of both the molecular weights of newly synthesized DNA and the rated of synthesis per cell. At the same ultraviolet dose, both molecular weights and rates of synthesis were inhibited more in XP than in normal cells. This indicates that excision repair plays a role in minimizing the inhibition of chain growth, possibly by excision of dimers ahead of the growing point. The ability to synthesize normal-sized DNA recovered more rapidly than rates of synthesis in normal cells, but both parameters recovered in phase in XP cells. During recovery in normal cells there are therefore fewer actively replicating clusters of replicons because the single-strand breaks involved in the excision of dimers inhibit replicon initiation. XP cells have few excision repair events and therefore fewer breaks to interfere with initiation, but chain growth is blocked by unexcised dimers. In both cell types recovery of the ability to synthesize normal-sized DNA was prevented by growing cells in caffeine after irradiation, possibly because of competition between the DNA binding properties of caffeine and replication proteins. These observations imply that excision repair and semiconservative replication interact strongly in irradiated cells to produce a complex spectrum of changes in DNA replication which may be confused with parts of alternative systems such as post-replication repair. (author)

  16. Analysis of single nucleotide polymorphisms in case-control studies.

    Science.gov (United States)

    Li, Yonghong; Shiffman, Dov; Oberbauer, Rainer

    2011-01-01

    Single nucleotide polymorphisms (SNPs) are the most common type of genetic variants in the human genome. SNPs are known to modify susceptibility to complex diseases. We describe and discuss methods used to identify SNPs associated with disease in case-control studies. An outline on study population selection, sample collection and genotyping platforms is presented, complemented by SNP selection, data preprocessing and analysis.

  17. Effects of Dietary Nucleotides on Growth Rate and Disease ...

    African Journals Online (AJOL)

    Nucleotides are low molecular weight biological compounds, which are ... nutrition and disease aspects of crustaceans (Overton and Bland 1981 .... additives on growth and disease resistance. Effects of ... metabolically active cells during stressful conditions ... in humans supplemented with Uracyl, which resulted in optimal ...

  18. Excision technique in constrained formulations of Einstein equations: collapse scenario

    International Nuclear Information System (INIS)

    Cordero-Carrión, I; Vasset, N; Novak, J; Jaramillo, J L

    2015-01-01

    We present a new excision technique used in constrained formulations of Einstein equations to deal with black hole in numerical simulations. We show the applicability of this scheme in several scenarios. In particular, we present the dynamical evolution of the collapse of a neutron star to a black hole, using the CoCoNuT code and this excision technique. (paper)

  19. Revenue and Health Impacts of Restructuring Tobacco Excise Tax ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Revenue and Health Impacts of Restructuring Tobacco Excise Tax in the Philippines. A proposed law in the Philippines to increase the excise tax on tobacco by 215% will likely have implications for tobacco control and consumption, and public health, not just for that country but for the region. Although half of deaths due to ...

  20. Circumareolar Incision‑subdermal Tunneling Dissection for Excision ...

    African Journals Online (AJOL)

    Excision of multiple fibroadenomas (MF) in separate breast quadrants presents difficulties of number and location of incision(s) and extent of tissue dissection and may be associated with more complications and poorer cosmetic outcome. This is a report of excision of MF in multiple quadrants of the breast using a ...

  1. Effects of burn wound excision on bacterial colonization and invasion

    NARCIS (Netherlands)

    Barret, JP; Herndon, DN

    Rates of survival after thermal injury have improved in the past two decades, and rates of wound infections and sepsis have decreased during the same period. Early excision has been advocated as one of the major factors, but its safety and efficacy and the exact timing of burn excision are still

  2. P element excision in drosophila melanogaster and related drosophilids

    Science.gov (United States)

    The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species, Drosophila melanogaster, D. virilis, and Chymomyza procnemis. A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlik...

  3. 76 FR 52862 - Time for Payment of Certain Excise Taxes, and Quarterly Excise Tax Payments for Small Alcohol...

    Science.gov (United States)

    2011-08-24

    ... 40 Cigars and cigarettes, Claims, Electronic fund transfers, Excise taxes, Labeling, Packaging and... that are not required to pay taxes through electronic funds transfer (EFT), this first payment period..., Electronic funds transfers, Excise taxes, Exports, Food additives, Fruit juices, Labeling, Liquors, Packaging...

  4. Mitochondria as determinant of nucleotide pools and chromosomal stability

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Munch-Petersen, Birgitte; Stevnsner, Tinna

    2007-01-01

    Mitochondrial function plays an important role in multiple human diseases and mutations in the mitochondrial genome have been detected in nearly every type of cancer investigated to date. However, the mechanism underlying the interrelation is unknown. We used human cell lines depleted of mitochon...... mitochondrial activity. Our results suggest that mitochondria are central players in maintaining genomic stability and in controlling essential nuclear processes such as upholding a balanced supply of nucleotides....

  5. Exonuclease of human DNA polymerase gamma disengages its strand displacement function.

    Science.gov (United States)

    He, Quan; Shumate, Christie K; White, Mark A; Molineux, Ian J; Yin, Y Whitney

    2013-11-01

    Pol γ, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo(-) (3'-5' exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol γA alone, even when exo(-), is unable to synthesize by strand displacement, making this the only known reaction of Pol γ holoenzyme that has an absolute requirement for the accessory subunit Pol γB. © 2013. Published by Elsevier B.V.

  6. Xeroderma pigmentosum and other diseases of human premature aging and DNA repair: Molecules to patients

    Science.gov (United States)

    Niedernhofer, Laura J.; Bohr, Vilhelm A.; Sander, Miriam; Kraemer, Kenneth H.

    2012-01-01

    A workshop1 to share, consider and discuss the latest developments in understanding xeroderma pigmentosum and other human diseases caused by defects in nucleotide excision repair (NER) of DNA damage was held on September 21–24, 2010 in Virginia. It was attended by approximately 100 researchers and clinicians, as well as several patients and representatives of patient support groups. This was the third in a series of workshops with similar design and goals: to emphasize discussion and interaction among participants as well as open exchange of information and ideas. The participation of patients, their parents and physicians was an important feature of this and the preceding two workshops. Topics discussed included the natural history and clinical features of the diseases, clinical and laboratory diagnosis of these rare diseases, therapeutic strategies, mouse models of neurodegeneration, molecular analysis of accelerated aging, impact of transcriptional defects and mitochondrial dysfunction on neurodegeneration, and biochemical insights into mechanisms of NER and base excision repair. PMID:21708183

  7. Diode Laser Excision of Oral Benign Lesions.

    Science.gov (United States)

    Mathur, Ena; Sareen, Mohit; Dhaka, Payal; Baghla, Pallavi

    2015-01-01

    Lasers have made tremendous progress in the field of dentistry and have turned out to be crucial in oral surgery as collateral approach for soft tissue surgery. This rapid progress can be attributed to the fact that lasers allow efficient execution of soft tissue procedures with excellent hemostasis and field visibility. When matched to scalpel, electrocautery or high frequency devices, lasers offer maximum postoperative patient comfort. Four patients agreed to undergo surgical removal of benign lesions of the oral cavity. 810 nm diode lasers were used in continuous wave mode for excisional biopsy. The specimens were sent for histopathological examination and patients were assessed on intraoperative and postoperative complications. Diode laser surgery was rapid, bloodless and well accepted by patients and led to complete resolution of the lesions. The excised specimen proved adequate for histopathological examination. Hemostasis was achieved immediately after the procedure with minimal postoperative problems, discomfort and scarring. We conclude that diode lasers are rapidly becoming the standard of care in contemporary dental practice and can be employed in procedures requiring excisional biopsy of oral soft tissue lesions with minimal problems in histopathological diagnosis.

  8. Lingual Thyroid Excision with Transoral Robotic Surgery

    Directory of Open Access Journals (Sweden)

    Elif Ersoy Callıoglu

    2015-01-01

    Full Text Available Ectopic thyroid gland may be detected at any place between foramen caecaum and normal thyroid localization due to inadequacy of the embryological migration of the thyroid gland. It has a prevalence varying between 1/10.000 and 1/100000 in the community. Usually follow-up without treatment is preferred except for obstructive symptoms, bleeding, and suspicion of malignity. Main symptoms are dysphagia, dysphonia, bleeding, dyspnea, and obstructive sleep apnea. In symptomatic cases, the first described method in surgical treatment is open approach since it is a region difficult to have access to. However, this approach has an increased risk of morbidity and postoperative complications. Transoral robotic surgery, which is a minimally invasive surgical procedure, has advantages such as larger three-dimensional point of view and ease of manipulation due to robotic instruments. In this report, a case at the age of 49 who presented to our clinic with obstructive symptoms increasing within the last year and was found to have lingual thyroid and underwent excision of ectopic thyroid tissue by da Vinci surgical system is presented.

  9. Nasal encephalocele: endoscopic excision with anesthetic consideration.

    Science.gov (United States)

    Abdel-Aziz, Mosaad; El-Bosraty, Hussam; Qotb, Mohamed; El-Hamamsy, Mostafa; El-Sonbaty, Mohamed; Abdel-Badie, Hazem; Zynabdeen, Mustapha

    2010-08-01

    Nasal encephalocele may presents as a nasal mass, its treatment is surgical and it should be done early in life. When removal is indicated, there are multiple surgical approaches; including lateral rhinotomy, a transnasal approach and a coronal flap approach. However, the treatment of a basal intranasal encephalocele using transnasal endoscopic approach could obviates the possible morbidity associated with other approaches. The aim of this study was to evaluate the efficacy of endoscopic removal of intranasal encephalocele, also to document the role of anesthetist in the operative and postoperative periods. Nine cases with nasal encephalocele were included in this study; CT and/or MRI were used in their examination. The lesions were removed via transnasal endoscopic approach. Preoperative evaluation, intervention and postoperative follow-up were presented with discussion of anesthesia used for those children. The lesions of all patients were removed successfully with no recurrence through the follow-up period of at least 21 months. No cases showed morbidity or mortality intra- or post-operatively. Endoscopic excision of intranasal encephalocele is an effective method with high success rate. Anesthetist plays an important role in the operative and postoperative period, even during the endoscopic follow up; sedation of the children is usually needed. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

  10. Uptake of benzyladenine by excised watermelon cotyledons.

    Science.gov (United States)

    Lampugnani, M G; Fantelli, R; Longo, G P; Longo, C P; Rossi, G

    1981-07-01

    The uptake of 8-[(14)C]N(6)-benzyladenine (BA) was studied in excised watermelon (Citrullus vulgaris Schrad.) cotyledons 24 hours after the start of imbibition. The passive nature of this uptake is suggested by the following evidence: (a) no sign of saturation on increasing external concentration of BA; (b) no decrease in uptake under conditions that inhibit ATP synthesis; (c) no change in amount of radioactivity absorbed when cotyledons are frozen and thawed before the uptake test. About two-thirds of the radioactivity taken up is released after 12 hours of washing. If the washing is performed at 2 C very little radioactivity is released.There seems to be a correlation between the level of radioactivity (i.e. of BA + derivatives) present in the cotyledons and the magnitude of hormonal responses that are observed four days after uptake. This relationship holds regardless of whether a given level of radioactivity has been reached after a short period of uptake or after a long period of uptake followed by washing.

  11. The Strategy of Excise Taxation of Tobacco Products in Ukraine

    Directory of Open Access Journals (Sweden)

    Pasichnyi Mykola D.

    2017-06-01

    Full Text Available The article is aimed at disclosing and improving approaches to the development of a strategy of excise tax policy in Ukraine, taking into account the foreign experience of harmonizing tax legislation in this sphere. An analysis of the implementation of the EU directives on the regulation of the minimum excise tax liability for the payment of excise taxes on tobacco products in the countries with transformational economies has been carried out. It has been found that, in cases of excessive tax pressure, the equilibrium of the market is disrupted, its shadow component is growing, and the overall economic efficiency level decreases. It has been determined that for the period of 2007-2016 the availability index for cigarettes in Ukraine decreased 2,3 times, which in some way demotivated their consumption. However, the change in the approach of calculation of ad valorem rate for the excise tax and introduction of the excise tax on the sub-excise goods sold by the retailers led to manipulative actions by the major actors in the market concerning the price of cigarettes, which impacted both the increase in the availability of cigarettes in 2016 and the decline in budget revenues. Regulation of the minimum excise duty is the most effective instrument of fiscal policy to achieve goals in the area of limitation of smoking.

  12. Nasal base narrowing: the combined alar base excision technique.

    Science.gov (United States)

    Foda, Hossam M T

    2007-01-01

    To evaluate the role of the combined alar base excision technique in narrowing the nasal base and correcting excessive alar flare. The study included 60 cases presenting with a wide nasal base and excessive alar flaring. The surgical procedure combined an external alar wedge resection with an internal vestibular floor excision. All cases were followed up for a mean of 32 (range, 12-144) months. Nasal tip modification and correction of any preexisting caudal septal deformities were always completed before the nasal base narrowing. The mean width of the external alar wedge excised was 7.2 (range, 4-11) mm, whereas the mean width of the sill excision was 3.1 (range, 2-7) mm. Completing the internal excision first resulted in a more conservative external resection, thus avoiding any blunting of the alar-facial crease. No cases of postoperative bleeding, infection, or keloid formation were encountered, and the external alar wedge excision healed with an inconspicuous scar that was well hidden in the depth of the alar-facial crease. Finally, the risk of notching of the alar rim, which can occur at the junction of the external and internal excisions, was significantly reduced by adopting a 2-layered closure of the vestibular floor (P = .01). The combined alar base excision resulted in effective narrowing of the nasal base with elimination of excessive alar flare. Commonly feared complications, such as blunting of the alar-facial crease or notching of the alar rim, were avoided by using simple modifications in the technique of excision and closure.

  13. Laparoscopic excision of a newborn rectal duplication cyst.

    Science.gov (United States)

    Hartin, Charles W; Lau, Stanley T; Escobar, Mauricio A; Glick, Philip L

    2008-08-01

    Congenital rectal duplication cyst is a rare entity treated with surgical excision. Without treatment, a rectal duplication cyst may cause a variety of complications, most notably, transforming into a malignancy. We report on a 7-week-old girl who was found to have a rectal duplication cyst. The rectal duplication cyst was successfully excised laparoscopically. Rectal duplication cysts are rare alimentary tract anomalies generally discovered during childhood. Complications include symptoms arising from the cyst and the possibility of malignant degeneration. They are typically managed by surgical excision.

  14. Quantitative imaging of excised osteoarthritic cartilage using spectral CT

    Energy Technology Data Exchange (ETDEWEB)

    Rajendran, Kishore; Bateman, Christopher J.; Younis, Raja Aamir; De Ruiter, Niels J.A.; Ramyar, Mohsen; Anderson, Nigel G. [University of Otago - Christchurch, Department of Radiology, Christchurch (New Zealand); Loebker, Caroline [University of Otago, Christchurch Regenerative Medicine and Tissue Engineering Group, Department of Orthopaedic Surgery and Musculoskeletal Medicine, Christchurch (New Zealand); University of Twente, Department of Developmental BioEngineering, Enschede (Netherlands); Schon, Benjamin S.; Hooper, Gary J.; Woodfield, Tim B.F. [University of Otago, Christchurch Regenerative Medicine and Tissue Engineering Group, Department of Orthopaedic Surgery and Musculoskeletal Medicine, Christchurch (New Zealand); Chernoglazov, Alex I. [University of Canterbury, Human Interface Technology Laboratory New Zealand, Christchurch (New Zealand); Butler, Anthony P.H. [University of Otago - Christchurch, Department of Radiology, Christchurch (New Zealand); European Organisation for Nuclear Research (CERN), Geneva (Switzerland); MARS Bioimaging, Christchurch (New Zealand)

    2017-01-15

    To quantify iodine uptake in articular cartilage as a marker of glycosaminoglycan (GAG) content using multi-energy spectral CT. We incubated a 25-mm strip of excised osteoarthritic human tibial plateau in 50 % ionic iodine contrast and imaged it using a small-animal spectral scanner with a cadmium telluride photon-processing detector to quantify the iodine through the thickness of the articular cartilage. We imaged both spectroscopic phantoms and osteoarthritic tibial plateau samples. The iodine distribution as an inverse marker of GAG content was presented in the form of 2D and 3D images after applying a basis material decomposition technique to separate iodine in cartilage from bone. We compared this result with a histological section stained for GAG. The iodine in cartilage could be distinguished from subchondral bone and quantified using multi-energy CT. The articular cartilage showed variation in iodine concentration throughout its thickness which appeared to be inversely related to GAG distribution observed in histological sections. Multi-energy CT can quantify ionic iodine contrast (as a marker of GAG content) within articular cartilage and distinguish it from bone by exploiting the energy-specific attenuation profiles of the associated materials. (orig.)

  15. Technique for laparoscopic autonomic nerve preserving total mesorectal excision.

    Science.gov (United States)

    Breukink, S O; Pierie, J P E N; Hoff, C; Wiggers, T; Meijerink, W J H J

    2006-05-01

    With the introduction of total mesorectal excision (TME) for treatment of rectal cancer, the prognosis of patients with rectal cancer is improved. With this better prognosis, there is a growing awareness about the quality of life of patients after rectal carcinoma. Laparoscopic total mesorectal excision (LTME) for rectal cancer offers several advantages in comparison with open total mesorectal excision (OTME), including greater patient comfort and an earlier return to daily activities while preserving the oncologic radicality of the procedure. Moreover, laparoscopy allows good exposure of the pelvic cavity because of magnification and good illumination. The laparoscope seems to facilitate pelvic dissection including identification and preservation of critical structures such as the autonomic nervous system. The technique for laparoscopic autonomic nerve preserving total mesorectal excision is reported. A three- or four-port technique is used. Vascular ligation, sharp mesorectal dissection and identification and preservation of the autonomic pelvic nerves are described.

  16. Development of excise on automotive fuels in the neighbouring countries

    International Nuclear Information System (INIS)

    Bleijenberg, A.N.; Janse, P.

    1994-04-01

    The political options for the Netherlands to implement the pricing policy for road traffic by means of an increase of excise, as formulated in the Second Transport Structure Plan (SVV-2), are limited by the excise level in neighbouring countries. Therefore, a study on the title subject has been carried out. First, an overview is given of the historical development of sales prices since 1980 for Belgium, Germany, Luxembourg and France with special attention for the large price differences. Next, insight into the effects of a price difference at the borders is given. Subsequently the development in the policy of the European Union with regard to harmonization of excise tariffs is discussed. Environmental organizations in Belgium and Luxembourg were contacted to learn about the expected development of excises in those countries. 5 figs., 6 tabs., 4 appendices, 10 refs

  17. Impacts of restructuring tabacco excise tax in the Philippines

    International Development Research Centre (IDRC) Digital Library (Canada)

    A recently approved law restructuring tobacco taxes in the. Philippines includes a steep increase in the excise tax (the tax paid by producers which ... part of Canada's International Development Research Centre. (IDRC), a Canadian Crown ...

  18. Comparison between preoperative biopsy and post-excision ...

    African Journals Online (AJOL)

    Comparison between preoperative biopsy and post-excision histology results in sarcoma: Experience at Chris Hani Baragwanath Academic Hospital, Johannesburg, South Africa. KG Panda, MJ Hale, D Kruger, TE Luvhengo ...

  19. Cyclic nucleotide specific phosphodiesterases of Leishmania major

    Directory of Open Access Journals (Sweden)

    Linder Markus

    2006-03-01

    Full Text Available Abstract Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range

  20. Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time

    OpenAIRE

    Kong, Muwen; Beckwitt, Emily C.; Springall, Luke; Kad, Neil M.; Van Houten, Bennett

    2017-01-01

    Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesion...

  1. Polymorphisms in RAI and in genes of nucleotide and base excision repair are not associated with risk of testicular cancer.

    Science.gov (United States)

    Laska, Magdalena J; Nexø, Bjørn A; Vistisen, Kirsten; Poulsen, Henrik Enghusen; Loft, Steffen; Vogel, Ulla

    2005-07-28

    Testicular cancer has been suggested to be primed in utero and there is familiar occurrence, particularly brothers and sons of men with testicular cancer have increased risk. Although no specific causative genotoxic agents have been identified, variations in DNA repair capacity could be associated with the risk of testicular cancer. A case-control study of 184 testicular cancer cases and 194 population-based controls living in the Copenhagen Greater Area in Denmark was performed. We found that neither polymorphisms in several DNA repair genes nor alleles of several polymorphisms in the chromosomal of region 19q13.2-3, encompassing the genes ASE, ERCC1, RAI and XPD, were associated with risk of testicular cancer in Danish patients. This is in contrast to other cancers, where we reported strong associations between polymorphisms in ERCC1, ASE and RAI and occurrence of basal cell carcinoma, breast cancer and lung. To our knowledge this is the first study of DNA repair gene polymorphisms and risk of testicular cancer.

  2. Genotypic and antimicrobial characterisation of Propionibacterium acnes isolates from surgically excised lumbar disc herniations

    DEFF Research Database (Denmark)

    Rollason, Jess; McDowell, Andrew; Albert, Hanne B

    2013-01-01

    The anaerobic skin commensal Propionibacterium acnes is an underestimated cause of human infections and clinical conditions. Previous studies have suggested a role for the bacterium in lumbar disc herniation and infection. To further investigate this, five biopsy samples were surgically excised...... from each of 64 patients with lumbar disc herniation. P. acnes and other bacteria were detected by anaerobic culture, followed by biochemical and PCR-based identification. In total, 24/64 (38%) patients had evidence of P. acnes in their excised herniated disc tissue. Using recA and mAb typing methods...... isolate collection (63%) suggests that the role of P. acnes in lumbar disc herniation should not be readily dismissed....

  3. Excision of Mucocele Using Diode Laser in Lower Lip

    Directory of Open Access Journals (Sweden)

    Subramaniam Ramkumar

    2016-01-01

    Full Text Available Mucoceles are nonneoplastic cystic lesions of major and minor salivary glands which result from the accumulation of mucus. These lesions are most commonly seen in children. Though usually these lesions can be treated by local surgical excision, in our case, to avoid intraoperative surgical complications like bleeding and edema and to enable better healing, excision was done using a diode laser in the wavelength of 940 nm.

  4. Determining Factors for Fast Corneal Sensitivity Recovery After Pterygium Excision

    OpenAIRE

    Julio Morán, Gemma; Campos, Pamela; Pujol Vives, Pere; Munguia, Aitana; Mas Aixalà, Enric

    2016-01-01

    Purpose: To establish determining factors for fast corneal sensitivity (CS) recovery after pterygium excision. Methods: Thirty-two eyes of 14 males and 18 females with primary nasal pterygium were recruited. Differences in CS (in the 4 quadrants and the center using a Cochet–Bonnet esthesiometer), pterygium corneal area (PCA), tear osmolarity, tear break-up time, Schirmer test, and ocular symptoms were analyzed before and 1 month after lesion excision. The relationship between CS recovery...

  5. Genomic island excisions in Bordetella petrii

    Directory of Open Access Journals (Sweden)

    Levillain Erwan

    2009-07-01

    Full Text Available Abstract Background Among the members of the genus Bordetella B. petrii is unique, since it is the only species isolated from the environment, while the pathogenic Bordetellae are obligately associated with host organisms. Another feature distinguishing B. petrii from the other sequenced Bordetellae is the presence of a large number of mobile genetic elements including several large genomic regions with typical characteristics of genomic islands collectively known as integrative and conjugative elements (ICEs. These elements mainly encode accessory metabolic factors enabling this bacterium to grow on a large repertoire of aromatic compounds. Results During in vitro culture of Bordetella petrii colony variants appear frequently. We show that this variability can be attributed to the presence of a large number of metastable mobile genetic elements on its chromosome. In fact, the genome sequence of B. petrii revealed the presence of at least seven large genomic islands mostly encoding accessory metabolic functions involved in the degradation of aromatic compounds and detoxification of heavy metals. Four of these islands (termed GI1 to GI3 and GI6 are highly related to ICEclc of Pseudomonas knackmussii sp. strain B13. Here we present first data about the molecular characterization of these islands. We defined the exact borders of each island and we show that during standard culture of the bacteria these islands get excised from the chromosome. For all but one of these islands (GI5 we could detect circular intermediates. For the clc-like elements GI1 to GI3 of B. petrii we provide evidence that tandem insertion of these islands which all encode highly related integrases and attachment sites may also lead to incorporation of genomic DNA which originally was not part of the island and to the formation of huge composite islands. By integration of a tetracycline resistance cassette into GI3 we found this island to be rather unstable and to be lost from

  6. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions

    Science.gov (United States)

    Coari, Kristin M.; Martin, Rebecca C.; Jain, Kopal; McGown, Linda B.

    2017-09-01

    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  7. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions.

    Science.gov (United States)

    Coari, Kristin M; Martin, Rebecca C; Jain, Kopal; McGown, Linda B

    2017-09-01

    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  8. 29 CFR 779.262 - Excise taxes at the retail level.

    Science.gov (United States)

    2010-07-01

    ... AS APPLIED TO RETAILERS OF GOODS OR SERVICES Employment to Which the Act May Apply; Enterprise Coverage Excise Taxes § 779.262 Excise taxes at the retail level. (a) Federal excise taxes are imposed at... 29 Labor 3 2010-07-01 2010-07-01 false Excise taxes at the retail level. 779.262 Section 779.262...

  9. Exploiting nucleotide composition to engineer promoters.

    Directory of Open Access Journals (Sweden)

    Manfred G Grabherr

    Full Text Available The choice of promoter is a critical step in optimizing the efficiency and stability of recombinant protein production in mammalian cell lines. Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. Here, we show how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs: by overlapping motifs that occur infrequently in the genome, we constructed contiguous sequence that is rich in GC and CpGs, both features of known promoters, but lacking homology to real promoters. We show that snippets from this sequence, at 100 base pairs or longer, drive gene expression in vitro in a number of mammalian cells, and are thus candidates for use in protein production. We further show that expression is driven by the general transcription factors TFIIB and TFIID, both being ubiquitously present across cell types, which results in less tissue- and species-specific regulation compared to the viral promoter SV40. We lastly found that the strength of a promoter can be tuned up and down by modulating the counts of GC and CpGs in localized regions. These results constitute a "proof-of-concept" for custom-designing promoters that are suitable for biotechnological and medical applications.

  10. Total excision of intramedullary epidermoid cyst in one case

    Directory of Open Access Journals (Sweden)

    PU Ke

    2013-12-01

    Full Text Available Clinical experience of total excision in a 14-year-old female with intramedullary epidermoid cyst was reported. The patient with L3-4 intramedullary epidermoid cyst underwent total excision through posterior median approach under microscopy. The patient was admitted for progressive strephexopodia and urinary and fecal incontinence. Preoperative imaging examination showed scoliosis, incontionous L4-S1 and abnormal signal of L3-4. Total excision and spinal remodeling were performed under intraoperative neurophysiological monitoning. Epidermoid cyst and its membrane were totally removed without aseptic meningitis after surgery, and the neurologic symptoms of the patient were gradually improved. Completely removing the membrane of epidermoid cyst is the key point to prevent recurrence and aseptic meningitis postoperatively. Dissection should be strictly in accordance with the boundaries of the membrane and the spinal cord, in order to avoid spinal cord injury.

  11. Excision of oral mucocele by different wavelength lasers

    Directory of Open Access Journals (Sweden)

    Umberto Romeo

    2013-01-01

    Full Text Available Background: Mucocele is a common benign neoplasm of oral soft tissues and the most common after fibroma. It generally occurs in the lower lip and its treatment includes excision of cyst and the responsible salivary gland, in order to prevent recurrences. Aims: To evaluate the capability of three different lasers in performing the excision of labial mucocele with two different techniques. Materials and Methods: In the presented cases, excision was performed using two different techniques (circumferential incision technique and mucosal preservation technique and three different laser wavelengths (Er,Cr:YSGG 2780 nm, diode 808 nm, and KTP 532 nm. Results: All the tested lasers, regardless of wavelength, showed many advantages (bloodless surgical field, no postoperative pain, relative speed, and easy execution. The most useful surgical technique depends on clinical features of the lesion. Conclusion: Tested lasers, with both techniques, are helpful in the management of labial mucocele.

  12. CELL POPULATION KINETICS OF EXCISED ROOTS OF PISUM SATIVUM

    Science.gov (United States)

    Van't Hof, Jack

    1965-01-01

    The cell population kinetics of excised, cultured pea roots was studied with the use of tritiated thymidine and colchicine to determine (1) the influence of excision, (2) the influence of sucrose concentration, (3) the average mitotic cycle duration, and (4) the duration of mitosis and the G 1, S, and G 2 periods of interphase.1 The results indicate that the process of excision causes a drop in the frequency of mitotic figures when performed either at the beginning of the culture period or after 100 hours in culture. This initial decrease in frequency of cell division is independent of sucrose concentration, but the subsequent rise in frequency of division, after 12 hours in culture, is dependent upon sucrose concentration. Two per cent sucrose maintains the shortest mitotic cycle duration. The use of colchicine indicated an average cycle duration of 20 hours, whereas the use of tritiated thymidine produced an average cycle duration of 17 hours. PMID:5857253

  13. Nucleotide sequence of the triosephosphate isomerase gene from Macaca mulatta

    Energy Technology Data Exchange (ETDEWEB)

    Old, S.E.; Mohrenweiser, H.W. (Univ. of Michigan, Ann Arbor (USA))

    1988-09-26

    The triosephosphate isomerase gene from a rhesus monkey, Macaca mulatta, charon 34 library was sequenced. The human and chimpanzee enzymes differ from the rhesus enzyme at ASN 20 and GLU 198. The nucleotide sequence identity between rhesus and human is 97% in the coding region and >94% in the flanking regions. Comparison of the rhesus and chimp genes, including the intron and flanking sequences, does not suggest a mechanism for generating the two TPI peptides of proliferating cells from hominoids and a single peptide from the rhesus gene.

  14. Differential role of base excision repair proteins in mediating cisplatin cytotoxicity.

    Science.gov (United States)

    Sawant, Akshada; Floyd, Ashley M; Dangeti, Mohan; Lei, Wen; Sobol, Robert W; Patrick, Steve M

    2017-03-01

    Interstrand crosslinks (ICLs) are covalent lesions formed by cisplatin. The mechanism for the processing and removal of ICLs by DNA repair proteins involves nucleotide excision repair (NER), homologous recombination (HR) and fanconi anemia (FA) pathways. In this report, we monitored the processing of a flanking uracil adjacent to a cisplatin ICL by the proteins involved in the base excision repair (BER) pathway. Using a combination of extracts, purified proteins, inhibitors, functional assays and cell culture studies, we determined the specific BER proteins required for processing a DNA substrate with a uracil adjacent to a cisplatin ICL. Uracil DNA glycosylase (UNG) is the primary glycosylase responsible for the removal of uracils adjacent to cisplatin ICLs, whereas other uracil glycosylases can process uracils in the context of undamaged DNA. Repair of the uracil adjacent to cisplatin ICLs proceeds through the classical BER pathway, highlighting the importance of specific proteins in this redundant pathway. Removal of uracil is followed by the generation of an abasic site and subsequent cleavage by AP endonuclease 1 (APE1). Inhibition of either the repair or redox domain of APE1 gives rise to cisplatin resistance. Inhibition of the lyase domain of Polymerase β (Polβ) does not influence cisplatin cytotoxicity. In addition, lack of XRCC1 leads to increased DNA damage and results in increased cisplatin cytotoxicity. Our results indicate that BER activation at cisplatin ICLs influences crosslink repair and modulates cisplatin cytotoxicity via specific UNG, APE1 and Polβ polymerase functions. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Union Women, the Tobacco Industry, and Excise Taxes

    Science.gov (United States)

    Balbach, Edith D.; Campbell, Richard B.

    2009-01-01

    Between 1987 and 1997, the tobacco industry used the issue of cigarette excise tax increases to create a political partnership with the Coalition of Labor Union Women (CLUW), a group representing female trade unionists in the U.S. This paper documents how the industry created this relationship and the lessons tobacco-control advocates can learn from the industry’s example, in order to mitigate possible unintended consequences of advocating excise tax increases In 1998, under the terms of the Master Settlement Agreement, the tobacco industry began making documents produced in litigation available publicly. Currently, approximately 50 million pages are available online, including substantial documentation of the industry–CLUW relationship. For this study, a comprehensive search of these documents was conducted. The tobacco industry encouraged CLUW’s opposition to excise tax increases by emphasizing the economic regressivity of these taxes, discussing excise taxes generically to deflect attention from cigarettes, and encouraging opposition to earmarking cigarette taxes to pay for specific programs. In addition, CLUW received at least $221,500 in financial support between 1987 and 1997 and in-kind support for its conferences, membership materials, and other services. Excise tax increases, if pursued without considering the impacts they may have on low-SES populations, may have unintended consequences. In this case, such proposals may have helped to create a relationship between CLUW and the tobacco industry. Because excise taxes are endorsed in the Framework Convention on Tobacco Control, tobacco-control advocates must understand how to build relationships with low-SES populations and mitigate potential alliances with the tobacco industry. PMID:19591750

  16. Black-hole excision with multiple grid patches

    International Nuclear Information System (INIS)

    Thornburg, Jonathan

    2004-01-01

    When using black-hole excision to numerically evolve a black-hole spacetime with no continuous symmetries, most 3 + 1 finite differencing codes use a Cartesian grid. It is difficult to do excision on such a grid because the natural r = constant excision surface must be approximated either by a very different shape such as a contained cube, or by an irregular and non-smooth 'LEGO 1 sphere' which may introduce numerical instabilities into the evolution. In this paper I describe an alternate scheme which uses multiple {r x (angular coordinates)} grid patches, each patch using a different (nonsingular) choice of angular coordinates. This allows excision on a smooth r = constant 2-sphere. I discuss the key design choices in such a multiple-patch scheme, including the choice of ghost-zone versus internal-boundary treatment of the interpatch boundaries (I use a ghost-zone scheme), the number and shape of the patches (I use a 6-patch 'inflated-cube' scheme), the details of how the ghost zones are 'synchronized' by interpolation from neighbouring patches, the tensor basis for the Einstein equations in each patch, and the handling of non-tensor field variables such as the BSSN Γ-tilde i (I use a scheme which requires ghost zones which are twice as wide for the BSSN conformal factor φ as for Γ-tilde i and the other BSSN field variables). I present sample numerical results from a prototype implementation of this scheme. This code simulates the time evolution of the (asymptotically flat) spacetime around a single (excised) black hole, using fourth-order finite differencing in space and time. Using Kerr initial data with J/m 2 = 0.6, I present evolutions to t ∼> 1500m. The lifetime of these evolutions appears to be limited only by outer boundary instabilities, not by any excision instabilities or by any problems inherent to the multiple-patch scheme

  17. Recurred Myofibroblastoma of Breast After Excision: A Case Report

    International Nuclear Information System (INIS)

    Cha, Yoon Ki; Kim, Ji Young; Jeong, Myeong Ja; Kim, Jae Hyung; Kim, Soung Hee; Kim, Soo Hyun; Jun, Woo Sun; Park, Kyeong Mee; Yang, Keun Ho

    2010-01-01

    Myofibroblastoma of the breast is a rare benign mesenchymal tumor that is known to occur in middle-aged and elderly men, yet there are some recent reports showing no certain difference for the gender distribution of this malady. Localized mass excision can usually provide a complete cure. To the best of our knowledge, there have been no reports of metastasis or recurrence of this tumor. Here we describe the sonographic findings of a case of recurrent myofibroblastoma after surgical excision for suspected fibroadenomas in both breasts of a 25-year-old woman

  18. A seventh complementation group in excision-deficient xeroderma pigmentosum

    International Nuclear Information System (INIS)

    Keijzer, W.; Jaspers, N.G.J.; Bootsma, D.; Abrahams, P.J.; Taylor, A.M.R.; Arlett, C.F.; Zelle, B.; Kinmont, P.D.S.

    1979-01-01

    Cells from a xeroderma pigmentosum patient XP2B1 who has reached 17 years of age with no keratoses or skin tumours constitute a new, 7th complementation group G. These cells exhibit a low residual level of excision repair, 2% of normal after a UV dose of 5 J/m 2 and an impairment of post-replication repair characteristic of excision-defective XPs. They are also sensitive to the lethal effects of UV and defective in host-cell reactivation of UV-irradiated SV40 DNA. (Auth.)

  19. Nucleotide diversity and phylogenetic relationships among ...

    Indian Academy of Sciences (India)

    2017-03-03

    Mar 3, 2017 ... 2Department of Botany, D. S. B. Campus, Kumaun University, Nainital 263 001, India ... Rana T. S. 2017 Nucleotide diversity and phylogenetic relationships ... Anderson and Park 1989). ..... Edgewood Press, Edgewood, USA.

  20. Ionic charge transport between blockages: Sodium cation conduction in freshly excised bulk brain tissue

    Energy Technology Data Exchange (ETDEWEB)

    Emin, David, E-mail: emin@unm.edu [Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM 87131 (United States); Akhtari, Massoud [Semple Institutes for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095 (United States); Ellingson, B. M. [Department of Radiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095 (United States); Mathern, G. W. [Department of Neurosurgery, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095 (United States)

    2015-08-15

    We analyze the transient-dc and frequency-dependent electrical conductivities between blocking electrodes. We extend this analysis to measurements of ions’ transport in freshly excised bulk samples of human brain tissue whose complex cellular structure produces blockages. The associated ionic charge-carrier density and diffusivity are consistent with local values for sodium cations determined non-invasively in brain tissue by MRI (NMR) and diffusion-MRI (spin-echo NMR). The characteristic separation between blockages, about 450 microns, is very much shorter than that found for sodium-doped gel proxies for brain tissue, >1 cm.

  1. Results of surgical excision of urethral prolapse in symptomatic patients.

    Science.gov (United States)

    Hall, Mary E; Oyesanya, Tola; Cameron, Anne P

    2017-11-01

    Here, we present the clinical presentation and surgical outcomes of women with symptomatic urethral prolapse presenting to our institution over 20 years, and seek to provide treatment recommendations for management of symptomatic urethral prolapse and caruncle. A retrospective review of medical records from female patients who underwent surgery for symptomatic urethral prolapse from June 1995 to August 2015 was performed. Surgical technique consisted of a four-quadrant excisional approach for repair of urethral prolapse. A total of 26 patients were identified with a mean age of 38.8 years (range 3-81). The most common presentations were vaginal bleeding, hematuria, pain, and dysuria. All patients underwent surgical excision of urethral prolapse via a standard approach. Follow-up data was available in 24 patients. Six patients experienced temporary postoperative bleeding, and one patient required placement of a Foley catheter for tamponade. One patient experienced temporary postoperative urinary retention requiring Foley catheter placement. Three patients had visible recurrence of urethral prolapse, for which one later underwent re-excision. Surgical excision of urethral prolapse is a reasonable treatment option in patients who have tried conservative management without relief, as well as in those who present with severe symptoms. Possible complications following excision include postoperative bleeding and recurrence, and patients must be counseled accordingly. In this work, we propose a treatment algorithm for symptomatic urethral prolapse. © 2017 Wiley Periodicals, Inc.

  2. Revenue and Health Impacts of Restructuring Tobacco Excise Tax ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    A proposed law in the Philippines to increase the excise tax on tobacco by ... developing countries to reduce demand for tobacco through price and tax measures. Southeast Asian countries are struggling with how to implement the main provisions of the Convention. ... Far East Asia, Philippines, Central Asia, South Asia ...

  3. Positive malignant margins in clinically diagnosed and excised be ...

    African Journals Online (AJOL)

    This study was aimed at utilizing retrospective descriptive data to evaluate the percentage of clini-cally benign breast lumps that turned out to be histologically malignant and the prevalence of posi-tive tumour margins among the malignant cases. A total of 2,917 registered cases of excised breast lump at the Department of ...

  4. Comparison between preoperative biopsy and post-excision ...

    African Journals Online (AJOL)

    peripheral nerve sheath tumour (6%).[6] Soft-tissue sarcomas most frequently affect the extremities and include MFH (40%), lipo- sarcoma (25%), synovial sarcoma and fibrosarcoma.[7]. Appropriate management is reliant on an accurate preoperative histology result. Excision biopsy is recommended for tumours. <3 cm in ...

  5. The safety of complete mesocolic excision once again confirmed

    DEFF Research Database (Denmark)

    Bertelsen, C A; Neuenschwander, A U

    2018-01-01

    We would like to commend Bernhoff and colleagues (1) for publishing further evidence supporting complete mesocolic excision (CME) as a safe approach in colon cancer surgery. They report no increased risk of 30-day mortality or reoperation after right-sided CME in a nested case-control study...

  6. 26 CFR 25.2512-7 - Effect of excise tax.

    Science.gov (United States)

    2010-04-01

    ... by a taxpayer and made the subject of gifts within a reasonable time after purchase, the purchase...-7 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) ESTATE AND GIFT TAXES GIFT TAX; GIFTS MADE AFTER DECEMBER 31, 1954 Transfers § 25.2512-7 Effect of excise tax. If...

  7. Peri-operative management for excision of plexiform ...

    African Journals Online (AJOL)

    Case report: A 28-year-old female weighing 78 kilograms presented at the University of Port Harcourt Teaching Hospital (UPTH) with a huge left thigh mass, nodules and brownish skin patches (café-au-lait spots) all over her body. Plexiform neurofibromatosis was diagnosed. The mass was subsequently excised under ...

  8. Excise Taxes and the Price Elasticity of Demand.

    Science.gov (United States)

    Gamble, Ralph C., Jr.

    1989-01-01

    Points out that, although the analysis of the imposition of an excise tax is widely used in economics courses, the consequences of a change in the tax rate are different and ignored. This article presents an effective way to teach about such a change. (GG)

  9. Modeling base excision repair in Escherichia coli bacterial cells

    International Nuclear Information System (INIS)

    Belov, O.V.

    2011-01-01

    A model describing the key processes in Escherichia coli bacterial cells during base excision repair is developed. The mechanism is modeled of damaged base elimination involving formamidopyrimidine DNA glycosylase (the Fpg protein), which possesses several types of activities. The modeling of the transitions between DNA states is based on a stochastic approach to the chemical reaction description

  10. A Method To Determine Adhesion Of Suppository Mass On Excised ...

    African Journals Online (AJOL)

    A method to determine adhesion of suppository mass to intestinal tissue was developed using excised pig intestine. The method which employs the principe of drainage unto and subsequent detachment from the mucosa, of an adherent suppository mass is simple, inexpensive and accurate. Fully optimised, it can be used ...

  11. A Rat Excised Larynx Model of Vocal Fold Scar

    Science.gov (United States)

    Welham, Nathan V.; Montequin, Douglas W.; Tateya, Ichiro; Tateya, Tomoko; Choi, Seong Hee; Bless, Diane M.

    2009-01-01

    Purpose: To develop and evaluate a rat excised larynx model for the measurement of acoustic, aerodynamic, and vocal fold vibratory changes resulting from vocal fold scar. Method: Twenty-four 4-month-old male Sprague-Dawley rats were assigned to 1 of 4 experimental groups: chronic vocal fold scar, chronic vocal fold scar treated with 100-ng basic…

  12. Papillary lesions of the breast: To excise or observe?

    Science.gov (United States)

    Khan, Sidrah; Diaz, Adrian; Archer, Kellie J; Lehman, Rebecca R; Mullins, Tiffany; Cardenosa, Gilda; Bear, Harry D

    2018-05-01

    Papillary lesions of the breast range from benign to atypical to malignant. Although papillomas without frank cancer are benign, their management remains controversial. When a core needle biopsy of a lesion yields a diagnosis of intraductal papilloma with atypia, excision is generally recommended to rule out a concurrent malignant neoplasm. For intraductal papillomas without atypia, however, recommendations for excision versus observation are variable. The aims of this study are to evaluate the rate of concurrent malignancies for intraductal papilloma diagnosed on core needle biopsy and to assess the long-term risk of developing cancer after the diagnosis of a papillary lesion. This single institution retrospective study analyzed 259 patients that were diagnosed with intraductal papilloma (IDP) by core needle biopsy from 1995 to 2010. Patients were grouped by initial diagnosis into three groups (papilloma without atypia, papilloma with atypia, and papilloma with atypical duct hyperplasia or atypical lobular hyperplasia (ADH/ALH) and followed up for long-term outcomes. After a core needle biopsy showing IDP with atypia or IDP + ADH/ALH, surgical excision yielded a diagnosis of concomitant invasive or ductal in situ cancer in greater that 30% of cases. For intraductal papilloma without atypia, the likelihood of cancer was much lower. Moreover, even with excision, the finding of intraductal papilloma with atypia carries a significant risk of developing cancer long-term, and such patients should be followed carefully and perhaps should be considered for chemoprevention. © 2017 Wiley Periodicals, Inc.

  13. 76 FR 46677 - Indoor Tanning Services; Cosmetic Services Excise Taxes

    Science.gov (United States)

    2011-08-03

    ... DEPARTMENT OF THE TREASURY Internal Revenue Service 26 CFR Parts 40 and 49 [REG-112841-10] RIN 1545-BJ40 Indoor Tanning Services; Cosmetic Services Excise Taxes AGENCY: Internal Revenue Service (IRS), Treasury. ACTION: Notice of public hearing on proposed rulemaking. SUMMARY: This document provides notice...

  14. 75 FR 33740 - Indoor Tanning Services; Cosmetic Services; Excise Taxes

    Science.gov (United States)

    2010-06-15

    ... 1545-BJ40 Indoor Tanning Services; Cosmetic Services; Excise Taxes AGENCY: Internal Revenue Service.... 7805. * * * Par. 5. Section 49.0-3 is added to read as follows: Sec. 49.0-3 Introduction; cosmetic...--Cosmetic Services Sec. 49.5000B-1 Indoor tanning services. [The text of this proposed Sec. 49.5000B-1 is...

  15. of lung metastases carcinoma of the cervix Surgical excision from ...

    African Journals Online (AJOL)

    of lung metastases carcinoma of the cervix. Surgical excision from squamous. A report of 2 cases. N. G. DE MOOR, A. V. BERRY, M. M. NISSENBAUM. These2casereportsservetoemphasizetwoimpor- tant points concerning carcinoma of the cervix: (i) blood-borne metastases are now frequently encountered in this disease; ...

  16. Clinical trial comparing excision and primary closure with modified ...

    African Journals Online (AJOL)

    The aim of this study was to compare modified Limberg flap procedure with excision and primary closure in the treatment of uncomplicated pilonidal disease. Methods: This study was conducted on 120 patients with uncomplicated sacrococcygeal pilonidal disease that were randomly allocated into two groups: group I ...

  17. Microendoscopic excision of C2 osteoid osteoma: a technical report.

    Science.gov (United States)

    Kulkarni, Arvind G; Dhruv, Abhilash N; Bassi, Anupreet J

    2013-09-01

    Case report and description of technique. To describe a microendoscopic posterior approach for excision of an osteoid osteoma of C2. Microendoscopic techniques are widely used in the management of degenerative disorders of the spine. This is the first report of their use in the management of an osteoid osteoma via the posterior approach. A 12-year-old-boy presented with left-sided neck pain of 3-month duration. Investigations revealed an osteoid osteoma of C2 lamina-lateral mass complex. The patient underwent a posterior microendoscopic excision using 18-mm diameter METRx system (Medtronic Sofamor Danek, Memphis, TN) of tubular retractors. A postoperative computed tomographic scan was done and preoperative and postoperative visual analogue scale and Neck Disability Index were evaluated. The patient was periodically followed up for 1 year. The postoperative computed tomographic scan revealed complete excision of the tumor. The visual analogue scale score for neck pain improved from 3/5 (preoperative) to 0/5 (postoperative) and Neck Disability Index from 33.33 (preoperative) to 0 (postoperative) at 1-year follow-up. Microendoscopic techniques can be extended to excise lesions of the spine. It is a safe procedure in experienced hands. The advantages are minimal morbidity, minimal postoperative pain and discomfort, less analgesic dependence, and better cosmesis. The authors recommend this technique for accessible lesions involving the spine.

  18. Principles of Periocular Reconstruction following Excision of Cutaneous Malignancy

    International Nuclear Information System (INIS)

    Hayano, S. M.; Whipple, K. M.; Korn, B. S.; Kikkawa, D. O.

    2012-01-01

    Reconstruction of periocular defects following excision of cutaneous malignancy can present difficulties for oculofacial and reconstructive surgeons. The intricate anatomy of the eyelids and face requires precise restoration in order to avoid postoperative functional anesthetic concerns. Various reconstructive procedures based on common principles, location and size of the defect, can be applied to achieve restoration with the best possible functional and aesthetic outcomes.

  19. Surgical excision of lung metastases from squamous carcinoma of ...

    African Journals Online (AJOL)

    These 2 case reports serveto emphasizetwo important points concerning carcinoma of the cervix: (i) blood-borne metastases are now frequently encountered in this disease; and (ii). in selected cases surgical excision of a secondary deposit in the lung is the treatment of choice and may even result -in cure.

  20. Excision Les jeunes changent l'Afrique par les TIC

    International Development Research Centre (IDRC) Digital Library (Canada)

    Comité Inter-Africain sur les Pratiques Traditionnelles ayant effet sur la santé des ... Planche 7 - Perception genrée de la citoyenneté : les dire des femmes. 90 ...... dans le monde avec les migrations internationales, l'excision ne semble pas être ...... En utilisant les TIC pour exprimer leurs attentes et leurs besoins, les jeunes, ...

  1. Obturator foramen dissection for excision of symptomatic transobturator mesh.

    Science.gov (United States)

    Reynolds, W Stuart; Kit, Laura Chang; Kaufman, Melissa R; Karram, Mickey; Bales, Gregory T; Dmochowski, Roger R

    2012-05-01

    Groin pain after transobturator synthetic mesh placement can be recalcitrant to conservative therapy and ultimately requires surgical excision. We describe our experiences with and technique of obturator foramen dissection for mesh excision. The records of 8 patients treated from 2005 to 2010, were reviewed. Obturator dissection was performed via a lateral groin incision over the inferior pubic ramus at the level of the obturator foramen, typically in conjunction with orthopedic surgery. Five patients had transobturator mid urethral sling surgery for stress urinary incontinence, 2 had mid urethral sling and trocar based anterior vaginal wall mesh kits with transobturator passage of mesh arms for stress urinary incontinence and pelvic organ prolapse, and 1 had an anterior vaginal wall mesh kit for pelvic organ prolapse. Patients had 0 to 2 prior transvaginal mesh excisions before obturator surgery. All patients presented with intractable pain in the area of the obturator foramen and/or medial groin for which conservative treatment measures had failed. Six patients underwent concurrent vaginal and obturator dissection and 2 underwent obturator dissection alone. In all cases residual mesh (3 to 11 cm) was identified and excised from the obturator foramen. Mesh was closely associated to or traversing the adductor longus muscle and tendon with significant fibrous reaction in all cases. Postoperatively 5 patients were cured of pain and/or infection, and 3 reported no or some improvement at a mean followup of 6 months (range 1 to 12). Our experience suggests that surgical excision of residual mesh can alleviate many of the symptoms in many patients. In all cases mesh remnants were identified and removed, and typically involved neuromuscular structures adjacent to the obturator foramen. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  2. Outcome of excision of megarectum in children with anorectal malformation.

    Science.gov (United States)

    Keshtgar, Alireza S; Ward, Harry C; Richards, Catherine; Clayden, Graham S

    2007-01-01

    Megarectum in association with anorectal malformation contributes to chronic constipation and fecal incontinence. Resection of megarectum in anorectal malformation improves bowel function, but neuropathy and poor sphincter quality may affect the outcome of fecal continence adversely. The aim of this study was to evaluate the benefits of resection of megarectum in anorectal malformation and to ascertain the impact of anal sphincter quality and neuropathy on the outcome. We studied 62 children with intractable fecal incontinence after repair of anorectal malformation between January 1991 and January 2005. All patients were investigated with anorectal manometry and anal endosonography under ketamine anesthesia. On endosonography, an intact or scarred internal anal sphincter (IAS) was classified as good and a fragmented or absent IAS as poor. On manometry, a resting anal sphincter pressure equal to or more than 30 mm Hg was classified as good and a lower pressure as poor. Functional assessment of fecal continence was done before and after excision of megarectum using a modified Wingfield scores. Sixteen children had excision of megarectum with median age of 9 years (range, 2-15 years) and postoperative follow-up of 5 years (range, 1-10 years). Seven had formation of antegrade continent enema stoma before excision of megarectum. Children were classified into three groups of anomalies: low (n = 6), intermediate (n = 4), and high (n = 6). All children were incontinent of feces. After excision of megarectum, of the 9 children with good IAS and no neuropathy, 7 became continent of feces. Of the remaining 7 children, 4 had poor IAS and 3 had neuropathy, 5 of whom required an antegrade continent enema stoma to be clean. Excision of megarectum in children who had previous repair of anorectal malformation results in fecal continence in the presence of a good IAS and absence of neuropathy. Patients with a poor IAS or neuropathy will often require artificial means of fecal

  3. Scambio, a novel guanine nucleotide exchange factor for Rho

    Directory of Open Access Journals (Sweden)

    Groffen John

    2004-04-01

    Full Text Available Abstract Background Small GTPases of the Rho family are critical regulators of various cellular functions including actin cytoskeleton organization, activation of kinase cascades and mitogenesis. For this reason, a major objective has been to understand the mechanisms of Rho GTPase regulation. Here, we examine the function of a novel protein, Scambio, which shares homology with the DH-PH domains of several known guanine nucleotide exchange factors for Rho family members. Results Scambio is located on human chromosome 14q11.1, encodes a protein of around 181 kDa, and is highly expressed in both heart and skeletal muscle. In contrast to most DH-PH-domain containing proteins, it binds the activated, GTP-bound forms of Rac and Cdc42. However, it fails to associate with V14RhoA. Immunofluorescence studies indicate that Scambio and activated Rac3 colocalize in membrane ruffles at the cell periphery. In accordance with these findings, Scambio does not activate either Rac or Cdc42 but rather, stimulates guanine nucleotide exchange on RhoA and its close relative, RhoC. Conclusion Scambio associates with Rac in its activated conformation and functions as a guanine nucleotide exchange factor for Rho.

  4. Genetics of human sensitivity to ultraviolet radiation

    Science.gov (United States)

    Cleaver, James E.

    1994-07-01

    the major human health effects of solar and artificial UV light occur from the UVB and UVC wavelength ranges and involve a variety of short-term and long-term deleterious changes to the skin and eyes. the more important initial damage to cellular macromolecules involves dimerization of adjacent pyrimidines in DNA to produce cyclobutane pyrimidine dimes, (6-4) pyrimidine- pyrimidone, and (6-4) dewar photoproducts. these photoproducts can be repaired by a genetically regulated enzyme system (nucleotide excision repair) which removes oligonucleotides 29-30 nucleotides long that contain the photoproducts, and synthesizes replacement patches. At least a dozen gene products are involved in the process of recognizing photoproducts in DNA, altering local DNA helicity and cleaving the polynucleotide chain at defined positions either side of a photoproduct. Hereditary mutations in many of these genes are recognized in the human genetic disorders xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). Several of the gene products have other functions involving the regulation of gene transcription which accounts for the complex clinical presentation of repair deficient diseases that involve sensitivity of the skin and eyes to UV light, increased solar carcinogenesis (in XP), demyelination, and ganglial calcification (in CS), hair abnormalities (in TTD), and developmental and neurological abnormalities

  5. 76 FR 3502 - Time for Payment of Certain Excise Taxes, and Quarterly Excise Tax Payments for Small Alcohol...

    Science.gov (United States)

    2011-01-20

    ..., Surety bonds, Virgin Islands, Warehouses. 27 CFR Part 40 Cigars and cigarettes, Claims, Electronic fund... required to pay taxes through electronic funds transfer (EFT), this first payment period ends on September... Part 19 Caribbean Basin Initiative, Claims, Electronic funds transfers, Excise taxes, Exports, Gasohol...

  6. 76 FR 3584 - Time for Payment of Certain Excise Taxes, and Quarterly Excise Tax Payments for Small Alcohol...

    Science.gov (United States)

    2011-01-20

    ... 40 Cigars and cigarettes, Claims, Electronic fund transfers, Excise taxes, Labeling, Packaging and..., Regulations.gov , we will post, and you may view, copies of this notice, any electronic or mailed comments we... material that we consider unsuitable for posting. You also may view copies of this notice, any electronic...

  7. A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

    Science.gov (United States)

    Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

    2012-01-01

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

  8. A nucleotide phosphatase activity in the nucleotide binding domain of an orphan resistance protein from rice.

    Science.gov (United States)

    Fenyk, Stepan; Campillo, Alba de San Eustaquio; Pohl, Ehmke; Hussey, Patrick J; Cann, Martin J

    2012-02-03

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.

  9. Robotic transanal total mesorectal excision for rectal cancer: experience with a first case

    NARCIS (Netherlands)

    Verheijen, P.M.; Consten, E.C.J.; Broeders, Ivo Adriaan Maria Johannes

    2014-01-01

    Background: A transanal approach for total mesorectal excision (TME) using a single incision port is feasible. The disadvantages are technical difficulties associated with limited manoeuvrability. Methods: We present our first experience with robotic-assisted transanal total mesorectal excision. A

  10. Black-hole excision with multiple grid patches

    Energy Technology Data Exchange (ETDEWEB)

    Thornburg, Jonathan [Max-Planck-Institut fuer Gravitationsphysik, Albert-Einstein-Institut, Am Muehlenberg 1, D-14476 Golm (Germany)

    2004-08-07

    When using black-hole excision to numerically evolve a black-hole spacetime with no continuous symmetries, most 3 + 1 finite differencing codes use a Cartesian grid. It is difficult to do excision on such a grid because the natural r = constant excision surface must be approximated either by a very different shape such as a contained cube, or by an irregular and non-smooth 'LEGO{sup 1} sphere' which may introduce numerical instabilities into the evolution. In this paper I describe an alternate scheme which uses multiple {l_brace}r x (angular coordinates){r_brace} grid patches, each patch using a different (nonsingular) choice of angular coordinates. This allows excision on a smooth r = constant 2-sphere. I discuss the key design choices in such a multiple-patch scheme, including the choice of ghost-zone versus internal-boundary treatment of the interpatch boundaries (I use a ghost-zone scheme), the number and shape of the patches (I use a 6-patch 'inflated-cube' scheme), the details of how the ghost zones are 'synchronized' by interpolation from neighbouring patches, the tensor basis for the Einstein equations in each patch, and the handling of non-tensor field variables such as the BSSN {gamma}-tilde{sup i} (I use a scheme which requires ghost zones which are twice as wide for the BSSN conformal factor {phi} as for {gamma}-tilde{sup i} and the other BSSN field variables). I present sample numerical results from a prototype implementation of this scheme. This code simulates the time evolution of the (asymptotically flat) spacetime around a single (excised) black hole, using fourth-order finite differencing in space and time. Using Kerr initial data with J/m{sup 2} = 0.6, I present evolutions to t {approx}> 1500m. The lifetime of these evolutions appears to be limited only by outer boundary instabilities, not by any excision instabilities or by any problems inherent to the multiple-patch scheme.

  11. Resolution of vitiligo following excision of halo congenital melanocytic nevus: a rare case report.

    Science.gov (United States)

    Wang, Kai; Wang, Zhi; Huang, Weiqing

    2016-05-01

    Halo congenital melanocytic nevus (CMN) associated with vitiligo is rare, especially with regard to CMN excision. Only two reports of excision of halo CMN following repigmentation of vitiligo are found in the literature. We present a case of a girl with halo CMN and periorbital vitiligo. The halo CMN was excised and followed by spontaneous improvement of vitiligo. The result suggests excision of the inciting lesion may be a promising way to control vitiligo. © 2015 Wiley Periodicals, Inc.

  12. 29 CFR 779.263 - Excise taxes not at the retail level.

    Science.gov (United States)

    2010-07-01

    ... ACT AS APPLIED TO RETAILERS OF GOODS OR SERVICES Employment to Which the Act May Apply; Enterprise Coverage Excise Taxes § 779.263 Excise taxes not at the retail level. There are also a wide variety of... 29 Labor 3 2010-07-01 2010-07-01 false Excise taxes not at the retail level. 779.263 Section 779...

  13. First observation of excision and integration in Class 1 integron in ...

    African Journals Online (AJOL)

    So in this study, we tested in S. aureus, the class 1 integron mediated excision and integration. We first asked 8 plasmids from previous studies, then established some transformants and perform the excision and integration reaction. As the results revealed, we observed positive excision assay, which had been confirmed by ...

  14. Excise Tax Avoidance: The Case of State Cigarette Taxes

    Science.gov (United States)

    DeCicca, Philip; Kenkel, Donald; Liu, Feng

    2013-01-01

    We conduct an applied welfare economics analysis of cigarette tax avoidance. We develop an extension of the standard formula for the optimal Pigouvian corrective tax to incorporate the possibility that consumers avoid the tax by making purchases in nearby lower-tax jurisdictions. To provide a key parameter for our formula, we estimate a structural endogenous switching regression model of border-crossing and cigarette prices. In illustrative calculations, we find that for many states, after taking into account tax avoidance the optimal tax is at least 20 percent smaller than the standard Pigouvian tax that simply internalizes external costs. Our empirical estimate that tax avoidance strongly responds to the price differential is the main reason for this result. We also use our results to examine the benefits of replacing avoidable state excise taxes with a harder-to-avoid federal excise tax on cigarettes. PMID:24140760

  15. Transanal vs laparoscopic total mesorectal excision for rectal cancer

    DEFF Research Database (Denmark)

    Perdawood, Sharaf; Al Khefagie, Ghalib Ali Abod

    2016-01-01

    BACKGROUND: Laparoscopic total mesorectal excision (LaTME) has improved short-term outcomes of rectal cancer surgery with comparable oncological results to open approach. LaTME can be difficult in the lower most part of the rectum, leading potentially to higher rates of complications, conversion...... to open surgery and probably suboptimal oncological quality. Transanal TME (TaTME) can potentially solve these problems. The aim of this study was to compare the short-term results after TaTME with those after LaTME. METHODS: A prospectively collected database of consecutive patients who underwent Ta......TME was maintained. Results were compared with those underwent LaTME in the preceding period. Patients who underwent low anterior resection or intersphincteric abdominoperineal excision (APE) were included. Primary end-points were radical resection and specimen quality. Secondary end-points were complications, rates...

  16. Excise tax avoidance: the case of state cigarette taxes.

    Science.gov (United States)

    DeCicca, Philip; Kenkel, Donald; Liu, Feng

    2013-12-01

    We conduct an applied welfare economics analysis of cigarette tax avoidance. We develop an extension of the standard formula for the optimal Pigouvian corrective tax to incorporate the possibility that consumers avoid the tax by making purchases in nearby lower tax jurisdictions. To provide a key parameter for our formula, we estimate a structural endogenous switching regression model of border-crossing and cigarette prices. In illustrative calculations, we find that for many states, after taking into account tax avoidance the optimal tax is at least 20% smaller than the standard Pigouvian tax that simply internalizes external costs. Our empirical estimate that tax avoidance strongly responds to the price differential is the main reason for this result. We also use our results to examine the benefits of replacing avoidable state excise taxes with a harder-to-avoid federal excise tax on cigarettes. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Isolated myocardial hydatid cyst: Managed with total curative excision

    Directory of Open Access Journals (Sweden)

    Santosh Kumar Sinha

    2016-01-01

    Full Text Available Hydatid disease is still prevalent in developing countries, and isolated cardiac hydatid cysts are the rarest presentation. We report a 40-year-old nondiabetic, nonhypertensive female who presented with low-grade fever for 2 months shortness of breath and orthopnea for 2 weeks. Transthoracic echocardiography revealed a large, round cystic lesion with multiple daughter cysts without any obvious intraluminal detached membranes with mass effect on the left ventricular outflow tract. After total excision, residual tissue was closed with Teflon patch. Germinative membrane and hundreds of daughter cysts were seen. Following total excision of the cyst from myocardium, myocardial cavity was washed thoroughly with 10% Betadine solution. Pathological examination confirmed the diagnosis of hydatid cyst. Preoperatively started albendazole was continued for 4 weeks even after the operation. On follow-up after 4 weeks, the patient is doing well and cardiac imaging showed normal contours of the heart.

  18. Principles of Periocular Reconstruction following Excision of Cutaneous Malignancy

    Directory of Open Access Journals (Sweden)

    Scott M. Hayano

    2012-01-01

    Full Text Available Reconstruction of periocular defects following excision of cutaneous malignancy can present difficulties for oculofacial and reconstructive surgeons. The intricate anatomy of the eyelids and face requires precise restoration in order to avoid postoperative functional anesthetic concerns. Various reconstructive procedures based on common principles, location and size of the defect, can be applied to achieve restoration with the best possible functional and aesthetic outcomes.

  19. Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition

    International Nuclear Information System (INIS)

    Jarvis, P.; Belzile, F.; Page, T.; Dean, C.

    1997-01-01

    The maize transposable element Ac is highly active in the heterologous hosts tobacco and tomato, but shows very much reduced levels of activity in Arabidopsis. A mutagenesis experiment was undertaken with the aim of identifying Arabidopsis host factors responsible for the observed low levels of Ac activity. Seed from a line carrying a single copy of the Ac element inserted into the streptomycin phosphotransferase (SPT) reporter fusion, and which displayed typically low levels of Ac activity, were mutagenized using gamma rays. Nineteen mutants displaying high levels of somatic Ac activity, as judged by their highly variegated phenotypes, were isolated after screening the M2 generation on streptomycin-containing medium. The mutations fall into two complementation groups, iae1 and iae2, are unlinked to the SPT::Ac locus and segregate in a Mendelian fashion. The iae1 mutation is recessive and the iae2 mutation is semi-dominant. The iae1 and iae2 mutants show 550- and 70-fold increases, respectively, in the average number of Ac excision sectors per cotyledon. The IAE1 locus maps to chromosome 2, whereas the SPT::Ac reporter maps to chromosome 3. A molecular study of Ac activity in the iae1 mutant confirmed the very high levels of Ac excision predicted using the phenotypic assay, but revealed only low levels of Ac re-insertion. Analyses of germinal transposition in the iae1 mutant demonstrated an average germinal excision frequency of 3% and a frequency of independent Ac re-insertions following germinal excision of 22%. The iae mutants represents a possible means of improving the efficiency of Ac/Ds transposon tagging systems in Arabidopsis, and will enable the dissection of host involvement in Ac transposition and the mechanisms employed for controlling transposable element activity

  20. Norwegian NOx emissions excise duty hits shipping and offshore industry

    International Nuclear Information System (INIS)

    Gulseth, Oeystein Arff; Fjeld-Nielsen, Siri

    2007-01-01

    The regulatory framework concerning the new excise duty appears as complex and unfinished, raising more questions than providing answers. The duty will entail a significant expense item for the businesses liable to register and it is therefore important to have an overview of how the regulatory framework should be understood. Errors made since 1 January 2007 may lead to future reassessments of duties, with interest (author) (ml)

  1. European Union Harmonized Excise Taxation : Occasional Importation Process

    OpenAIRE

    Tanhua, Taina

    2013-01-01

    This thesis was written with the intent to compile the information related to occasional importation process and European Union harmonized taxation into a single package. The process is based on European Union legislation and the aim of it is to unify the taxation within the internal market area. The national excise duties are not part of the occasional importation process but are partly linked to it. The first part of the thesis discusses the occasional importation of goods subject to ha...

  2. Temporal flap approach for preauricular epidermal cyst excision

    OpenAIRE

    Harshavardhan N Reddy; D R Srinivas; Konappa E Reddy; C Chandrakiran

    2011-01-01

    A 40-year-old male presented with a fluctuant swelling in front of the right ear. His past history is significant for having undergone surgery for the swelling four times in the past 6 years with recurrence each time. We excised the swelling by using a preauricular incision with reverse question mark type extension into the temporal area. The advantages are complete unhindered exposure, excellent cosmesis, and prevention of damage to the temporal branch of facial nerve.

  3. Endoscope-assisted approach to excision of branchial cleft cysts.

    Science.gov (United States)

    Teng, Stephanie E; Paul, Benjamin C; Brumm, John D; Fritz, Mark; Fang, Yixin; Myssiorek, David

    2016-06-01

    The purpose of this study is to describe an endoscope-assisted surgical technique for the excision of branchial cleft cysts and compare it to the standard approach. Retrospective case series review. Twenty-seven cases described as branchial cleft excisions performed by a single surgeon at one academic medical center were identified between 2007 and 2014. Twenty-five cases (8 endoscopic, 17 standard approach) were included in the study. Cases were excluded if final pathology was malignant. Patient charts were reviewed, and two techniques were compared through analysis of incision size, operative time, and surgical outcomes. This study showed that the length of incision required for the endoscopic approach (mean = 2.13 ± 0.23) was significantly less than that of the standard approach (mean = 4.10 ± 1.46, P = 0.008) despite the fact that there was no significant difference in cyst size between the two groups (P = 0.09). The other variables examined, including operative time and surgical outcomes, were not significantly different between the two groups. This transcervical endoscope-assisted approach to branchial cleft cyst excision is a viable option for uncomplicated cases. It provides better cosmetic results than the standard approach and does not negatively affect outcomes, increase operative time, or result in recurrence. 4. Laryngoscope, 126:1339-1342, 2016. © 2015 The American Laryngological, Rhinological and Otological Society, Inc.

  4. Do alcohol excise taxes affect traffic accidents? Evidence from Estonia.

    Science.gov (United States)

    Saar, Indrek

    2015-01-01

    This article examines the association between alcohol excise tax rates and alcohol-related traffic accidents in Estonia. Monthly time series of traffic accidents involving drunken motor vehicle drivers from 1998 through 2013 were regressed on real average alcohol excise tax rates while controlling for changes in economic conditions and the traffic environment. Specifically, regression models with autoregressive integrated moving average (ARIMA) errors were estimated in order to deal with serial correlation in residuals. Counterfactual models were also estimated in order to check the robustness of the results, using the level of non-alcohol-related traffic accidents as a dependent variable. A statistically significant (P traffic accidents was disclosed under alternative model specifications. For instance, the regression model with ARIMA (0, 1, 1)(0, 1, 1) errors revealed that a 1-unit increase in the tax rate is associated with a 1.6% decrease in the level of accidents per 100,000 population involving drunk motor vehicle drivers. No similar association was found in the cases of counterfactual models for non-alcohol-related traffic accidents. This article indicates that the level of alcohol-related traffic accidents in Estonia has been affected by changes in real average alcohol excise taxes during the period 1998-2013. Therefore, in addition to other measures, the use of alcohol taxation is warranted as a policy instrument in tackling alcohol-related traffic accidents.

  5. The Impact of Pterygium Excision on Corneal Astigmatism

    International Nuclear Information System (INIS)

    Khan, F. A.; Niazi, S. P. K.; Khan, D. A.

    2014-01-01

    Objective: To compare the corneal astigmatism before and after the excision of pterygium and also to determine the correlation of pterygium size with the postoperative corneal astigmatism. Study Design: Cross-sectional interventional study. Place and Duration of Study: Eye Department, Combined Military Hospital, Abbottabad, from May 2011 to March 2012. Methodology: Thirty patients underwent pterygium excision. Pre-operatively Snellen visual acuity, manifest refraction and slit lamp examination was done. The size of the pterygium was recorded in mm by projecting a horizontal slit lamp beam from the limbus to the apex. All the pterygium were equal to or greater than 2.5 mm. Keratometry was performed with an automated keratometer. Keratometric data was recorded pre-operatively and at 28 days postoperatively. Wilcoxon signed rank test was used for comparing the pre-operative and the postoperative corneal astigmatism. Spearman's rank order was calculated to observe correlation of pterygium size with the postoperative astigmatism. Results: The median (mean rank) pre-operative astigmatism of 2.25 (15.50) reduced to a median (mean rank) postoperative astigmatism of 1.30 (14.96). This decrease in the postoperative astigmatism was statistically significant (p < 0.001). There was a statistically non-significant correlation between the postoperative astigmatism and the pterygium size (rs = -0.29, p = 0.12). Conclusion: Pterygium excision caused significant reduction in corneal astigmatism. (author)

  6. Distinctive features of single nucleotide alterations in induced pluripotent stem cells with different types of DNA repair deficiency disorders

    Science.gov (United States)

    Okamura, Kohji; Sakaguchi, Hironari; Sakamoto-Abutani, Rie; Nakanishi, Mahito; Nishimura, Ken; Yamazaki-Inoue, Mayu; Ohtaka, Manami; Periasamy, Vaiyapuri Subbarayan; Alshatwi, Ali Abdullah; Higuchi, Akon; Hanaoka, Kazunori; Nakabayashi, Kazuhiko; Takada, Shuji; Hata, Kenichiro; Toyoda, Masashi; Umezawa, Akihiro

    2016-01-01

    Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications. PMID:27197874

  7. Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand.

    Science.gov (United States)

    Reis, António M C; Mills, Wilbur K; Ramachandran, Ilangovan; Friedberg, Errol C; Thompson, David; Queimado, Lurdes

    2012-01-01

    Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process.

  8. Excision of x-ray-induced thymine damage in chromatin from heated cells

    International Nuclear Information System (INIS)

    Warters, R.L.; Roti Roti, J.L.

    1979-01-01

    Experiments were performed to distinguish between two possible modes of hyperthermia-induced inhibition of thymine base damage excision from the DNA of CHO cells: (1) heat denaturation of excision enzyme(s) or (2) heat-induced alteration of the substrate for damage excision (chromatin). While hyperthermia (45 0 C, 15 min) had no apparent effect on the capacity of the excision enzymes to excise damage from DNA it had a dramatic effect (ca. 80% inhibition) on the ability of chromatin to serve as a substrate for unheated enzymes. These results suggest that hyperthermia-induced radiosensitization of CHO cells may be due primarily to lesions in the cellular chromatin

  9. Pulsed terahertz imaging of breast cancer in freshly excised murine tumors

    Science.gov (United States)

    Bowman, Tyler; Chavez, Tanny; Khan, Kamrul; Wu, Jingxian; Chakraborty, Avishek; Rajaram, Narasimhan; Bailey, Keith; El-Shenawee, Magda

    2018-02-01

    This paper investigates terahertz (THz) imaging and classification of freshly excised murine xenograft breast cancer tumors. These tumors are grown via injection of E0771 breast adenocarcinoma cells into the flank of mice maintained on high-fat diet. Within 1 h of excision, the tumor and adjacent tissues are imaged using a pulsed THz system in the reflection mode. The THz images are classified using a statistical Bayesian mixture model with unsupervised and supervised approaches. Correlation with digitized pathology images is conducted using classification images assigned by a modal class decision rule. The corresponding receiver operating characteristic curves are obtained based on the classification results. A total of 13 tumor samples obtained from 9 tumors are investigated. The results show good correlation of THz images with pathology results in all samples of cancer and fat tissues. For tumor samples of cancer, fat, and muscle tissues, THz images show reasonable correlation with pathology where the primary challenge lies in the overlapping dielectric properties of cancer and muscle tissues. The use of a supervised regression approach shows improvement in the classification images although not consistently in all tissue regions. Advancing THz imaging of breast tumors from mice and the development of accurate statistical models will ultimately progress the technique for the assessment of human breast tumor margins.

  10. [Technical background of data collection for parametric observation of total mesorectal excision (TME) in rectal cancer].

    Science.gov (United States)

    Bláha, M; Hoch, J; Ferko, A; Ryška, A; Hovorková, E

    Improvement in any human activity is preconditioned by inspection of results and providing feedback used for modification of the processes applied. Comparison of experts experience in the given field is another indispensable part leading to optimisation and improvement of processes, and optimally to implementation of standards. For the purpose of objective comparison and assessment of the processes, it is always necessary to describe the processes in a parametric way, to obtain representative data, to assess the achieved results, and to provide unquestionable and data-driven feedback based on such analysis. This may lead to a consensus on the definition of standards in the given area of health care. Total mesorectal excision (TME) is a standard procedure of rectal cancer (C20) surgical treatment. However, the quality of performed procedures varies in different health care facilities, which is given, among others, by internal processes and surgeons experience. Assessment of surgical treatment results is therefore of key importance. A pathologist who assesses the resected tissue can provide valuable feedback in this respect. An information system for the parametric assessment of TME performance is described in our article, including technical background in the form of a multicentre clinical registry and the structure of observed parameters. We consider the proposed system of TME parametric assessment as significant for improvement of TME performance, aimed at reducing local recurrences and at improving the overall prognosis of patients. rectal cancer total mesorectal excision parametric data clinical registries TME registry.

  11. Effect of the FSH receptor single nucleotide polymorphisms (FSHR 307/680) on the follicular fluid hormone profile and the granulosa cell gene expression in human small antral follicles

    DEFF Research Database (Denmark)

    Borgbo, Tanni Kjær; Jeppesen, J V; Lindgren, I

    2014-01-01

    ), by evaluating the hormone and gene expression profiles of human small antral follicles collected under physiological conditions in connection with fertility preservation. In total 69 women at various time during the menstrual cycle were included in this study. The intrafollicular hormone content of 179...... was significantly increased, whereas AMH gene expression was significantly reduced for the G/G genotype. In follicles >6 mm, estradiol and CYP19A1 gene expression levels were significantly higher for the G/G genotype. In conclusion, significant changes were observed between the FSHR 307/680 polymorphisms in human......The most pronounced effects of FSH signalling are potentially displayed in the follicle fluid, which acts as a reservoir for FSH-induced granulosa cell (GC) secreted hormones. This study investigates the effects of two common polymorphisms of FSHR, FSHR 307 (rs6165) and FSHR 680 (rs6166...

  12. Increased cell division but not thymic dysfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection

    NARCIS (Netherlands)

    Hazenberg, Mette D.; Borleffs, J.C.C.; Otto, S.A.; Cohen Stuart, J.W.T. (James Willem Theodoor); Verschuren, M.C.M. (Martie); Boucher, C.A.B.; Coutinho, R.A.; Lange, Joep M.A.; Rinke de Wit, T.F. (Tobias); Tsegaye, A. (Aster); Dongen, J.J.M. (Jaques) van; Hamann, D. (Dörte); Boer, R.J. de; Miedema, F.

    2000-01-01

    Recent thymic emigrants can be identified by T cell receptor excision circles (TRECs) formed during T-cell receptor rearrangement. Decreasing numbers of TRECs have been observed with aging and in human immunodeficiency virus (HIV)-1 infected individuals, suggesting for thymic impairment. Here,

  13. Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Konze-Thomas, B.; Hazard, R.M.; Maher, V.M.; McCormick, J.J. (Michigan State Univ., East Lansing (USA). Carcinogenesis Lab.)

    1982-01-01

    Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G/sub 0/ state. Autoradiography studies of cells released from G/sub 0/ after 72 h and replated at lower densities (3-9 x 10/sup 3/ cells/cm/sup 2/) in fresh medium showed that semiconservative DNA synthesis (S phase) began approx. equal to 24 h after the replating. The task was to determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts.

  14. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping

    Science.gov (United States)

    Hendershot, Jenna M.; O'Brien, Patrick J.

    2014-01-01

    Nucleotide flipping is a common feature of DNA-modifying enzymes that allows access to target sites within duplex DNA. Structural studies have identified many intercalating amino acid side chains in a wide variety of enzymes, but the functional contribution of these intercalating residues is poorly understood. We used site-directed mutagenesis and transient kinetic approaches to dissect the energetic contribution of intercalation for human alkyladenine DNA glycosylase, an enzyme that initiates repair of alkylation damage. When AAG flips out a damaged nucleotide, the void in the duplex is filled by a conserved tyrosine (Y162). We find that tyrosine intercalation confers 140-fold stabilization of the extrahelical specific recognition complex, and that Y162 functions as a plug to slow the rate of unflipping by 6000-fold relative to the Y162A mutant. Surprisingly, mutation to the smaller alanine side chain increases the rate of nucleotide flipping by 50-fold relative to the wild-type enzyme. This provides evidence against the popular model that DNA intercalation accelerates nucleotide flipping. In the case of AAG, DNA intercalation contributes to the specific binding of a damaged nucleotide, but this enhanced specificity comes at the cost of reduced speed of nucleotide flipping. PMID:25324304

  15. Higher Volume at Time of Breast Conserving Surgery Reduces Re-Excision in DCIS

    Directory of Open Access Journals (Sweden)

    J. H. Wolf

    2011-01-01

    Full Text Available Purpose. The purpose of this study was to compare the surgical and pathological variables which impact rate of re-excision following breast conserving therapy (BCS with or without concurrent additional margin excision (AM. Methods. The pathology database was queried for all patients with DCIS from January 2004 to September 2008. Pathologic assessment included volume of excision, subtype, size, distance from margin, grade, necrosis, multifocality, calcifications, and ER/PR status. Results. 405 cases were identified and 201 underwent BCS, 151-BCS-AM, and 53-mastectomy. Among the 201 BCS patients, 190 underwent re-excision for close or involved margins. 129 of these were treated with BCS and 61 with BCS-AM (P<.0001. The incidence of residual DCIS in the re-excision specimens was 32% (n=65 for BCS and 22% (n=33 for BCS-AM (P<.05. For both the BCS and the BCS-AM cohorts, volume of tissue excised is inversely correlated to the rate of re-excision (P=.0284. Multifocality (P=.0002 and ER status (P=.0382 were also significant predictors for rate of re-excision and variation in surgical technique was insignificant. Conclusions. The rate of positive margins, re-excision, and residual disease was significantly higher in patients with lower volume of excision. The performance of concurrent additional margin excision increases the efficacy of BCS for DCIS.

  16. Human DNA repair and recombination genes

    International Nuclear Information System (INIS)

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs

  17. Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells.

    Science.gov (United States)

    Lee, Hyun-Wook; Wang, Hsiang-Tsui; Weng, Mao-wen; Hu, Yu; Chen, Wei-sheng; Chou, David; Liu, Yan; Donin, Nicholas; Huang, William C; Lepor, Herbert; Wu, Xue-Ru; Wang, Hailin; Beland, Frederick A; Tang, Moon-shong

    2014-06-15

    Tobacco smoke (TS) is a major cause of human bladder cancer (BC). Two components in TS, 4-aminobiphenyl (4-ABP) and acrolein, which also are environmental contaminants, can cause bladder tumor in rat models. Their role in TS related BC has not been forthcoming. To establish the relationship between acrolein and 4-ABP exposure and BC, we analyzed acrolein-deoxyguanosine (dG) and 4-ABP-DNA adducts in normal human urothelial mucosa (NHUM) and bladder tumor tissues (BTT), and measured their mutagenicity in human urothelial cells. We found that the acrolein-dG levels in NHUM and BTT are 10-30 fold higher than 4-ABP-DNA adduct levels and that the acrolein-dG levels in BTT are 2 fold higher than in NHUM. Both acrolein-dG and 4-ABP-DNA adducts are mutagenic; however, the former are 5 fold more mutagenic than the latter. These two types of DNA adducts induce different mutational signatures and spectra. We found that acrolein inhibits nucleotide excision and base excision repair and induces repair protein degradation in urothelial cells. Since acrolein is abundant in TS, inhaled acrolein is excreted into urine and accumulates in the bladder and because acrolein inhibits DNA repair and acrolein-dG DNA adducts are mutagenic, we propose that acrolein is a major bladder carcinogen in TS.

  18. The International Nucleotide Sequence Database Collaboration.

    Science.gov (United States)

    Cochrane, Guy; Karsch-Mizrachi, Ilene; Nakamura, Yasukazu

    2011-01-01

    Under the International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org), globally comprehensive public domain nucleotide sequence is captured, preserved and presented. The partners of this long-standing collaboration work closely together to provide data formats and conventions that enable consistent data submission to their databases and support regular data exchange around the globe. Clearly defined policy and governance in relation to free access to data and relationships with journal publishers have positioned INSDC databases as a key provider of the scientific record and a core foundation for the global bioinformatics data infrastructure. While growth in sequence data volumes comes no longer as a surprise to INSDC partners, the uptake of next-generation sequencing technology by mainstream science that we have witnessed in recent years brings a step-change to growth, necessarily making a clear mark on INSDC strategy. In this article, we introduce the INSDC, outline data growth patterns and comment on the challenges of increased growth.

  19. Bacterial nucleotide-based second messengers.

    Science.gov (United States)

    Pesavento, Christina; Hengge, Regine

    2009-04-01

    In all domains of life nucleotide-based second messengers transduce signals originating from changes in the environment or in intracellular conditions into appropriate cellular responses. In prokaryotes cyclic di-GMP has emerged as an important and ubiquitous second messenger regulating bacterial life-style transitions relevant for biofilm formation, virulence, and many other bacterial functions. This review describes similarities and differences in the architecture of the cAMP, (p)ppGpp, and c-di-GMP signaling systems and their underlying signaling principles. Moreover, recent advances in c-di-GMP-mediated signaling will be presented and the integration of c-di-GMP signaling with other nucleotide-based signaling systems will be discussed.

  20. Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions.

    Science.gov (United States)

    Hesketh, Andy; Vergnano, Marta; Wan, Chris; Oliver, Stephen G

    2017-07-25

    We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. IMPORTANCE During infections, pathogenic bacteria can release nucleotides into the cells of their eukaryotic hosts. These nucleotides are recognized as signals that contribute to the initiation of defensive immune responses that help the infected

  1. Nucleotide Manipulatives to Illustrate the Central Dogma

    OpenAIRE

    Sonja B. Yung; Todd P. Primm

    2015-01-01

    The central dogma is a core concept that is critical for introductory biology and microbiology students to master. However, students often struggle to conceptualize the processes involved, and fail to move beyond simply memorizing the basic facts. To encourage critical thinking, we have designed a set of magnetic nucleotide manipulatives that allow students to model DNA structure, along with the processes of replication, transcription, and translation.

  2. Nucleotide Manipulatives to Illustrate the Central Dogma

    Directory of Open Access Journals (Sweden)

    Sonja B. Yung

    2015-08-01

    Full Text Available The central dogma is a core concept that is critical for introductory biology and microbiology students to master. However, students often struggle to conceptualize the processes involved, and fail to move beyond simply memorizing the basic facts. To encourage critical thinking, we have designed a set of magnetic nucleotide manipulatives that allow students to model DNA structure, along with the processes of replication, transcription, and translation.

  3. Pyrrolidine nucleotide analogs with a tunable conformation

    Czech Academy of Sciences Publication Activity Database

    Poštová Slavětínská, Lenka; Rejman, Dominik; Pohl, Radek

    2014-01-01

    Roč. 10, Aug 22 (2014), s. 1967-1980 ISSN 1860-5397 R&D Projects: GA ČR GA13-24880S Institutional support: RVO:61388963 Keywords : conformation * NMR * nucleic acids * nucleotide analog * phosphonic acid * pseudorotation * pyrrolidine Subject RIV: CC - Organic Chemistry Impact factor: 2.762, year: 2014 http://www.beilstein-journals.org/bjoc/single/articleFullText.htm?publicId=1860-5397-10-205

  4. Vacuum ultraviolet photoionization of carbohydrates and nucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Joong-Won, E-mail: jshin@govst.edu [Division of Science, Governors State University, University Park, Illinois 60484-0975 (United States); Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872 (United States); Bernstein, Elliot R., E-mail: erb@lamar.colostate.edu [Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872 (United States)

    2014-01-28

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5{sup ′}-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

  5. Vacuum ultraviolet photoionization of carbohydrates and nucleotides

    International Nuclear Information System (INIS)

    Shin, Joong-Won; Bernstein, Elliot R.

    2014-01-01

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5 ′ -monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results

  6. Descriptive Study of Patients Receiving Excision and Radiotherapy for Keloids

    International Nuclear Information System (INIS)

    Speranza, Giovanna; Sultanem, Khalil M.D.; Muanza, Thierry

    2008-01-01

    Purpose: To review and describe our institution's outcomes in patients treated with external beam radiotherapy after keloid excision. Methods and Materials: This was a retrospective study. Patients who received radiotherapy between July 1994 and January 2004 after keloid excision were identified. A questionnaire was mailed regarding sociodemographic factors, early and late radiation toxicities, the need for additional therapy, and satisfaction level. All patients had received a total of 15 Gy in three daily 5-Gy fractions. Treatment started within 24 h after surgery and was delivered on a Siemens orthovoltage machine. The data were analyzed using the STATA statistical package. Results: A total of 234 patients were approached. The response rate was 41%, and 75% were female. The mean age was 36.5 years (range, 16-69 years). The patients were mainly of European (53.1%) or African (19.8%) descent. For early toxicity outcomes, 54.2% reported skin redness and 24% reported skin peeling. For late toxicity outcomes, 27% reported telangiectasia and 62% reported permanent skin color changes. No association was found with gender, skin color, or age for the late toxicity outcomes. Of the patients responding, 14.6% required adjuvant treatment. On a visual scale of 1-10 for the satisfaction level, 60% reported a satisfaction level of ≥8. Telangiectasia was the most significant predictor of a low satisfaction level (≤3, p < 0.005). Conclusion: The results of our study have shown that orthovoltage-based radiotherapy after surgical excision for keloids is a good method for the prevention of relapse. It is well tolerated, causes little toxicity, and leads to a high patient satisfaction level

  7. Inverted 'V' osteotomy excision arthroplasty for bony ankylosed elbows

    Directory of Open Access Journals (Sweden)

    Rex Chadrabose

    2011-12-01

    Full Text Available Abstract Background Bony ankylosis of elbow is challenging and difficult problem to treat. The options are excision arthroplasty and total elbow replacement. We report our midterm results on nine patients, who underwent inverted 'V' osteotomy excision arthroplasty in our hospital with good functional results. Materials Our case series includes 9 patients (seven males and two females with the mean age of 34 years (13-56 years. Five patients had trauma, two had pyogenic arthritis, one had tuberculous arthritis, and one had pyogenic arthritis following surgical fixation. Results The average duration of follow up is 65 months (45 months-80 months. The mean Mayo's elbow performance score (MEPS preoperatively was 48 (35-70. The MEPS at final follow up was 80 (60-95. With no movement at elbow and fixed in various degrees of either flexion or extension preoperatively, the mean preoperative position of elbow was 64°(30°to 100°. The mean post operative range of motion at final follow up was 27°of extension (20-500, 116°of flexion (1100-1300, and the arc of motion was 88°(800-1000. One patient had ulnar nerve neuropraxia and another patient developed median nerve neuropraxia, and both recovered completely in six weeks. No patient had symptomatic instability of the elbow. All patients were asymptomatic except one patient, who had pain mainly on heavy activities. Conclusion We conclude that inverted 'V' osteotomy excision arthroplasty is a viable option in the treatment of bony ankylosis of the elbow in young patients.

  8. Female urogenital dysfunction following total mesorectal excision for rectal cancer

    Directory of Open Access Journals (Sweden)

    Raja Ashraf

    2006-01-01

    Full Text Available Abstract Background The effect of Total Mesorectal Excision (TME on sexual function in the male is well documented. However, there is little literature in female patients. The aim of this study was to review the pelvic autonomic nervous anatomy in the female and to perform a retrospective audit of urinary and sexual function in women following surgery for rectal cancer where TME had been performed. Urogenital dysfunction was assessed through interview and questionnaire. Method Twenty-three questionnaires, eighteen returned, were sent to women with a mean age 65.5 yrs (range 34–86. All had undergone total mesorectal excision for rectal cancer between 1998–2001. Mean follow-up was 18.8 months (range 3–35. Results Preoperatively 5/18 (28% were sexually active, 3/18 (17% of patients described urinary frequency and nocturia and 7/18 (39% described symptoms of stress incontinence prior to surgery. Postoperatively all sexually active patients remained active although all described some discomfort with penetration. Two of the patients sexually active described reduced libido secondary to the stoma. Postoperative urinary symptoms developed with 59% reporting the development of nocturia, 18% developed stress incontinence and one patient required a permanent catheter. Of those with symptoms, 80% persisted longer than three months from surgery. Symptoms were predominant in those patients with low rectal cancers, particularly those undergoing abdomino-perineal excision and in those who had previously undergone abdominal hysterectomy. Conclusion The treatment of rectal cancer involves surgery to the pelvic floor. Despite nerve preservation this is associated with the development of worsening nocturia and stress incontinence. This is most marked in those patients who had previously undergone a hysterectomy. Further studies are warranted to assess the interaction with previous gynaecological surgery.

  9. Abscisic Acid Stimulates Elongation of Excised Pea Root Tips

    Science.gov (United States)

    Gaither, Douglas H.; Lutz, Donald H.; Forrence, Leonard E.

    1975-01-01

    Excised Pisum sativum L. root tips were incubated in a pH 5.2 sucrose medium containing abscisic acid. Elongation growth was inhibited by 100 μm abscisic acid. However, decreasing the abscisic acid concentration caused stimulation of elongation, the maximum response (25% to 30%) occurring at 1 μm abscisic acid. Prior to two hours, stimulation of elongation by 1 μm abscisic acid was not detectable. Increased elongation did not occur in abscisic acid-treated root tips of Lens culinaris L., Phaseolus vulgaris L., or Zea mays L. PMID:16659198

  10. Use of Preputial Skin as Cutaneous Graft after Nevus Excision

    Directory of Open Access Journals (Sweden)

    A. D'Alessio

    2010-01-01

    Full Text Available We report a four-year-old boy with a nevus covering all the plantar side of his second finger on the left foot. He was also affected by congenital phimosis. Surgical excision of the nevus was indicated, but the skin defect would have been too large to be directly closed. The foreskin was taken as a full-thickness skin graft to cover the cutaneous defect of the finger. The graft intake was favourable and provided a functional repair with good aesthetic characteristic.

  11. Base excision repair mechanisms and relevance to cancer susceptibility

    International Nuclear Information System (INIS)

    Dogliotti, E.; Wilson, S.H.

    2009-01-01

    The base excision repair (BER) pathway is considered the predominant DNA repair system in mammalian cells for eliminating small DNA lesions generated at DNA bases either exogenously by environmental agents or endogenously by normal cellular metabolic processes (e.g. production of oxyradical species, alkylating agents, etc). The main goal of this project is the understanding of the involvement of BER in genome stability and in particular in sporadic cancer development associated with inflammation such as gastric cancer (GC). A major risk factor of GC is the infection by Helicobacter pylori, which causes oxidative stress. Oxidative DNA damage is mainly repaired by BER

  12. Pure transvaginal excision of mesh erosion involving the bladder.

    Science.gov (United States)

    Firoozi, Farzeen; Goldman, Howard B

    2013-06-01

    We present a pure transvaginal approach to the removal of eroded mesh involving the bladder secondary to placement of transvaginal mesh for management of pelvic organ prolapse (POP) using a mesh kit. Although technically challenging, we demonstrate the feasibility of a purely transvaginal approach, avoiding a potentially more morbid transabdominal approach. The video presents the surgical technique of pure transvaginal excision of mesh erosion involving the bladder after mesh placement using a prolapse kit was performed. This video shows that purely transvaginal removal of mesh erosion involving the bladder can be done safely and is feasible.

  13. Identification of cyclic nucleotide gated channels using regular expressions

    KAUST Repository

    Zelman, Alice K.; Dawe, Adam Sean; Berkowitz, Gerald A.

    2013-01-01

    Cyclic nucleotide-gated channels (CNGCs) are nonselective cation channels found in plants, animals, and some bacteria. They have a six-transmembrane/one- pore structure, a cytosolic cyclic nucleotide-binding domain, and a cytosolic calmodulin

  14. Effects of hypokinesia on cyclic nucleotides and hormonal regulation ...

    African Journals Online (AJOL)

    PTH), calcitonin (CT), cyclic nucleotides (cAMP, cGMP) and calcium in the blood of rats, while in urine - phosphate, calcium and cyclic nucleotides. Design: Laboratory based experiment. Setting: Laboratory in the Department of Biochemistry, ...

  15. 26 CFR 55.4981-2 - Imposition of excise tax with respect to certain undistributed income of real estate investment...

    Science.gov (United States)

    2010-04-01

    ... certain undistributed income of real estate investment trusts; calendar years beginning after December 31... (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) EXCISE TAX ON REAL ESTATE INVESTMENT TRUSTS AND REGULATED INVESTMENT COMPANIES Excise Tax on Real Estate Investment Trusts § 55.4981-2 Imposition of excise tax with...

  16. A new strategy to inhibit the excision reaction catalysed by HIV-1 reverse transcriptase: compounds that compete with the template–primer

    Science.gov (United States)

    Cruchaga, Carlos; Anso, Elena; Font, María; Martino, Virginia S.; Rouzaut, Ana; Martinez-Irujo, Juan J.

    2007-01-01

    Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PPi-dependent phosphorolysis catalysed by wild-type and AZT (3′-azido-3′-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template–primer (Kd=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template–primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues. PMID:17355225

  17. Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA

    Science.gov (United States)

    Loh, Yuin-Han; Yang, Jimmy Chen; De Los Angeles, Alejandro; Guo, Chunguang; Cherry, Anne; Rossi, Derrick J.; Park, In-Hyun; Daley, George Q.

    2012-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This report describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. PMID:22605648

  18. Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy

    Directory of Open Access Journals (Sweden)

    Stylianos Michalakis

    2018-03-01

    Full Text Available The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP or cyclic adenosine monophosphate (cAMP. Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN channels and voltage-gated potassium channels (KCN. In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.

  19. Cloning human DNA repair genes

    International Nuclear Information System (INIS)

    Jeggo, P.A.; Carr, A.M.; Lehmann, A.R.

    1994-01-01

    Many human genes involved in the repair of UV damage have been cloned using different procedures and they have been of great value in assisting the understanding of the mechanism of nucleotide excision-repair. Genes involved in repair of ionizing radiation damage have proved more difficult to isolate. Positional cloning has localized the XRCC5 gene to a small region of chromosome 2q33-35, and a series of yeast artificial chromosomes covering this region have been isolated. Very recent work has shown that the XRCC5 gene encodes the 80 kDa subunit of the Ku DNA-binding protein. The Ku80 gene also maps to this region. Studies with fission yeast have shown that radiation sensitivity can result not only from defective DNA repair but also from abnormal cell cycle control following DNA damage. Several genes involved in this 'check-point' control in fission yeast have been isolated and characterized in detail. It is likely that a similar checkpoint control mechanism exists in human cells. (author)

  20. Repair of 3-methyladenine and abasic sites by base excision repair mediates glioblastoma resistance to temozolomide

    Energy Technology Data Exchange (ETDEWEB)

    Bobola, Michael S.; Kolstoe, Douglas D.; Blank, A. [Department of Neurological Surgery, University of Washington Medical Center, Seattle, WA (United States); Chamberlain, Marc C. [Department of Neurological Surgery, University of Washington Medical Center, Seattle, WA (United States); Department of Neurology, University of Washington Medical Center, Seattle, WA (United States); Silber, John R., E-mail: jrsilber@u.washington.edu [Department of Neurological Surgery, University of Washington Medical Center, Seattle, WA (United States)

    2012-11-30

    Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma (GBM). More recently, inclusion of temozolomide (TMZ), an orally administered methylating agent with low systemic toxicity, during and after radiotherapy has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O{sup 6}-methylguanine (O{sup 6}-meG) mediates GBM cell killing. Moreover, low or absent expression of O{sup 6}-methylguanine-DNA methyltransferase (MGMT), the sole human repair protein that removes O{sup 6}-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O{sup 6}-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA) and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O{sup 6}-meG, is mediated by the base excision repair (BER) pathway, and occurs independently of removal of O{sup 6}-meG. These observations indicate that BER activities are also potential targets for strategies to potentiate TMZ cytotoxicity. Here we review the evidence that 3-meA and abasic sites mediate killing of GBM cells. We also present in vitro and in vivo evidence that alkyladenine-DNA glycosylase, the sole repair activity that excises 3-meA from DNA, and Ape1, the major human abasic site endonuclease, mediate TMZ resistance in GBMs and represent potential anti-resistance targets.

  1. Framing the policy debate over spirits excise tax in Poland.

    Science.gov (United States)

    Zatonski, Mateusz; Hawkins, Benjamin; McKee, Martin

    2018-06-01

    Industry lobbying remains an obstacle to effective health-oriented alcohol policy. In 2013, an increase in excise tax on spirits was announced by the Polish government. This article presents a qualitative analysis of the public debate that ensued on the potential economic, health and social effects of the policy. It focuses on how competing groups, including industry actors, framed their position and sought to dominate the debate. Online archives of five Polish national newspapers, two spirits trade associations, and parliamentary and ministerial archives were searched. A thematic content analysis of the identified sources was conducted. The overall findings were compared with existing research on the framing of the Minimum Unit Pricing (MUP) debate in the UK. A total of 155 sources were analysed. Two main frames were identified: health, and economic. The spirits industry successfully promoted the economic frame in their own publications and in the media. The debate was dominated by arguments about potential growth of the grey market and losses in tax revenue that might result from the excise tax increase. The framing of the debate in Poland differed from the framing of the MUP debate in the United Kingdom. The Polish public health community was unsuccessful in making health considerations a significant element of the alcohol policy debate. The strategies pursued by UK health advocates offer lessons for how to make a more substantial impact on media coverage and promote health-oriented legislation.

  2. Validating excised rodent lungs for functional hyperpolarized xenon-129 MRI.

    Directory of Open Access Journals (Sweden)

    David M L Lilburn

    Full Text Available Ex vivo rodent lung models are explored for physiological measurements of respiratory function with hyperpolarized (hp (129Xe MRI. It is shown that excised lung models allow for simplification of the technical challenges involved and provide valuable physiological insights that are not feasible using in vivo MRI protocols. A custom designed breathing apparatus enables MR images of gas distribution on increasing ventilation volumes of actively inhaled hp (129Xe. Straightforward hp (129Xe MRI protocols provide residual lung volume (RV data and permit for spatially resolved tracking of small hp (129Xe probe volumes during the inhalation cycle. Hp (129Xe MRI of lung function in the excised organ demonstrates the persistence of post mortem airway responsiveness to intravenous methacholine challenges. The presented methodology enables physiology of lung function in health and disease without additional regulatory approval requirements and reduces the technical and logistical challenges with hp gas MRI experiments. The post mortem lung functional data can augment histological measurements and should be of interest for drug development studies.

  3. Economic Impact of Imposing Excise Tax on Plastic Bottles of Drinks

    Directory of Open Access Journals (Sweden)

    Eugenia Mardanugraha

    2018-03-01

    Full Text Available This research simulates the effect of imposing excise tax on plastic container of drinks towards economic performance of beverage industry in Indonesia and governmentâ˘A ´Zs tax revenue. The results showed that by imposing excise tax on plastic cups and plastic bottles the government would lose tax revenue from value added tax (PPN and corporate income tax (PPh badan more than they gain additional revenue from excise tax. Hence, imposing excise tax on drink containers should serve a clear purpose and an undeniable reason. This paper recommends the government to develop proper excise infrastructure to extend the goods or services to be taxed. This paper also recommends the required stages for extending the excise tax.

  4. Sekuen Nukleotida Gene Shiga like toxin-2 dari Isolat Lokal Escherichia coli O157:H7 asal Hewan dan Manusia (NUCLEOTIDES SQUENCES OF SHIGA-LIKE TOXIN 2 GENES OF ESCHERICHIA COLI O157:H7 LOCAL ISOLATES ORIGINATED FROM ANIMALS AND HUMAN

    Directory of Open Access Journals (Sweden)

    I Wayan Suardana

    2017-04-01

    Full Text Available Animals/livestock, especially cattle, are known as the main reservoir of Escherichia coli O157: H7. As the only one of zoonotic E. coli, the pathogenicity of these bacteria is determined by its ability to produce one or more very potent cytotoxin known as Shiga-like toxin (Stx or verocytotoxin, particularly of the Stx2 type that is closely related to the incidence of hemolytic uremic syndrome (HUS in humans. This study analyzed the nucleotide sequences of stx2 gene between isolates from animals and humans in an effort to assess the potential zoonoses of the agent. The research activity was initiated by cultivating 20 isolates of E. coli O157:H7 collection based on result in the previous study i.e. 2 isolates originated from cattle feces, 2 isolates originated from beef, 2 isolates originated from chicken feces, 2 isolates originated from human feces, and 12 non-clinical isolates originated from human fecal who were suffering with renal failure. All isolates were confirmed on selective medium Sorbitol MacConkey Agar (SMAC followed by testing on aglutination O157 latex test, and H7 antisera. Molecular analysis of stx2 gene covering open reading frame (ORF of the stx2 gene was performed using the primer which was designed by researcher i.e. Stx2 (F/Stx2 (R. The results showed, there were 2 isolates i.e. KL-48 (2 originated from human feces and SM-25 (1 originated from cattle feces were positive for carrying a stx2 gene, which was marked by the 1587 bp PCR product. Analysis of sequencing showed both isolates had identical to stx2 nucleotide squences with E. phaga 933 as well as E. coli ATCC 933. These results indicate the both local isolates are potential as zoonotic agents with clinical effects similar to E. phaga 933 and E. coli ATCC 43894. ABSTRAK Hewan ternak khususnya sapi, dikenal sebagai reservoir utama Escherichia coli O157:H7. Sebagai satu-satunya serotipe E. coli yang bersifat zoonosis, patogenitas bakteri ini ditentukan oleh kemampuannya

  5. Multifactor dimensionality reduction analysis identifies specific nucleotide patterns promoting genetic polymorphisms

    Directory of Open Access Journals (Sweden)

    Arehart Eric

    2009-03-01

    Full Text Available Abstract Background The fidelity of DNA replication serves as the nidus for both genetic evolution and genomic instability fostering disease. Single nucleotide polymorphisms (SNPs constitute greater than 80% of the genetic variation between individuals. A new theory regarding DNA replication fidelity has emerged in which selectivity is governed by base-pair geometry through interactions between the selected nucleotide, the complementary strand, and the polymerase active site. We hypothesize that specific nucleotide combinations in the flanking regions of SNP fragments are associated with mutation. Results We modeled the relationship between DNA sequence and observed polymorphisms using the novel multifactor dimensionality reduction (MDR approach. MDR was originally developed to detect synergistic interactions between multiple SNPs that are predictive of disease susceptibility. We initially assembled data from the Broad Institute as a pilot test for the hypothesis that flanking region patterns associate with mutagenesis (n = 2194. We then confirmed and expanded our inquiry with human SNPs within coding regions and their flanking sequences collected from the National Center for Biotechnology Information (NCBI database (n = 29967 and a control set of sequences (coding region not associated with SNP sites randomly selected from the NCBI database (n = 29967. We discovered seven flanking region pattern associations in the Broad dataset which reached a minimum significance level of p ≤ 0.05. Significant models (p Conclusion The present study represents the first use of this computational methodology for modeling nonlinear patterns in molecular genetics. MDR was able to identify distinct nucleotide patterning around sites of mutations dependent upon the observed nucleotide change. We discovered one flanking region set that included five nucleotides clustered around a specific type of SNP site. Based on the strongly associated patterns identified in

  6. Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens.

    Science.gov (United States)

    Moulin, Morgane; Alguacil, Javier; Gu, Siyi; Mehtougui, Asmaa; Adams, Erin J; Peyrottes, Suzanne; Champagne, Eric

    2017-12-01

    Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

  7. Genome-wide patterns of nucleotide polymorphism in domesticated rice

    DEFF Research Database (Denmark)

    Caicedo, Ana L; Williamson, Scott H; Hernandez, Ryan D

    2007-01-01

    Domesticated Asian rice (Oryza sativa) is one of the oldest domesticated crop species in the world, having fed more people than any other plant in human history. We report the patterns of DNA sequence variation in rice and its wild ancestor, O. rufipogon, across 111 randomly chosen gene fragments......, and use these to infer the evolutionary dynamics that led to the origins of rice. There is a genome-wide excess of high-frequency derived single nucleotide polymorphisms (SNPs) in O. sativa varieties, a pattern that has not been reported for other crop species. We developed several alternative models...... to explain contemporary patterns of polymorphisms in rice, including a (i) selectively neutral population bottleneck model, (ii) bottleneck plus migration model, (iii) multiple selective sweeps model, and (iv) bottleneck plus selective sweeps model. We find that a simple bottleneck model, which has been...

  8. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    Science.gov (United States)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  9. Excision repair in MUT-mutants of Proteus mirabilis after UV-irradiation

    International Nuclear Information System (INIS)

    Stoerl, K.; Mund, C.

    1977-01-01

    The behaviour of MUT-mutants of P.mirabilis to perform certain steps of excision repair after U.V.-irradiation is described. MUT-mutants introduce single-strand breaks in the DNA immediately after U.V.-irradiation, but their ability to excise pyrimidine dimers from the DNA is very diminished. Moreover, they are not able to accomplish the excision repair by rejoining of the single-strand breaks. The connection between the incomplete excision repair and the mutator phenotype of these mutants is discussed. (author)

  10. Total mesorectal excision for the treatment of rectal cancer.

    Science.gov (United States)

    Zedan, Ali; Salah, Tareq

    2015-12-01

    In the surgical treatment of rectal cancer, a clear circumferential resection margin and distal resection margin should be obtained. The aim of this study was to determine the morbidity, mortality, survival outcome, and local failure after total mesorectal excision (TME) in the surgical treatment of rectal cancer. This retrospective study was conducted on 101 patients treated for rectal cancer using low anterior resection (LAR), abdominoperinial resection (APR), or Hartmaan's technique. In all operative procedures, total mesorectal excisions (TMEs) were done. The patients were treated from November 2000 to April 2011 in the South Egypt Cancer Institute (SECI) of Assuit University (Egypt). Neo-adjuvant therapy was given to those patients with serosalin filtration, lymph node involvement, and sexual and urinary function impairment. Data were analyzed using IBM-SPSS version 21, and survival rates were estimated using the Kaplan-Meier method. One hundred one patients were evaluable (61 males, 40 females). Regarding the operative procedure used, it was: (APR), LAR, Hartmaan's technique in 15.8%, 71.3%, and 12.9% of patients, respectively. Operation-related mortality during the 30 days after surgery was 3%. The operations resulted in morbidity in 25% of the patients, anastomotic site leak in 5.9% of the patients, urinary dysfynction in 9.9% of the patients, and erectile dysfunction in 15.8% of the male patients. Regarding safety margin, the median distances were distal/radial margin, 23/12 mm, distal limit 7 cm. Median lymph nodes harvest 19 nodes. Primary tumor locations were anteriorly 23.8%, laterally 13.9%, posteriorly 38.6%, and circumferential 23.8%. Protective stoma 16.8%. Primary Tumor TNM classification (T1, T2, T3, and T4; 3, 28.7, 55.4, and 12.9%, respectively). Nodes Metastases (N0, N1, and N2; 57.4, 31.7, and 10.9%, respectively). TNM staging (I, II, III, and IV; 15.8, 29.7, 46.5, and 7.9%, respectively). Chemotherapy was administered to 67.3% of the

  11. Detecção do DNA do papilomavírus humano após excisão da zona de transformação com alça diatérmica para tratamento de neoplasia intra-epitelial cervical Human papillomavirus DNA detection after large loop excision of the transformation zone for the treatment of cervical intraepithelial neoplasia

    Directory of Open Access Journals (Sweden)

    Priscila Garcia Figueirêdo

    2003-02-01

    Full Text Available OBJETIVO: avaliar a presença do DNA do papilomavírus humano (HPV de alto risco oncológico antes e quatro meses após excisão da zona de transformação com alça diatérmica em mulheres com neoplasia intra-epitelial cervical (NIC. MÉTODOS: neste estudo clínico prospectivo foram incluídas 78 mulheres submetidas à excisão da zona de transformação tratadas no período de fevereiro a dezembro de 2001. Todas foram submetidas a colposcopia, citologia oncológica e captura híbrida II (CH II antes da cirurgia e após 4±1,25 meses. Para análise estatística utilizou-se o cálculo do odds ratio (OR com intervalo de confiança de 95% (IC 95%. RESULTADOS: antes da excisão, 67 (86% mulheres apresentavam CH II positiva para DNA-HPV de alto risco oncológico e destas, apenas 22 (33% mantiveram a CH II positiva quatro meses após. A detecção do DNA-HPV após o tratamento não se relacionou com a carga viral prévia, presença de doença nas margens da peça cirúrgica ou idade da mulher. Após quatro meses, a detecção do DNA-HPV associou-se significativamente com a presença de alterações citológicas (OR = 4,8; IC 95% = 1,7-13,7, porém não se relacionou com doença residual ou recidiva histológica (OR = 6,0; IC 95% = 0,8-52,3. CONCLUSÃO: após o tratamento da NIC, a detecção do DNA-HPV diminuiu significativamente porém não se observou relação com a presença de doença residual ou recidiva histológica.PURPOSE: to evaluate the detection of high oncogenic risk human papillomavirus DNA (HPV-DNA immediately before and 4±1.25 months after large loop excision of the transformation zone (LLETZ in the treatment of cervical intraepithelial neoplasia (CIN. METHODS: in this clinical prospective study, 78 patients submitted to LLETZ from February to December 2001 were enrolled. All patients were submitted to colposcopic evaluation and had Pap smear and hybrid capture II (HC II specimens collected immediately before LLETZ and four months

  12. Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions

    Directory of Open Access Journals (Sweden)

    Andy Hesketh

    2017-07-01

    Full Text Available We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP, cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection.

  13. Classifying Coding DNA with Nucleotide Statistics

    Directory of Open Access Journals (Sweden)

    Nicolas Carels

    2009-10-01

    Full Text Available In this report, we compared the success rate of classification of coding sequences (CDS vs. introns by Codon Structure Factor (CSF and by a method that we called Universal Feature Method (UFM. UFM is based on the scoring of purine bias (Rrr and stop codon frequency. We show that the success rate of CDS/intron classification by UFM is higher than by CSF. UFM classifies ORFs as coding or non-coding through a score based on (i the stop codon distribution, (ii the product of purine probabilities in the three positions of nucleotide triplets, (iii the product of Cytosine (C, Guanine (G, and Adenine (A probabilities in the 1st, 2nd, and 3rd positions of triplets, respectively, (iv the probabilities of G in 1st and 2nd position of triplets and (v the distance of their GC3 vs. GC2 levels to the regression line of the universal correlation. More than 80% of CDSs (true positives of Homo sapiens (>250 bp, Drosophila melanogaster (>250 bp and Arabidopsis thaliana (>200 bp are successfully classified with a false positive rate lower or equal to 5%. The method releases coding sequences in their coding strand and coding frame, which allows their automatic translation into protein sequences with 95% confidence. The method is a natural consequence of the compositional bias of nucleotides in coding sequences.

  14. Laser excision of a mucocele: A case report

    Directory of Open Access Journals (Sweden)

    Rajashree Ganguly

    2015-01-01

    Full Text Available A mucous cyst (mucocele, mucous retention cyst, ranula, and epulis is usually a painless, thin sac on the inner surface of the lips. It contains clear fluid. It is painless but can be bothersome. The cyst is thought to be caused due to sucking of the lip membranes between the teeth. A mucous cyst is common and harmless. However, if left untreated, it can organize and form a permanent bump on the inner surface of the lip. A mucous cyst is called ranula when it occurs on the floor of the mouth and epulis when it occurs on the gums. The sac may also be formed around the jewellery (piercings that has been inserted into the lips or the tongue. This article highlights the use of lasers in the treatment of the excision of mucocele.

  15. [A modified technique of liposuction with excision for gynecomastia].

    Science.gov (United States)

    Yang, Hong-Yan; Xu, Jun; Yan, Xiao-Qing; You, Lei

    2007-11-20

    To introduce a modified technique of liposuction with excision for gynecomastia. From 2003 to 2007, 32 cases of gynecomastia were treated with the modified technique: the operative region was divided into central and periphery parts, liposuction was performed only in the periphery part and the deep layer of the central part, while the breast gland in the superficial layer of the central part underwent sharp dissection, the subcutaneous tissue of the central part was conserved, and blood supply was reserved and saucer deformity was avoided. Follow-up was conducted for 3.0 - 18.5 months. Normal men breast appearance was achieved. No complication happened such as hematoma, seroma, saucer deformity, and necrosis in nipple and areola. This modified operative technique for gynecomastia proves to be an excellent and effective technique.

  16. Harmonization of chosen excise duty in the EU

    Directory of Open Access Journals (Sweden)

    Petr David

    2008-01-01

    Full Text Available The level and structure of cigarettes taxation are very much in the news these days. In the field of tax on cigarettes in the European Union there is the question of whether specific rate or ad valorem rate should be used. The choice between these two rates depends on primary aim of tax policy in each European Union member state. The founding is that differential excises could be used as pro­tec­tio­nist trade barriers. Decision about tax rate on cigarettes can bring some other related effects. That is the reason why European Union established some restrictions in the field of level and structure of cigarettes taxation.

  17. Reverse-hybrid robotic mesorectal excision for rectal cancer.

    Science.gov (United States)

    Park, In Ja; You, Y Nancy; Schlette, Erika; Nguyen, Sa; Skibber, John M; Rodriguez-Bigas, Miguel A; Chang, George J

    2012-02-01

    The robotic system offers potential technical advantages over laparoscopy for total mesorectal excision with radical lymphadenectomy for rectal cancer. However, the requirement for fixed docking limits its utility when the working volume is large or patient repositioning is required. The purpose of this study was to evaluate short-term outcomes associated with a novel setup to perform total mesorectal excision and radical lymphadenectomy for rectal cancer by the use of a "reverse" hybrid robotic-laparoscopic approach. This is a prospective consecutive cohort observational study of patients who underwent robotic rectal cancer resection from January 2009 to March 2011. During the study period, a technique of reverse-hybrid robotic-laparoscopic rectal resection with radical lymphadenectomy was developed. This technique involves reversal of the operative sequence with lymphovascular and rectal dissection to precede proximal colonic mobilization. This technique evolved from a conventional-hybrid resection with laparoscopic vascular control, colonic mobilization, and robotic pelvic dissection. Perioperative and short-term oncologic outcomes were analyzed. Thirty patients underwent reverse-hybrid resection. Median tumor location was 5 cm (interquartile range 3-9) from the anal verge. Median BMI was 27.6 (interquartile range 25.0-32.1 kg/m). Twenty (66.7%) received neoadjuvant chemoradiation. There were no conversions. Median blood loss was 100 mL (interquartile range 75-200). Total operation time was a median 369 (interquartile range 306-410) minutes. Median docking time was 6 (interquartile range 5-8) minutes, and console time was 98 (interquartile range 88-140) minutes. Resection was R0 in all patients; no patients had an incomplete mesorectal resection. Six patients (20%) underwent extended lymph node dissection or en bloc resection. Reverse-hybrid robotic surgery for rectal cancer maximizes the therapeutic applicability of the robotic and conventional laparoscopic

  18. Evaluation of type II thyroplasty on phonatory physiology in an excised canine larynx model

    Science.gov (United States)

    Devine, Erin E.; Hoffman, Matthew R.; McCulloch, Timothy M.; Jiang, Jack J.

    2016-01-01

    Objective Type II thyroplasty is an alternative treatment for spasmodic dysphonia, addressing hyperadduction by incising and lateralizing the thyroid cartilage. We quantified the effect of lateralization width on phonatory physiology using excised canine larynges. Methods Normal closure, hyperadduction, and type II thyroplasty (lateralized up to 5mm at 1mm increments with hyperadducted arytenoids) were simulated in excised larynges (N=7). Aerodynamic, acoustic, and videokymographic data were recorded at three subglottal pressures relative to phonation threshold pressure (PTP). One-way repeated measures ANOVA assessed effect of condition on aerodynamic parameters. Random intercepts linear mixed effects models assessed effects of condition and subglottal pressure on acoustic and videokymographic parameters. Results PTP differed across conditions (p<0.001). Condition affected percent shimmer (p<0.005) but not percent jitter. Both pressure (p<0.03) and condition (p<0.001) affected fundamental frequency. Pressure affected vibratory amplitude (p<0.05) and intra-fold phase difference (p<0.05). Condition affected phase difference between the vocal folds (p<0.001). Conclusions Hyperadduction increased PTP and worsened perturbation compared to normal, with near normal physiology restored with 1mm lateralization. Further lateralization deteriorated voice quality and increased PTP. Acoustic and videokymographic results indicate that normal physiologic relationships between subglottal pressure and vibration are preserved at optimal lateralization width, but then degrade with further lateralization. The 1mm optimal width observed here is due to the small canine larynx size. Future human trials would likely demonstrate a greater optimal width, with patient-specific value potentially determined based on larynx size and symptom severity. PMID:27223665

  19. Impact of cigarette taxation policy on excise revenues and cigarette consumption in Uzbekistan

    Directory of Open Access Journals (Sweden)

    Konstantin S. Krasovsky

    2013-05-01

    Full Text Available BACKGROUND: In 2012, Uzbekistan ratified the Framework Convention on Tobacco Control, which states that price and tax measures are an effective means of reducing tobacco consumption. We aimed to explore the effect of taxation policies on revenues and cigarette consumption. METHODS: Data on tax rates, revenues, cigarette sales were taken from national reports. To forecast potential revenues, a scenario analysis was performed. RESULTS: In 1991-2004, ad valorem excise system was in place in Uzbekistan, which was later replaced by the specific excise system. In 1997-2011, the nominal average excise has increased by a factor of twenty, but in real terms, after a sharp increase in 1999, average excise declined annually and increased only in 2010-2011. Annual cigarette sales per capita of adult population in 1999-2007 constituted 17-25 cigarette packs, while in 2008-2011 it increased to 30-37 packs. Four scenarios of excise tax increases in 2012 were developed: one actual scenario based on the rates effective in Uzbekistan in 2012, and three hypothetical ones anticipating excise rates increase by 1.5, 2 and 3-fold. With actual excise increase in 2012, the inflation-adjusted budget revenues would grow by 5%, and with three hypothetical - by 17%, 35% and 66% respectively, despite the decline of tax-paid cigarette sales. CONCLUSION: Stabilization or reduction in cigarette excises in Uzbekistan in 2002-2008 led to a decline in real excise revenues and the growth of cigarette sales. In 1999 and 2010-2011, excises were significantly increased and the real revenues have risen, despite the decline in cigarette sales. As cigarette prices are low, the illegal outflow of cigarettes from Uzbekistan apparently exceeds the illegal inflow. A significant increase in cigarette excise (1.5-3 fold can both increase budget revenues and reduce cigarette consumption, with greater increase yielding more benefits.

  20. Changes in nuclear protein acetylation in u.v.-damaged human cells

    International Nuclear Information System (INIS)

    Ramanathan, B.; Smerdon, M.J.

    1986-01-01

    The levels of nuclear protein acetylation in u.v.-irradiated human fibroblasts have been investigated. Initially, we measured the levels of acetylation in total acid-soluble nuclear proteins and observed two distinct differences between the irradiated and unirradiated (control) cells. Immediately after irradiation, there is a 'wave' of protein hyperacetylation that lasts for 2-6 h, followed by a hypoacetylation phase, lasting for many hours, and the total level of acetylation does not return to that of control cells until 24-72 h after u.v. damage. Both the magnitude and duration of each phase is dependent on the dose of u.v. light used. The wave of hyperacetylation is more pronounced at low u.v. doses, while the wave of hypoacetylation is more pronounced at higher u.v. doses. Furthermore, the duration of each phase is prolonged when cells are exposed to 2 mM hydroxyurea, an agent which retards the rate of excision repair at u.v.-damaged sites. Examinations of the acetylation levels of the individual nuclear proteins indicated that acetylation of the core histones follows the same pattern observed for the total acid-soluble protein fractions. Furthermore, these were the only major proteins in the total acid-soluble fraction observed to undergo the early, rapid hyperacetylation immediately following u.v. damage. These results raise the possibility that a causal relationship exists between nuclear protein acetylation and nucleotide excision repair of DNA in human cells. (author)

  1. Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats

    Directory of Open Access Journals (Sweden)

    Desirée Villahermosa

    2017-05-01

    Full Text Available Defective mismatch repair (MMR in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6, which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1, which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3 recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1. Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes.

  2. Oesophageal pseudodiverticulum after foregut duplication cyst excision: Case report and literature review

    Directory of Open Access Journals (Sweden)

    Iuliana D Bobanga

    2016-01-01

    Full Text Available Oesophageal pseudodiverticula rarely occur after excision of benign oesophageal neoplasms. While management and outcomes have been reported in the adult leiomyoma literature, sparse data exist on the occurrence and management of pseudodiverticula after foregut duplication cyst excision. We discuss our experience with a paediatric patient and review relevant literature regarding operative techniques and surgical outcomes.

  3. Aerodynamic and Nonlinear Dynamic Acoustic Analysis of Tension Asymmetry in Excised Canine Larynges

    Science.gov (United States)

    Devine, Erin E.; Bulleit, Erin E.; Hoffman, Matthew R.; McCulloch, Timothy M.; Jiang, Jack J.

    2012-01-01

    Purpose: To model tension asymmetry caused by superior laryngeal nerve paralysis (SLNP) in excised larynges and apply perturbation, nonlinear dynamic, and aerodynamic analyses. Method: SLNP was modeled in 8 excised larynges using sutures and weights to mimic cricothyroid (CT) muscle function. Weights were removed from one side to create tension…

  4. 77 FR 43157 - Disregarded Entities and the Indoor Tanning Services Excise Tax; Correction

    Science.gov (United States)

    2012-07-24

    ... Disregarded Entities and the Indoor Tanning Services Excise Tax; Correction AGENCY: Internal Revenue Service... tax. DATES: This correction is effective on July 24, 2012, and applies on and after June 25, 2012. FOR... Subjects in 26 CFR Part 301 Employment taxes, Estate taxes, Excise taxes, Gift taxes, Income taxes...

  5. Connective tissue: cancer patients’ attitudes towards medical research using excised (tumour) tissue

    NARCIS (Netherlands)

    Vermeulen, E.; Schmidt, M.K.; Cornel, M.C.; Knoppers, B.M.; van Leeuwen, F.E.; Aaronson, N.K.

    2011-01-01

    The objective of this article is to explore the views of Dutch cancer patients on the use of excised and stored (tumor) tissues in medical research. Excised tissues are routinely stored in hospitals for future diagnostic use. They are also important for scientific research. This article discusses

  6. Posterior Endoscopic Excision of Os Trigonum in Professional National Ballet Dancers.

    Science.gov (United States)

    Ballal, Moez S; Roche, Andy; Brodrick, Anna; Williams, R Lloyd; Calder, James D F

    2016-01-01

    Previous studies have compared the outcomes after open and endoscopic excision of an os trigonum in patients of mixed professions. No studies have compared the differences in outcomes between the 2 procedures in elite ballet dancers. From October 2005 to February 2010, 35 professional ballet dancers underwent excision of a symptomatic os trigonum of the ankle after a failed period of nonoperative treatment. Of the 35 patients, 13 (37.1%) underwent endoscopic excision and 22 (62.9%) open excision. We compared the outcomes, complications, and time to return to dancing. The open excision group experienced a significantly greater incidence of flexor hallucis longus tendon decompression compared with the endoscopic group. The endoscopic release group returned to full dance earlier at a mean of 9.8 (range 6.5 to 16.1) weeks and those undergoing open excision returned to full dance at a mean of 14.9 (range 9 to 20) weeks (p = .001). No major complications developed in either group, such as deep infection or nerve or vessel injury. We have concluded that both techniques are safe and effective in the treatment of symptomatic os trigonum in professional ballet dancers. Endoscopic excision of the os trigonum offers a more rapid return to full dance compared with open excision. Copyright © 2016 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

  7. Re-excision of margins before breast radiation-diagnostic or therapeutic?

    International Nuclear Information System (INIS)

    Chism, Derek B.; Freedman, Gary M.; Li, Tianyu; Anderson, Penny R.

    2006-01-01

    Purpose: To identify factors in breast cancer patients that predict the pathologic results of re-excision for close or positive margins and to determine the effect on local control. Methods and Materials: We divided 1,044 patients with Stage I-II breast cancer with a close (≤2 mm) or positive margin after initial excision into three groups. Group 1 included 199 patients without additional excision, Group 2 included 546 patients with re-excision found to be free of cancer, and Group 3 included 299 patients with re-excision and residual cancer. All patients were treated with radiotherapy with a median follow-up of 6.7 years. Results: The 10-year local control rate was 95% for Group 1 and 94% for Groups 2 and 3 (p = 0.788). Of the 846 patients, 65% had no residual disease on re-excision and 35% did have residual tumor. The factors significantly associated with positive re-excision findings were initial positive margins, positive nodes, Stage T2 tumor, and an extensive intraductal component. The 10-year local control rate was 95% for Group 2 vs. 91% for Group 3 (p = 0.038). Conclusion: The low recurrence rates seen in this study suggest that selected patients with non-negative margins, particularly those with a low risk of having residual disease at re-excision, may be treated with radiotherapy

  8. Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

    Directory of Open Access Journals (Sweden)

    Elaine C Thomas

    Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

  9. Human telomeres are hypersensitive to UV-induced DNA Damage and refractory to repair.

    Directory of Open Access Journals (Sweden)

    Patrick J Rochette

    2010-04-01

    Full Text Available Telomeric repeats preserve genome integrity by stabilizing chromosomes, a function that appears to be important for both cancer and aging. In view of this critical role in genomic integrity, the telomere's own integrity should be of paramount importance to the cell. Ultraviolet light (UV, the preeminent risk factor in skin cancer development, induces mainly cyclobutane pyrimidine dimers (CPD which are both mutagenic and lethal. The human telomeric repeat unit (5'TTAGGG/CCCTAA3' is nearly optimal for acquiring UV-induced CPD, which form at dipyrimidine sites. We developed a ChIP-based technique, immunoprecipitation of DNA damage (IPoD, to simultaneously study DNA damage and repair in the telomere and in the coding regions of p53, 28S rDNA, and mitochondrial DNA. We find that human telomeres in vivo are 7-fold hypersensitive to UV-induced DNA damage. In double-stranded oligonucleotides, this hypersensitivity is a property of both telomeric and non-telomeric repeats; in a series of telomeric repeat oligonucleotides, a phase change conferring UV-sensitivity occurs above 4 repeats. Furthermore, CPD removal in the telomere is almost absent, matching the rate in mitochondria known to lack nucleotide excision repair. Cells containing persistent high levels of telomeric CPDs nevertheless proliferate, and chronic UV irradiation of cells does not accelerate telomere shortening. Telomeres are therefore unique in at least three respects: their biophysical UV sensitivity, their prevention of excision repair, and their tolerance of unrepaired lesions. Utilizing a lesion-tolerance strategy rather than repair would prevent double-strand breaks at closely-opposed excision repair sites on opposite strands of a damage-hypersensitive repeat.

  10. Resistance of 3T3 mouse cells to UV light in relation to excision and transfer of dimers to daughter strands

    Energy Technology Data Exchange (ETDEWEB)

    Menck, C F.M.; Meneghini, R [Sao Paulo Univ. (Brazil). Inst. de Quimica

    1982-04-01

    Mouse cells (3T3 line) and human fibroblasts are equally sensitive to UV light. At fluences of 2.0-2.5 J/m/sup 2/ mouse cells excise only 20% of the pyrimidine dimers as compared to 80% excised by human fibroblasts. This fluence allows 37% survival in both cases. Hence, mouse cells are more resistant to the same burden of unexcised dimers. The reason for this increased tolerance to dimers does not seem to be due to a recombinational mechanism, as judged by the fact that only ca. 5% of the dimers are transferred from parental to daughter strands. The transfer of dimers was measured by the Micrococcus luteus UV endonuclease assay, irradiating cells at G/sub 1/ to avoid artifacts arising from introduction of dimers in nascent strands. The possibility of other mechanisms being involved in the process of tolerance to DNA lesions is discussed.

  11. Lower Lip Reconstruction after Wide Excision of a Malignancy with Barrel-Shaped Excision or the Webster Modification of the Bernard Operation

    Directory of Open Access Journals (Sweden)

    Hyung Joon Seo

    2013-01-01

    Full Text Available BackgroundBecause there are numerous methods for reconstruction of the lower lip, it is not easy to choose the optimal method. In choosing the surgical method for lower lip reconstruction, we obtained acceptable outcomes based on our treatment strategy, which included either a barrel-shaped excision or the Webster modification of the Bernard operation. We report on the surgical outcomes based on our treatment strategy.MethodsThis study included 26 patients who underwent lower lip reconstructive surgery from September 1996 to September 2010. The operation was done using either a barrel-shaped excision or the Webster modification, considering the location of the defect, the size of the defect, and the amount of residual tissue on the lateral side of the vermilion after excision.ResultsIn our series, 3 patients underwent a single barrel-shaped excision, and nine patients underwent a double barrel-shaped excision. In addition, the unilateral Webster modification was performed on in 6 patients, and there were eight cases of bilateral Webster modification. All of the patients except one were satisfied with the postoperative shape of the lip. In one case both recurrence and dehiscence occurred. One patient had a good postoperative lip shape, but had difficulty wearing a denture, and also underwent commissuroplasty. Furthermore, there were two patients who complained of drooling, and 4 with paresthesia.ConclusionsA soft tissue defect resulting from wide excision of a lower lip malignancy can be successfully reconstructed using only one of two surgical methods: the barrel-shaped excision or the Webster modification of the Bernard operation.

  12. DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Elena K Braithwaite

    2010-08-01

    Full Text Available Base excision repair (BER is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol beta is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol lambda, was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases lambda and beta in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol beta and pol lambda. Neutral red viability assays demonstrated that pol lambda and pol beta double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol lambda to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol lambda and pol beta interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.

  13. Post-operative outcomes following the excision of dorsal wrist ganglions with/without the use of Methylene Blue

    Directory of Open Access Journals (Sweden)

    Suleyman Tas

    2016-04-01

    Conclusions: The recurrence of cysts is rare after complete excision, particularly when rupture is prevented. Methylene blue injection is useful for ensuring complete excision and, therefore, to prevent recurrence. [Hand Microsurg 2016; 5(1.000: 1-6

  14. Retropharyngeal Schwannoma Excised Through a Transoral Approach: A Case Report

    Directory of Open Access Journals (Sweden)

    Chia-Ying Hsieh

    2006-09-01

    Full Text Available The contents of the retropharyngeal space are limited to fat and retropharyngeal nodes. Primary tumors originating from the retropharyngeal space are rare. More than 25% of schwannomas are found in the head and neck region, and they are rarely found in the retropharyngeal space. Here, we report the case of a 44-year-old woman with a schwannoma confined to the left retropharyngeal space, who presented with snoring and a mild lump in the throat sensation. Physical examination revealed anterior bulging of the left oropharyngeal wall, with intact mucosa. Magnetic resonance imaging showed a well-defined, encapsulated tumor in the left retropharyngeal space with bright signal intensity on T2-weighted images and low signal intensity on T1-weighted images, which was strongly enhanced after gadolinium administration. The tumor was removed through a transoral approach, resulting in a short postoperative recovery time without complications. The pathologic diagnosis was schwannoma. The patient has been well and free of tumor recurrence for 2 years. From anatomic and physiologic viewpoints, excision through a transoral approach is a good choice for a confined retropharyngeal schwannoma.

  15. Germline excision of transgenes in Aedes aegypti by homing endonucleases.

    Science.gov (United States)

    Aryan, Azadeh; Anderson, Michelle A E; Myles, Kevin M; Adelman, Zach N

    2013-01-01

    Aedes (Ae.) aegypti is the primary vector for dengue viruses (serotypes1-4) and chikungunya virus. Homing endonucleases (HEs) are ancient selfish elements that catalyze double-stranded DNA breaks (DSB) in a highly specific manner. In this report, we show that the HEs Y2-I-AniI, I-CreI and I-SceI are all capable of catalyzing the excision of genomic segments from the Ae. aegypti genome in a heritable manner. Y2-I-AniI demonstrated the highest efficiency at two independent genomic targets, with 20-40% of Y2-I-AniI-treated individuals producing offspring that had lost the target transgene. HE-induced DSBs were found to be repaired via the single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways in a manner dependent on the availability of direct repeat sequences in the transgene. These results support the development of HE-based gene editing and gene drive strategies in Ae. aegypti, and confirm the utility of HEs in the manipulation and modification of transgenes in this important vector.

  16. Implications of raising cigarette excise taxes in Peru

    Directory of Open Access Journals (Sweden)

    Martin Gonzalez-Rozada

    Full Text Available ABSTRACT Objective To assess how raising cigarette excise taxes in Peru might impact cigarette consumption, and to determine if higher taxes would be regressive. Methods Total demand price elasticity was estimated by income groups using two datasets: quarterly time-series data from 1993 – 2012 and data from a cross-sectional survey of income and expenses conducted in 2008 – 2009 . A functional form of the cigarette demand in Peru was specified using the quarterly data set, and the demand price elasticity was estimated for the short and long run. Using the second data set and Deaton methodology, the implementation of elasticity estimation and by groups’ elasticity was done in a two-step procedure. Results Demand price elasticity was −0.7, implying that a 10% price increase via a new tax would reduce consumption by 7%. Demand price elasticity estimations by income group suggested that poorer families are not more price sensitive than richer ones, which implies that increasing cigarette taxes could be regressive. Conclusions Increasing cigarette taxes is the most efficient policy for inducing a reduction in smoking. However, in the case of Peru, an increase in cigarette taxes could be regressive.

  17. A retrospective study of surgically excised phaeochromocytomas in Newfoundland, Canada

    Directory of Open Access Journals (Sweden)

    Joanna Holland

    2014-01-01

    Full Text Available Objective: A retrospective study detailing the circumstances surrounding diagnosis and treatment of pheochromocytomas with the associated genetic disorders. Materials and Methods: All patients with surgically excised pheochromocytomas in the Health Sciences Center, St. John′s, Newfoundland, Canada between January 2001 and December 2010 were retrospectively analyzed to determine associated familial syndromes, age, tumor size, symptomatology, and percentage of paragangliomas and bilateral pheochromocytomas. Pathology specimen reports, adrenalectomy lists and Meditech (electronic medical record diagnostic codes provided a comprehensive database for this study. Results: Twenty-four patients were studied; familial disorder patients comprised 42% (10/24. Average age at diagnosis was 57 among the sporadic and 34 in familial disorder groups (P = 0.006. Average tumor size was 4.5 cm in the sporadic group and 3 cm in the familial disorder group (P = 0.19. All atypical cases including bilateral or extra-adrenal tumors and malignancy occurred in familial disorder patients. Conclusions: The proportion of familial disorder patients (42% was higher in this study than would be expected, likely a result of the relatively high incidence of hereditary autosomal dominant disorders within Newfoundland. Among familial disorder patients, the average younger age at diagnosis and the smaller tumor size suggest syndromic pheochromocytomas may develop earlier, however they are more likely to be diagnosed sooner due to biochemical surveillance testing in known genetic disorder patients. We also demonstrate a relatively high incidence of surgically resected pheochromocytomas of 4.679/million/year in Newfoundland.

  18. Total Humeral Endoprosthetic Replacement following Excision of Malignant Bone Tumors

    Directory of Open Access Journals (Sweden)

    Suhel Kotwal

    2016-01-01

    Full Text Available Humerus is a common site for malignant tumors. Advances in adjuvant therapies and reconstructive methods provide salvage of the upper limb with improved outcomes. Reports of limb salvage with total humeral replacement in extensive humeral tumors are sparse. We undertook a retrospective study of 20 patients who underwent total humeral endoprosthetic replacement as limb salvage following excision of extensile malignant tumor from 1990 to 2011. With an average followup of 42.9, functional and oncological outcomes were analyzed. Ten patients were still alive at the time of review. Mean estimated blood loss was 1131 mL and duration of surgery was 314 minutes. Deep infection was encountered in one patient requiring debridement while mechanical loosening of ulnar component was identified in one patient. Subluxation of prosthetic humeral head was noted in 3 patients. Mean active shoulder abduction was 12.5° and active flexion was 15°. Incompetence of abduction mechanism was the major determinant of poor active functional outcome. Mean elbow flexion was 103.5° with 30.5° flexion contracture in 10 patients with good and useful hand function. Average MSTS score was 71.5%. Total humeral replacement is a reliable treatment option in restoring mechanical stability and reasonable functional results without compromising patient survival, with low complication rate.

  19. Assessment of single nucleotide polymorphisms in screening 52 DNA repair and cell cycle control genes in Fanconi anemia patients

    Directory of Open Access Journals (Sweden)

    Petrović Sandra

    2015-01-01

    Full Text Available Fanconi anemia (FA is a rare genetically heterogeneous disorder associated with bone marrow failure, birth defects and cancer susceptibility. Apart from the disease- causing mutations in FANC genes, the identification of specific DNA variations, such as single nucleotide polymorphisms (SNPs, in other candidate genes may lead to a better clinical description of this condition enabling individualized treatment with improvement of the prognosis. In this study, we have assessed 95 SNPs located in 52 key genes involved in base excision repair (BER, nucleotide excision repair (NER, mismatch repair (MMR, double strand break (DSB repair and cell cycle control using a DNA repair chip (Asper Biotech, Estonia which includes most of the common variants for the candidate genes. The SNP genotyping was performed in five FA-D2 patients and in one FA-A patient. The polymorphisms studied were synonymous (n=10, nonsynonymous (missense (n=52 and in non-coding regions of the genome (introns and 5 ‘and 3’ untranslated regions (UTR (n=33. Polymorphisms found at the homozygous state are selected for further analysis. Our results have shown a significant inter-individual variability among patients in the type and the frequency of SNPs and also elucidate the need for further studies of polymorphisms located in ATM, APEX APE 1, XRCC1, ERCC2, MSH3, PARP4, NBS1, BARD1, CDKN1B, TP53 and TP53BP1 which may be of great importance for better clinical description of FA. In addition, the present report recommends the use of SNPs as predictive and prognostic genetic markers to individualize therapy of FA patients. [Projekat Ministarstva nauke Republike Srbije, br. 173046

  20. Extralevator Abdominal Perineal Excision Versus Standard Abdominal Perineal Excision: Impact on Quality of the Resected Specimen and Postoperative Morbidity.

    Science.gov (United States)

    Habr-Gama, Angelita; São Julião, Guilherme P; Mattacheo, Adrian; de Campos-Lobato, Luiz Felipe; Aleman, Edgar; Vailati, Bruna B; Gama-Rodrigues, Joaquim; Perez, Rodrigo Oliva

    2017-08-01

    Abdominal perineal excision (APE) has been associated with a high risk of positive circumferential resection margin (CRM+) and local recurrence rates in the treatment of rectal cancer. An alternative extralevator approach (ELAPE) has been suggested to improve the quality of resection by avoiding coning of the specimen decreasing the risk of tumor perforation and CRM+. The aim of this study is to compare the quality of the resected specimen and postoperative complication rates between ELAPE and "standard" APE. All patients between 1998 and 2014 undergoing abdominal perineal excision for primary or recurrent rectal cancer at a single Institution were reviewed. Between 1998 and 2008, all patients underwent standard APE. In 2009 ELAPE was introduced at our Institution and all patients requiring APE underwent this alternative procedure (ELAPE). The groups were compared according to pathological characteristics, specimen quality (CRM status, perforation and failure to provide the rectum and anus in a single specimen-fragmentation) and postoperative morbidity. Fifty patients underwent standard APEs, while 22 underwent ELAPE. There were no differences in CRM+ (10.6 vs. 13.6%; p = 0.70) or tumor perforation rates (8 vs. 0%; p = 0.30) between APE and ELAPE. However, ELAPE were less likely to result in a fragmented specimen (42 vs. 4%; p = 0.002). Advanced pT-stage was also a risk factor for specimen fragmentation (p = 0.03). There were no differences in severe (Grade 3/4) postoperative morbidity (13 vs. 10%; p = 0.5). Perineal wound dehiscences were less frequent among ELAPE (52 vs 13%; p < 0.01). Despite short follow-up (median 21 mo.), 2-year local recurrence-free survival was better for patients undergoing ELAPE when compared to APE (87 vs. 49%; p = 0.04). ELAPE may be safely implemented into routine clinical practice with no increase in postoperative morbidity and considerable improvements in the quality of the resected specimen of patients with low rectal