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Sample records for membrane repair pathway

  1. RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin.

    Science.gov (United States)

    Los, Ferdinand C O; Kao, Cheng-Yuan; Smitham, Jane; McDonald, Kent L; Ha, Christine; Peixoto, Christina A; Aroian, Raffi V

    2011-02-17

    Pore-forming toxins (PFTs) secreted by pathogenic bacteria are the most common bacterial protein toxins and are important virulence factors for infection. PFTs punch holes in host cell plasma membranes, and although cells can counteract the resulting membrane damage, the underlying mechanisms at play remain unclear. Using Caenorhabditis elegans as a model, we demonstrate in vivo and in an intact epithelium that intestinal cells respond to PFTs by increasing levels of endocytosis, dependent upon RAB-5 and RAB-11, which are master regulators of endocytic and exocytic events. Furthermore, we find that RAB-5 and RAB-11 are required for protection against PFT and to restore integrity to the plasma membrane. One physical mechanism involved is the RAB-11-dependent expulsion of microvilli from the apical side of the intestinal epithelial cells. Specific vesicle-trafficking pathways thus protect cells against an attack by PFTs on plasma membrane integrity, via altered plasma membrane dynamics.

  2. PCNA Modifications for Regulation of Post-Replication Repair Pathways

    OpenAIRE

    2008-01-01

    Stalled DNA replication forks activate specific DNA repair mechanism called post-replication repair (PRR) pathways that simply bypass DNA damage. The bypassing of DNA damage by PRR prevents prolonged stalling of DNA replication that could result in double strand breaks (DSBs). Proliferating cell nuclear antigen (PCNA) functions to initiate and choose different bypassing pathways of PRR. In yeast, DNA replication forks stalled by DNA damage induces monoubiquitination of PCNA at K164, which is ...

  3. Microparticle Assembly Pathways on Lipid Membranes

    Science.gov (United States)

    van der Wel, Casper; Heinrich, Doris; Kraft, Daniela J.

    2017-09-01

    Understanding interactions between microparticles and lipid membranes is of increasing importance, especially for unraveling the influence of microplastics on our health and environment. Here, we study how a short-ranged adhesive force between microparticles and model lipid membranes causes membrane-mediated particle assembly. Using confocal microscopy, we observe the initial particle attachment to the membrane, then particle wrapping, and in rare cases spontaneous membrane tubulation. In the attached state, we measure that the particle mobility decreases by 26%. If multiple particles adhere to the same vesicle, their initial single-particle state determines their interactions and subsequent assembly pathways: 1) attached particles only aggregate when small adhesive vesicles are present in solution, 2) wrapped particles reversibly attract one another by membrane deformation, and 3) a combination of wrapped and attached particles form membrane-mediated dimers, which further assemble into a variety of complex structures. The experimental observation of distinct assembly pathways induced only by a short ranged membrane-particle adhesion, shows that a cellular cytoskeleton or other active components are not required for microparticle aggregation. We suggest that this membrane-mediated microparticle aggregation is a reason behind reported long retention times of polymer microparticles in organisms.

  4. Electronic Pathways in Photoactivated Repair of UV Mutated DNA

    Science.gov (United States)

    Bohr, Henrik; Jalkanen, K. J.; Bary Malik, F.

    An investigation of the physics, underlying the damage caused to DNA by UV radiation and its subsequent repair via a photoreactivation mechanism, is presented in this study. Electronic pathways, starting from the initial damage to the final repair process, are presented. UV radiation is absorbed to create a hole-excited thymine or other pyrimidine that subsequently is responsible for the formation of a dimer. The negative-ion of the cofactor riboflavin, FADH-, formed by the exposure of the photolyase protein to visible light, interacts with the hole-excited electronic orbital of the thymine dimer inducing a photon-less Auger transition, which restores the two thymines to the ground state, thereby detaching the lesion and repairing the DNA. Density functional theoretical calculations supporting the theory are presented. The mechanism involves the least amount of energy dissipation and is charge neutral. It also avoids radiation damage in the repair process. Recent experimental data are compatible with this theory.

  5. Microhomology directs diverse DNA break repair pathways and chromosomal translocations.

    Directory of Open Access Journals (Sweden)

    Diana D Villarreal

    Full Text Available Chromosomal structural change triggers carcinogenesis and the formation of other genetic diseases. The breakpoint junctions of these rearrangements often contain small overlapping sequences called "microhomology," yet the genetic pathway(s responsible have yet to be defined. We report a simple genetic system to detect microhomology-mediated repair (MHMR events after a DNA double-strand break (DSB in budding yeast cells. MHMR using >15 bp operates as a single-strand annealing variant, requiring the non-essential DNA polymerase subunit Pol32. MHMR is inhibited by sequence mismatches, but independent of extensive DNA synthesis like break-induced replication. However, MHMR using less than 14 bp is genetically distinct from that using longer microhomology and far less efficient for the repair of distant DSBs. MHMR catalyzes chromosomal translocation almost as efficiently as intra-chromosomal repair. The results suggest that the intrinsic annealing propensity between microhomology sequences efficiently leads to chromosomal rearrangements.

  6. Regulation of DNA double-strand break repair pathway choice

    Institute of Scientific and Technical Information of China (English)

    Meena Shrivastav; Leyma P De Haro; Jac A Nickoloff

    2008-01-01

    DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources includ-ing reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1 (XRS2) complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.

  7. Repair pathways evident in human liver organ slices

    NARCIS (Netherlands)

    Vickers, Alison E. M.; Fisher, Robyn; Olinga, Peter; Dial, Sharon

    2011-01-01

    The extension of human liver slice culture viability for several days broadens the potential of this ex vivo model for characterizing pathways of organ injury and repair, and allows for the multiple dosing of compounds. Extended viability is demonstrated by continued synthesis of GSH and ATP, and ma

  8. Herpes Simplex Virus Latency: The DNA Repair-Centered Pathway

    Directory of Open Access Journals (Sweden)

    Jay C. Brown

    2017-01-01

    Full Text Available Like all herpesviruses, herpes simplex virus 1 (HSV1 is able to produce lytic or latent infections depending on the host cell type. Lytic infections occur in a broad range of cells while latency is highly specific for neurons. Although latency suggests itself as an attractive target for novel anti-HSV1 therapies, progress in their development has been slowed due in part to a lack of agreement about the basic biochemical mechanisms involved. Among the possibilities being considered is a pathway in which DNA repair mechanisms play a central role. Repair is suggested to be involved in both HSV1 entry into latency and reactivation from it. Here I describe the basic features of the DNA repair-centered pathway and discuss some of the experimental evidence supporting it. The pathway is particularly attractive because it is able to account for important features of the latent response, including the specificity for neurons, the specificity for neurons of the peripheral compared to the central nervous system, the high rate of genetic recombination in HSV1-infected cells, and the genetic identity of infecting and reactivated virus.

  9. p53 downregulates the Fanconi anaemia DNA repair pathway.

    Science.gov (United States)

    Jaber, Sara; Toufektchan, Eléonore; Lejour, Vincent; Bardot, Boris; Toledo, Franck

    2016-04-01

    Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53(Δ31), a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53(Δ31/Δ31) fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53(Δ31/Δ31) fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop.

  10. Plasma membrane repair: the adaptable cell life-insurance.

    Science.gov (United States)

    Jimenez, Ana Joaquina; Perez, Franck

    2017-08-01

    The plasma membrane is the most basic element necessary for the cell to exist and be distinguishable from its environment. Regulated mechanisms allow tightly controlled communication between intacellular and extracellular medium allowing the maintenance of a specific biochemical environment, optimized for cellular functions. The anarchic and uncontrolled opening of a hole in the PM induces a change in the concentration of ions and oxidizing agents perturbing homeostasis. Fortunately, the cell possesses mechanisms that are capable of reacting to sudden extracellular medium entry and to block the leakage locally. Here we summarize the known mechanisms of membrane repair and how the size of the wound and the resulting calcium entry activates preferentially one or another mechanism adapted to the magnitude of the injury. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. A non-canonical mismatch repair pathway in prokaryotes

    Science.gov (United States)

    Castañeda-García, A.; Prieto, A. I.; Rodríguez-Beltrán, J.; Alonso, N.; Cantillon, D.; Costas, C.; Pérez-Lago, L.; Zegeye, E. D.; Herranz, M.; Plociński, P.; Tonjum, T.; García de Viedma, D.; Paget, M.; Waddell, S. J.; Rojas, A. M.; Doherty, A. J.; Blázquez, J.

    2017-01-01

    Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS–MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS–MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature. PMID:28128207

  12. High-Throughput Microplate-Based Assay to Monitor Plasma Membrane Wounding and Repair

    Directory of Open Access Journals (Sweden)

    Sarika Pathak-Sharma

    2017-07-01

    Full Text Available The plasma membrane of mammalian cells is susceptible to disruption by mechanical and biochemical damages that frequently occur within tissues. Therefore, efficient and rapid repair of the plasma membrane is essential for maintaining cellular homeostasis and survival. Excessive damage of the plasma membrane and defects in its repair are associated with pathological conditions such as infections, muscular dystrophy, heart failure, diabetes, and lung and neurodegenerative diseases. The molecular events that remodel the plasma membrane during its repair remain poorly understood. In the present work, we report the development of a quantitative high-throughput assay that monitors the efficiency of the plasma membrane repair in real time using a sensitive microplate reader. In this assay, the plasma membrane of living cells is perforated by the bacterial pore-forming toxin listeriolysin O and the integrity and recovery of the membrane are monitored at 37°C by measuring the fluorescence intensity of the membrane impermeant dye propidium iodide. We demonstrate that listeriolysin O causes dose-dependent plasma membrane wounding and activation of the cell repair machinery. This assay was successfully applied to cell types from different origins including epithelial and muscle cells. In conclusion, this high-throughput assay provides a novel opportunity for the discovery of membrane repair effectors and the development of new therapeutic compounds that could target membrane repair in various pathological processes, from degenerative to infectious diseases.

  13. Genetic Variation in Base Excision Repair Pathway Genes, Pesticide Exposure, and Prostate Cancer Risk

    National Research Council Canada - National Science Library

    Kathryn Hughes Barry; Stella Koutros; Sonja I. Berndt; Gabriella Andreotti; Jane A. Hoppin; Dale P. Sandler; Laurie A. Burdette; Meredith Yeager; Laura E. Beane Freeman; Jay H. Lubin; Xiaomei Ma; Tongzhang Zheng; Michael C. R. Alavanja

    2011-01-01

    .... OBJECTIVES: Because base excision repair (BER) is the predominant pathway involved in repairing oxidative damage, we evaluated interactions between 39 pesticides and 394 tag single-nucleotide polymorphisms (SNPs...

  14. DNA DSB repair pathway choice: an orchestrated handover mechanism.

    Science.gov (United States)

    Kakarougkas, A; Jeggo, P A

    2014-03-01

    DNA double strand breaks (DSBs) are potential lethal lesions but can also lead to chromosome rearrangements, a step promoting carcinogenesis. DNA non-homologous end-joining (NHEJ) is the major DSB rejoining process and occurs in all cell cycle stages. Homologous recombination (HR) can additionally function to repair irradiation-induced two-ended DSBs in G2 phase. In mammalian cells, HR predominantly uses a sister chromatid as a template for DSB repair; thus HR functions only in late S/G2 phase. Here, we review current insight into the interplay between HR and NHEJ in G2 phase. We argue that NHEJ represents the first choice pathway, repairing approximately 80% of X-ray-induced DSBs with rapid kinetics. However, a subset of DSBs undergoes end resection and repair by HR. 53BP1 restricts resection, thereby promoting NHEJ. During the switch from NHEJ to HR, 53BP1 is repositioned to the periphery of enlarged irradiation-induced foci (IRIF) via a BRCA1-dependent process. K63-linked ubiquitin chains, which also form at IRIF, are also repositioned as well as receptor-associated protein 80 (RAP80), a ubiquitin binding protein. RAP80 repositioning requires POH1, a proteasome component. Thus, the interfacing barriers to HR, 53BP1 and RAP80 are relieved by POH1 and BRCA1, respectively. Removal of RAP80 from the IRIF core is required for loss of the ubiquitin chains and 53BP1, and for efficient replication protein A foci formation. We propose that NHEJ is used preferentially to HR because it is a compact process that does not necessitate extensive chromatin changes in the DSB vicinity.

  15. New tools to study DNA double-strand break repair pathway choice.

    Directory of Open Access Journals (Sweden)

    Daniel Gomez-Cabello

    Full Text Available A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences.

  16. How SUMOylation Fine-Tunes the Fanconi Anemia DNA Repair Pathway

    Directory of Open Access Journals (Sweden)

    Kate eColeman

    2016-04-01

    Full Text Available Fanconi Anemia (FA is a rare human genetic disorder characterized by developmental defects, bone marrow failure and cancer predisposition, primarily due to a deficiency in the repair of DNA interstrand crosslinks (ICLs. ICL repair through the FA DNA repair pathway is a complicated multi-step process, involving at least 19 FANC proteins and coordination of multiple DNA repair activities, including homologous recombination (HR, nucleotide excision repair (NER and translesion synthesis (TLS. SUMOylation is a critical regulator of several DNA repair pathways, however, the role of this modification in controlling the FA pathway is poorly understood. Here, we summarize recent advances in the fine-tuning of the FA pathway by SUMO-targeted ubiquitin ligases (STUbLs and other SUMO-related interactions, and discuss the implications of these findings in the design of novel therapeutics for alleviating FA-associated condition, including cancer.

  17. The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

    Directory of Open Access Journals (Sweden)

    Erhong Meng

    2015-07-01

    Full Text Available Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair. Aberrant activation of the Hedgehog (Hh signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer.

  18. The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Erhong; Hanna, Ann; Samant, Rajeev S.; Shevde, Lalita A., E-mail: lsamant@uab.edu [Department of Pathology, Comprehensive Cancer Center, University of Alabama at Birmingham, WTI320D, 1824 6th Avenue South, Birmingham, AL 35233 (United States)

    2015-07-21

    Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair). Aberrant activation of the Hedgehog (Hh) signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer.

  19. Congenital diaphragmatic hernia: To repair on or off extracorporeal membrane oxygenation?

    NARCIS (Netherlands)

    R. Keijzer (Richard); D.E. Wilschut (Dorien); R.J.M. Houmes (Robert Jan); K. van de Ven (Kees); L. de Jongste-van den Hout (Lieke); I. Sluijter (Ilona); P. Rycus (Peter); N.M.A. Bax (Klaas); D. Tibboel (Dick)

    2012-01-01

    textabstractBackground: Congenital diaphragmatic hernia (CDH) can be repaired on or off extracorporeal membrane oxygenation (ECMO). In many centers, operating off ECMO is advocated to prevent bleeding complications. We aimed to compare surgery-related bleeding complications between repair on or off

  20. Repair of Pd/α-Al2O3 composite membrane with defects

    Institute of Scientific and Technical Information of China (English)

    李安武; 熊国兴

    1999-01-01

    A palladium composite membrane with a large number of defects was repaired using the electroless plating combined with the technique of osmosis. The loose structure of palladium film prepared by the conventional electroless plating was densified. Defects were repaired. Hydrogen selectivity was thus significantly increased without significantly increasing palladium film thickness and reducing hydrogen permeability.

  1. Congenital diaphragmatic hernia: To repair on or off extracorporeal membrane oxygenation?

    NARCIS (Netherlands)

    R. Keijzer (Richard); D.E. Wilschut (Dorien); R.J.M. Houmes (Robert Jan); K. van de Ven (Kees); L. de Jongste-van den Hout (Lieke); I. Sluijter (Ilona); P. Rycus (Peter); N.M.A. Bax (Klaas); D. Tibboel (Dick)

    2012-01-01

    textabstractBackground: Congenital diaphragmatic hernia (CDH) can be repaired on or off extracorporeal membrane oxygenation (ECMO). In many centers, operating off ECMO is advocated to prevent bleeding complications. We aimed to compare surgery-related bleeding complications between repair on or off

  2. Complex DNA repair pathways as possible therapeutic targets to overcome temozolomide resistance in glioblastoma

    Directory of Open Access Journals (Sweden)

    Koji eYoshimoto

    2012-12-01

    Full Text Available Many conventional chemotherapeutic drugs exert their cytotoxic function by inducing DNA damage in the tumor cell. Therefore, a cell-inherent DNA repair pathway, which reverses the DNA-damaging effect of the cytotoxic drugs, can mediate therapeutic resistance to chemotherapy. The monofunctional DNA-alkylating agent temozolomide (TMZ is a commonly used chemotherapeutic drug and the gold standard treatment for glioblastoma. Although the activity of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT has been described as the main modulator to determine the sensitivity of glioblastoma to TMZ, a subset of glioblastoma does not respond despite MGMT inactivation, suggesting that another DNA repair mechanism may also modulate the tolerance to TMZ. Considerable interest has focused on MGMT, mismatch repair (MMR, and the base-excision repair (BER pathway in the mechanism of mediating TMZ resistance, but emerging roles for the DNA strand-break repair pathway have been demonstrated. In the first part of this review article, we briefly review the significant role of MGMT, MMR, and the BER pathway in the tolerance to TMZ; in the last part, we review the recent publications that demonstrate possible roles of DNA strand-break repair pathways, such as single-strand break (SSB repair and double-strand break (DSB repair, as well as the Fanconi anemia pathway in the repair process after alkylating agent-based therapy. It is possible that all of these repair pathways have a potential to modulate the sensitivity to TMZ and aid in overcoming the therapeutic resistance in the clinic.

  3. Solid protein solder-doped biodegradable polymer membranes for laser-assisted tissue repair

    Science.gov (United States)

    Hodges, Diane E.; McNally-Heintzelman, Karen M.; Welch, Ashley J.

    2000-05-01

    Solid protein solder-doped polymer membranes have been developed for laser-assisted tissue repair. Biodegradable polymer films of controlled porosity were fabricated with poly(L-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) using a solvent-casting and particulate-leaching technique. The films provided a porous scaffold that readily absorbed the traditional protein solder mix composed of bovine serum albumin (BSA) and indocyanine green (ICG) dye. In vitro investigations were conducted to assess the influence of various processing parameters on the strength of tissue repairs formed using the new membranes. These parameters included the PLGA copolymer and PLGA/PEG blend ratio, the salt particle size, the initial bovine serum albumin (BSA) weight fraction, and the laser irradiance used to denature the solder. Altering the PLGA copolymer ratio had little effect on repair strength, however, it influenced the membrane degradation rate. Repair strength increased with increased membrane pore size and BSA concentration. The addition of PEG during the film casting stage increased the flexibility of the membranes but not necessarily the repair strength. The repair strength increased with increasing irradiance from 12 W/cm2 to 15 W/cm2. The new solder-doped polymer membranes provide all of the benefits associated with solid protein solders including high repair strength and improved edge coaptation. In addition, the flexible and moldable nature of the new membranes offer the capability of tailoring the membranes to a wide range of tissue geometries, and consequently, improved clinical applicability of laser- assisted tissue repair.

  4. Multiple-pathway analysis of double-strand break repair mutations in Drosophila.

    Directory of Open Access Journals (Sweden)

    Dena M Johnson-Schlitz

    2007-04-01

    Full Text Available The analysis of double-strand break (DSB repair is complicated by the existence of several pathways utilizing a large number of genes. Moreover, many of these genes have been shown to have multiple roles in DSB repair. To address this complexity we used a repair reporter construct designed to measure multiple repair outcomes simultaneously. This approach provides estimates of the relative usage of several DSB repair pathways in the premeiotic male germline of Drosophila. We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes. Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others. Perhaps surprisingly, none of the single mutants suppressed more than one pathway, but they varied widely in how the suppression was compensated. We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated. Taken as a whole, the data suggest that the choice of which repair pathway is used for a given DSB occurs by a two-stage "decision circuit" in which the DSB is first placed into one of two pools from which a specific pathway is then selected.

  5. Analysis of DNA double-strand break repair pathways in mice

    Energy Technology Data Exchange (ETDEWEB)

    Brugmans, Linda [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands); Kanaar, Roland [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands); Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam (Netherlands); Essers, Jeroen [Department of Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, PO Box 1738, Rotterdam 3015GE (Netherlands) and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam (Netherlands)]. E-mail: j.essers@erasmusmc.nl

    2007-01-03

    During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.

  6. Personalised pathway analysis reveals association between DNA repair pathway dysregulation and chromosomal instability in sporadic breast cancer.

    Science.gov (United States)

    Liu, Chao; Srihari, Sriganesh; Lal, Samir; Gautier, Benoît; Simpson, Peter T; Khanna, Kum Kum; Ragan, Mark A; Lê Cao, Kim-Anh

    2016-01-01

    The Homologous Recombination (HR) pathway is crucial for the repair of DNA double-strand breaks (DSBs) generated during DNA replication. Defects in HR repair have been linked to the initiation and development of a wide variety of human malignancies, and exploited in chemical, radiological and targeted therapies. In this study, we performed a personalised pathway analysis independently for four large sporadic breast cancer cohorts to investigate the status of HR pathway dysregulation in individual sporadic breast tumours, its association with HR repair deficiency and its impact on tumour characteristics. Specifically, we first manually curated a list of HR genes according to our recent review on this pathway (Liu et al., 2014), and then applied a personalised pathway analysis method named Pathifier (Drier et al., 2013) on the expression levels of the curated genes to obtain an HR score quantifying HR pathway dysregulation in individual tumours. Based on the score, we observed a great diversity in HR dysregulation between and within gene expression-based breast cancer subtypes, and by using two published HR-defect signatures, we found HR pathway dysregulation reflects HR repair deficiency. Furthermore, we identified a novel association between HR pathway dysregulation and chromosomal instability (CIN) in sporadic breast cancer. Although CIN has long been considered as a hallmark of most solid tumours, with recent extensive studies highlighting its importance in tumour evolution and drug resistance, the molecular basis of CIN in sporadic cancers remains poorly understood. Our results imply that HR pathway dysregulation might contribute to CIN in sporadic breast cancer.

  7. The Hemifused State on the Pathway to Membrane Fusion

    Science.gov (United States)

    Warner, Jason M.; O'Shaughnessy, Ben

    2012-04-01

    Fusion of compartments enclosed by membrane bilayers enables secretion and other vital cellular processes and is widely studied in model synthetic membrane systems. Experiments suggest the fusion pathway passes through a hemifused intermediate where only outer monolayers are fused. Here we show membrane tension and divalent cations drive vesicles to hemifused equilibrium with expanded hemifusion diaphragms (HDs) where inner monolayers engage. Predicted HD sizes agree with recent measurements of Nikolaus [Biophys. J. 98, 1192 (2010).BIOJAU0006-349510.1016/j.bpj.2009.11.042]. The fusion pathway is completed by HD lysis provided HD tension is sufficiently high.

  8. Repair Pathway Choices and Consequences at the Double-Strand Break.

    Science.gov (United States)

    Ceccaldi, Raphael; Rondinelli, Beatrice; D'Andrea, Alan D

    2016-01-01

    DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genomic integrity. Failure to repair a DSB has deleterious consequences, including genomic instability and cell death. Indeed, misrepair of DSBs can lead to inappropriate end-joining events, which commonly underlie oncogenic transformation due to chromosomal translocations. Typically, cells employ two main mechanisms to repair DSBs: homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). In addition, alternative error-prone DSB repair pathways, namely alternative end joining (alt-EJ) and single-strand annealing (SSA), have been recently shown to operate in many different conditions and to contribute to genome rearrangements and oncogenic transformation. Here, we review the mechanisms regulating DSB repair pathway choice, together with the potential interconnections between HR and the annealing-dependent error-prone DSB repair pathways.

  9. Lipid raft-dependent plasma membrane repair interferes with the activation of B lymphocytes.

    Science.gov (United States)

    Miller, Heather; Castro-Gomes, Thiago; Corrotte, Matthias; Tam, Christina; Maugel, Timothy K; Andrews, Norma W; Song, Wenxia

    2015-12-21

    Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca(2+)-dependent lysosome exocytosis. Acid sphingomyelinase (ASM) released from lysosomes induces endocytosis of injured membrane through caveolae, membrane invaginations from lipid rafts. How B lymphocytes, lacking any known form of caveolin, repair membrane injury is unknown. Here we show that B lymphocytes repair PM wounds in a Ca(2+)-dependent manner. Wounding induces lysosome exocytosis and endocytosis of dextran and the raft-binding cholera toxin subunit B (CTB). Resealing is reduced by ASM inhibitors and ASM deficiency and enhanced or restored by extracellular exposure to sphingomyelinase. B cell activation via B cell receptors (BCRs), a process requiring lipid rafts, interferes with PM repair. Conversely, wounding inhibits BCR signaling and internalization by disrupting BCR-lipid raft coclustering and by inducing the endocytosis of raft-bound CTB separately from BCR into tubular invaginations. Thus, PM repair and B cell activation interfere with one another because of competition for lipid rafts, revealing how frequent membrane injury and repair can impair B lymphocyte-mediated immune responses.

  10. Activation of the Notch signaling pathway promotes neurovascular repair after traumatic brain injury

    Directory of Open Access Journals (Sweden)

    Qi-shan Ran

    2015-01-01

    Full Text Available The Notch signaling pathway plays a key role in angiogenesis and endothelial cell formation, but it remains unclear whether it is involved in vascular repair by endothelial progenitor cells after traumatic brain injury. Therefore, in the present study, we controlled the Notch signaling pathway using overexpression and knockdown constructs. Activation of the Notch signaling pathway by Notch1 or Jagged1 overexpression enhanced the migration, invasiveness and angiogenic ability of endothelial progenitor cells. Suppression of the Notch signaling pathway with Notch1 or Jagged1 siRNAs reduced the migratory capacity, invasiveness and angiogenic ability of endothelial progenitor cells. Activation of the Notch signaling pathway in vivo in a rat model of mild traumatic brain injury promoted neurovascular repair. These findings suggest that the activation of the Notch signaling pathway promotes blood vessel formation and tissue repair after brain trauma.

  11. Repair of an oroantral communication by a human amniotic membrane: a novel technique.

    Science.gov (United States)

    Lakshmi, Subha; Bharani, Siva; Ambardar, Kalhan

    2015-08-01

    The amniotic membrane is the innermost layer of fetal membrane and is attached to the chorion in the placenta. This membrane has been used for nearly a century in varied fields such as ophthalmology, reconstructive surgery, and burn treatment. In this case report, we used a human amniotic membrane to repair an iatrogenic oroantral communication that occurred during the extraction of the patient's right upper second molar. A splint was given after the perforation was covered with human amniotic membrane and healing was clinically evaluated at various intervals. The outcome of the study revealed that the human amniotic membrane was an efficient graft material for repairing the defect caused by an iatrogenic oroantral communication following tooth extraction.

  12. Difference in membrane repair capacity between cancer cell lines and a normal cell line

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

    2016-01-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane...... repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p cell line (p membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher...

  13. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    Science.gov (United States)

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.

  14. Activation of the Notch signaling pathway promotes neurovascular repair after traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    Qi-shan Ran; Yun-hu Yu; Xiao-hong Fu; Yuan-chao Wen

    2015-01-01

    The Notch signaling pathway plays a key role in angiogenesis and endothelial cell formation, but it remains unclear whether it is involved in vascular repair by endothelial progenitor cells after traumatic brain injury. Therefore, in the present study, we controlled the Notch signaling path-way using overexpression and knockdown constructs. Activation of the Notch signaling pathway by Notch1 or Jagged1 overexpression enhanced the migration, invasiveness and angiogenic ability of endothelial progenitor cells. Suppression of the Notch signaling pathway with Notch1 or Jagged1 siRNAs reduced the migratory capacity, invasiveness and angiogenic ability of endo-thelial progenitor cells. Activation of the Notch signaling pathwayin vivo in a rat model of mild traumatic brain injury promoted neurovascular repair. These ifndings suggest that the activation of the Notch signaling pathway promotes blood vessel formation and tissue repair after brain trauma.

  15. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

    Science.gov (United States)

    Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

  16. Current advances in DNA repair: regulation of enzymes and pathways involved in maintaining genomic stability.

    Science.gov (United States)

    Neher, Tracy M; Turchi, John J

    2011-06-15

    Novel discoveries in the DNA repair field have lead to continuous and rapid advancement of our understanding of not only DNA repair but also DNA replication and recombination. Research in the field transcends numerous areas of biology, biochemistry, physiology, and medicine, making significant connections across these broad areas of study. From early studies conducted in bacterial systems to current analyses in eukaryotic systems and human disease, the innovative research into the mechanisms of repair machines and the consequences of ineffective DNA repair has impacted a wide scientific community. This Forum contains a select mix of primary research articles in addition to a number of timely reviews covering a subset of DNA repair pathways where recent advances and novel discoveries are improving our understanding of DNA repair, its regulation, and implications to human disease.

  17. Arsenic exposure disrupts the normal function of the FA/BRCA repair pathway.

    Science.gov (United States)

    Peremartí, Jana; Ramos, Facundo; Marcos, Ricard; Hernández, Alba

    2014-11-01

    Chronic arsenic exposure is known to enhance the genotoxicity/carcinogenicity of other DNA-damaging agents by inhibiting DNA repair activities. Interference with nucleotide excision repair and base excision repair are well documented, but interactions with other DNA repair pathways are poorly explored so far. The Fanconi anemia FA/BRCA pathway is a DNA repair mechanism required for maintaining genomic stability and preventing cancer. Here, interactions between arsenic compounds and the FA/BRCA pathway were explored by using isogenic FANCD2(-/-) (FA/BRCA-deficient) and FANCD2(+/+) (FA/BRCA-corrected) human fibroblasts. To study whether arsenic disrupts the normal FA/BRCA function, FANCD2(+/+) cells were preexposed to subtoxic concentrations of the trivalent arsenic compounds methylarsonous acid (MMA(III)) and arsenic trioxide (ATO) for 2 weeks. The cellular response to mitomicin-C, hydroxyurea, or diepoxybutane, typical inducers of the studied pathway, was then evaluated and compared to that of FANCD2(-/-) cells. Our results show that preexposure to the trivalent arsenicals MMA(III) and ATO induces in corrected cells, a cellular FA/BRCA-deficient phenotype characterized by hypersensitivity, enhanced accumulation in the G2/M compartment and increased genomic instability--measured as micronuclei. Overall, our data demonstrate that environmentally relevant arsenic exposures disrupt the normal function of the FA/BRCA activity, supporting a novel source of arsenic co- and carcinogenic effects. This is the first study linking arsenic exposure with the FA/BRCA DNA repair pathway.

  18. Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

    NARCIS (Netherlands)

    Sii-Felice, Karine; Etienne, Olivier; Hoffschir, Francoise; Mathieu, Celine; Riou, Lydia; Barroca, Vilma; Haton, Celine; Arwert, Fre; Fouchet, Pierre; Boussin, Francois D.; Mouthon, Marc-Andre

    2008-01-01

    Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway

  19. An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head.

    Science.gov (United States)

    Mace, Kimberly A; Pearson, Joseph C; McGinnis, William

    2005-04-15

    We used wounded Drosophila embryos to define an evolutionarily conserved pathway for repairing the epidermal surface barrier. This pathway includes a wound response enhancer from the Ddc gene that requires grainy head (grh) function and binding sites for the Grh transcription factor. At the signaling level, tyrosine kinase and extracellular signal-regulated kinase (ERK) activities are induced in epidermal cells near wounds, and activated ERK is required for a robust wound response. The conservation of this Grh-dependent pathway suggests that the repair of insect cuticle and mammal skin is controlled by an ancient, shared control system for constructing and healing the animal body surface barrier.

  20. Activation of the Notch signaling pathway promotes neurovascular repair after traumatic brain injury

    OpenAIRE

    2015-01-01

    The Notch signaling pathway plays a key role in angiogenesis and endothelial cell formation, but it remains unclear whether it is involved in vascular repair by endothelial progenitor cells after traumatic brain injury. Therefore, in the present study, we controlled the Notch signaling pathway using overexpression and knockdown constructs. Activation of the Notch signaling pathway by Notch1 or Jagged1 overexpression enhanced the migration, invasiveness and angiogenic ability of endothelial pr...

  1. Repair of a Perforated Sinus Membrane with a Subepithelial Palatal Conjunctive Flap: Technique Report and Evaluation

    OpenAIRE

    Gehrke, S. A.; S. Taschieri; Del Fabbro, M.; Corbella, S.

    2012-01-01

    The maxillary sinus grafting procedure has proven to be an acceptable modality for bone augmentation to provide a base for endosseous implants, routinely used for the rehabilitation of posterior maxilla. Perforation of the membrane is the most common complication in this type of procedure. This paper presents a technique for repairing a perforated Schneiderian membrane with a conjunctive connective tissue graft harvested from the palate and shows the histological and radiographic evaluation o...

  2. Calcium influx affects intracellular transport and membrane repair following nanosecond pulsed electric field exposure.

    Science.gov (United States)

    Thompson, Gary Lee; Roth, Caleb C; Dalzell, Danielle R; Kuipers, Marjorie; Ibey, Bennett L

    2014-05-01

    The cellular response to subtle membrane damage following exposure to nanosecond pulsed electric fields (nsPEF) is not well understood. Recent work has shown that when cells are exposed to nsPEF, ion permeable nanopores (2  nm) created by longer micro- and millisecond duration pulses. Nanoporation of the plasma membrane by nsPEF has been shown to cause a transient increase in intracellular calcium concentration within milliseconds after exposure. Our research objective is to determine the impact of nsPEF on calcium-dependent structural and repair systems in mammalian cells. Chinese hamster ovary (CHO-K1) cells were exposed in the presence and absence of calcium ions in the outside buffer to either 1 or 20, 600-ns duration electrical pulses at 16.2  kV/cm, and pore size was determined using propidium iodide and calcium green. Membrane organization was observed with morphological changes and increases in FM1-43 fluorescence. Migration of lysosomes, implicated in membrane repair, was followed using confocal microscopy of red fluorescent protein-tagged LAMP1. Microtubule structure was imaged using mEmerald-tubulin. We found that at high 600-ns PEF dosage, calcium-induced membrane restructuring and microtubule depolymerization coincide with interruption of membrane repair via lysosomal exocytosis.

  3. Self-Repair and Patterning of 2D Membrane-Like Peptoid Materials

    Energy Technology Data Exchange (ETDEWEB)

    Jiao, Fang [School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 China; Physical Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Chen, Yulin [Physical Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Jin, Haibao [Physical Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; He, Pingang [School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 China; Chen, Chun-Long [Physical Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; De Yoreo, James J. [Physical Sciences Division, Pacific Northwest National Laboratory, Richland WA 99352 USA; Department of Materials Science and Engineering, University of Washington, Seattle WA 98195 USA

    2016-08-31

    Two-dimensional materials are of increasing interest for use in filtration, sensing, nanoelectronics, and biomedical devices. Peptoids are a class of biomimetic sequence-defined polymers for which certain amphiphillic sequences self-assemble into 2D crystalline materials with properties that mimic those of cell membranes. Using in situ AFM to both dissect these membrane-like materials and image their subsequent behavior, we explore their ability to self-repair on a range of solid substrates. We show that, in a suitable range of pH, self-repair occurs on both negatively and positively charged substrates and can even occur in the absence of an underlying surface. Following dissection of pre-assembled peptoid membranes and upon introduction of a peptoid monomer solution, peptoids repair the damage by assembling at the newly created edges. The speed of the advancing edge depends on the edge orientation, reflecting the two-fold symmetry of the underlying peptoid lattice. Moreover, because the membranes are stabilized by hydrophobic interactions, if the solution contains peptoids possessing an identical sequence in the hydrophobic block but a distinct hydrophilic block, filling of the defects creates membranes that are patterned at the nanoscale. Consequently, we can utilize this ability to create nm-sized patterns of distinct functional groups within a single coherent membrane.

  4. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair.

    Directory of Open Access Journals (Sweden)

    Nicole Bennardo

    2008-06-01

    Full Text Available Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ, single-strand annealing (SSA, and homology directed repair (HDR/GC. Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI-induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy.

  5. Formaldehyde catabolism is essential in cells deficient for the Fanconi anemia DNA-repair pathway.

    Science.gov (United States)

    Rosado, Ivan V; Langevin, Frédéric; Crossan, Gerry P; Takata, Minoru; Patel, Ketan J

    2011-11-13

    Metabolism is predicted to generate formaldehyde, a toxic, simple, reactive aldehyde that can damage DNA. Here we report a synthetic lethal interaction in avian cells between ADH5, encoding the main formaldehyde-detoxifying enzyme, and the Fanconi anemia (FA) DNA-repair pathway. These results define a fundamental role for the combined action of formaldehyde catabolism and DNA cross-link repair in vertebrate cell survival.

  6. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair.

    Directory of Open Access Journals (Sweden)

    Nicole Bennardo

    2008-06-01

    Full Text Available Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ, single-strand annealing (SSA, and homology directed repair (HDR/GC. Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI-induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy.

  7. Extracorporeal membrane oxygenation for repair of tracheal injury during transhiatal esophagectomy

    Directory of Open Access Journals (Sweden)

    Lilibeth Fermin

    2017-01-01

    Full Text Available Extracorporeal Membrane Oxygenation (ECMO for repair of tracheal injury during transhiatal esophagectomy Tracheal injury is a rare but potentially fatal complication of esophagectomies requiring prompt recognition and treatment. We describe a case of tracheal injury recognized in the operative period of an open transhiatal esophagectomy for squamous cell carcinoma of the mid to distal esophagus. When injury was discovered, attempts to improve oxygenation and ventilation by conventional methods were unsuccessful. Therefore, peripheral ECMO was used to support oxygenation during the tracheal defect repair. The use of ECMO for the repair of a tracheal injury during esophagectomy is very uncommon but, in our case, provided adequate oxygenation and ventilation while the surgeon repaired the injury and the patient was able to be promptly weaned from ECMO support and extubated not long after.

  8. How to Relate Complex DNA Repair Genotypes to Pathway Function and, Ultimately, Health Risk

    Energy Technology Data Exchange (ETDEWEB)

    Jones, IM

    2002-01-09

    Exposure to ionizing radiation increases the incidence of cancer. However, predicting which individuals are at most risk from radiation exposure is a distant goal. Predictive ability is needed to guide policies that regulate radiation exposure and ensure that medical treatments have maximum benefit and minimum risk. Differences between people in susceptibility to radiation are largely based on their genotype, the genes inherited from their parents. Among the important genes are those that produce proteins that repair DNA damaged by radiation. Base Excision Repair (BER) proteins repair single strand breaks and oxidized bases in DNA. Double Strand Break Repair proteins repair broken chromosomes. Using technologies and information from the Human Genome Project, we have previously determined that the DNA sequence of DNA repair genes varies within the human population. An average of 3-4 different variants were found that affect the protein for each of 37 genes studied. The average frequency of these variants is 5%. Given the many genes in each DNA repair pathway and their many variants, technical ability to determine an individual's repair genotype greatly exceeds ability to interpret the information. A long-term goal is to relate DNA repair genotypes to health risk from radiation. This study focused on the BER pathway. The BER genes are known, variants of the genes have been identified at LLNL, and LLNL had recently developed an assay for BER function using white blood cells. The goal of this initial effort was to begin developing data that could be used to test the hypothesis that many different genotypes have similar DNA repair capacity phenotypes (function). Relationships between genotype and phenotype could then be used to group genotypes with similar function and ultimately test the association of groups of genotypes with health risk from radiation. Genotypes with reduced repair function are expected to increase risk of radiation-induced health effects. The

  9. Bacterial dynamin as a membrane puncture repair kit

    NARCIS (Netherlands)

    de Sousa Borges, Anabela; Scheffers, Dirk-Jan

    2016-01-01

    Dynamin(-like) proteins (DLPs) are widely distributed inbacteria, and both structural and biochemical data haveclearly demonstrated that, like their eukaryotic counter-parts, these proteins can function in membrane modelling.Although various functions for bacterial DLPs have beenhypothesized, a clea

  10. Genome engineering with TALENs and ZFNs: repair pathways and donor design.

    Science.gov (United States)

    Carroll, Dana; Beumer, Kelly J

    2014-09-01

    Genome engineering with targetable nucleases depends on cellular pathways of DNA repair after target cleavage. Knowledge of how those pathways work, their requirements and their active factors, can guide experimental design and improve outcomes. While many aspects of both homologous recombination (HR) and nonhomologous end joining (NHEJ) are shared by a broad range of cells and organisms, some features are specific to individual situations. This article reviews the influence of repair mechanisms on the results of gene targeting experiments, with an emphasis on lessons learned from experiments with Drosophila.

  11. Comparative acoustic performance and mechanical properties of silk membranes for the repair of chronic tympanic membrane perforations.

    Science.gov (United States)

    Allardyce, Benjamin J; Rajkhowa, Rangam; Dilley, Rodney J; Xie, Zhigang; Campbell, Luke; Keating, Adrian; Atlas, Marcus D; von Unge, Magnus; Wang, Xungai

    2016-12-01

    The acoustic and mechanical properties of silk membranes of different thicknesses were tested to determine their suitability as a repair material for tympanic membrane perforations. Membranes of different thickness (10-100μm) were tested to determine their frequency response and their resistance to pressure loads in a simulated ear canal model. Their mechanical rigidity to pressure loads was confirmed by tensile testing. These membranes were tested alongside animal cartilage, currently the strongest available myringoplasty graft as well as paper, which is commonly used for simpler procedures. Silk membranes showed resonant frequencies within the human hearing range and a higher vibrational amplitude than cartilage, suggesting that silk may offer good acoustic energy transfer characteristics. Silk membranes were also highly resistant to simulated pressure changes in the middle ear, suggesting they can resist retraction, a common cause of graft failure resulting from chronic negative pressures in the middle ear. Part of this strength can be explained by the substantially higher modulus of silk films compared with cartilage. This allows for the production of films that are much thinner than cartilage, with superior acoustic properties, but that still provide the same level of mechanical support as thicker cartilage. Together, these in vitro results suggest that silk membranes may provide good hearing outcomes while offering similar levels of mechanical support to the reconstructed middle ear.

  12. Bone Injury and Repair Trigger Central and Peripheral NPY Neuronal Pathways

    Science.gov (United States)

    Alencastre, Inês S.; Neto, Estrela; Ribas, João; Ferreira, Sofia; Vasconcelos, Daniel M.; Sousa, Daniela M.; Summavielle, Teresa; Lamghari, Meriem

    2016-01-01

    Bone repair is a specialized type of wound repair controlled by complex multi-factorial events. The nervous system is recognized as one of the key regulators of bone mass, thereby suggesting a role for neuronal pathways in bone homeostasis. However, in the context of bone injury and repair, little is known on the interplay between the nervous system and bone. Here, we addressed the neuropeptide Y (NPY) neuronal arm during the initial stages of bone repair encompassing the inflammatory response and ossification phases in femoral-defect mouse model. Spatial and temporal analysis of transcriptional and protein levels of NPY and its receptors, Y1R and Y2R, reported to be involved in bone homeostasis, was performed in bone, dorsal root ganglia (DRG) and hypothalamus after femoral injury. The results showed that NPY system activity is increased in a time- and space-dependent manner during bone repair. Y1R expression was trigged in both bone and DRG throughout the inflammatory phase, while a Y2R response was restricted to the hypothalamus and at a later stage, during the ossification step. Our results provide new insights into the involvement of NPY neuronal pathways in bone repair. PMID:27802308

  13. How Trypanosoma cruzi deals with oxidative stress: Antioxidant defence and DNA repair pathways.

    Science.gov (United States)

    Machado-Silva, Alice; Cerqueira, Paula Gonçalves; Grazielle-Silva, Viviane; Gadelha, Fernanda Ramos; Peloso, Eduardo de Figueiredo; Teixeira, Santuza Maria Ribeiro; Machado, Carlos Renato

    2016-01-01

    Trypanosoma cruzi, the causative agent of Chagas disease, is an obligatory intracellular parasite with a digenetic life cycle. Due to the variety of host environments, it faces several sources of oxidative stress. In addition to reactive oxygen species (ROS) produced by its own metabolism, T. cruzi must deal with high ROS levels generated as part of the host's immune responses. Hence, the conclusion that T. cruzi has limited ability to deal with ROS (based on the lack of a few enzymes involved with oxidative stress responses) seems somewhat paradoxical. Actually, to withstand such variable sources of oxidative stress, T. cruzi has developed complex defence mechanisms. This includes ROS detoxification pathways that are distinct from the ones in the mammalian host, DNA repair pathways and specialized polymerases, which not only protect its genome from the resulting oxidative damage but also contribute to the generation of genetic diversity within the parasite population. Recent studies on T. cruzi's DNA repair pathways as mismatch repair (MMR) and GO system suggested that, besides a role associated with DNA repair, some proteins of these pathways may also be involved in signalling oxidative damage. Recent data also suggested that an oxidative environment might be beneficial for parasite survival within the host cell as it contributes to iron mobilization from the host's intracellular storages. Besides contributing to the understanding of basic aspects of T. cruzi biology, these studies are highly relevant since oxidative stress pathways are part of the poorly understood mechanisms behind the mode of action of drugs currently used against this parasite. By unveiling new peculiar aspects of T. cruzi biology, emerging data on DNA repair pathways and other antioxidant defences from this parasite have revealed potential new targets for a much needed boost in drug development efforts towards a better treatment for Chagas disease.

  14. Targeting the DNA repair pathway in Ewing sarcoma.

    Science.gov (United States)

    Stewart, Elizabeth; Goshorn, Ross; Bradley, Cori; Griffiths, Lyra M; Benavente, Claudia; Twarog, Nathaniel R; Miller, Gregory M; Caufield, William; Freeman, Burgess B; Bahrami, Armita; Pappo, Alberto; Wu, Jianrong; Loh, Amos; Karlström, Åsa; Calabrese, Chris; Gordon, Brittney; Tsurkan, Lyudmila; Hatfield, M Jason; Potter, Philip M; Snyder, Scott E; Thiagarajan, Suresh; Shirinifard, Abbas; Sablauer, Andras; Shelat, Anang A; Dyer, Michael A

    2014-11-06

    Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.

  15. Common genetic variations in cell cycle and DNA repair pathways associated with pediatric brain tumor susceptibility

    DEFF Research Database (Denmark)

    Adel Fahmideh, Maral; Lavebratt, Catharina; Schüz, Joachim

    2016-01-01

    Knowledge on the role of genetic polymorphisms in the etiology of pediatric brain tumors (PBTs) is limited. Therefore, we investigated the association between single nucleotide polymorphisms (SNPs), identified by candidate gene-association studies on adult brain tumors, and PBT risk.The study...... cycle and DNA repair pathways variations associated with susceptibility to adult brain tumors also seem to be associated with PBT risk, suggesting pediatric and adult brain tumors might share similar etiological pathways....

  16. Common genetic variations in cell cycle and DNA repair pathways associated with pediatric brain tumor susceptibility

    DEFF Research Database (Denmark)

    Fahmideh, Maral Adel; Lavebratt, Catharina; Schüz, Joachim

    2016-01-01

    Knowledge on the role of genetic polymorphisms in the etiology of pediatric brain tumors (PBTs) is limited. Therefore, we investigated the association between single nucleotide polymorphisms (SNPs), identified by candidate gene-association studies on adult brain tumors, and PBT risk. The study...... cycle and DNA repair pathways variations associated with susceptibility to adult brain tumors also seem to be associated with PBT risk, suggesting pediatric and adult brain tumors might share similar etiological pathways....

  17. Genetic polymorphisms in the nucleotide excision repair pathway and lung cancer risk: A meta-analysis

    Directory of Open Access Journals (Sweden)

    Chikako Kiyohara, Kouichi Yoshimasu

    2007-01-01

    Full Text Available Various DNA alterations can be caused by exposure to environmental and endogenous carcinogens. Most of these alterations, if not repaired, can result in genetic instability, mutagenesis and cell death. DNA repair mechanisms are important for maintaining DNA integrity and preventing carcinogenesis. Recent lung cancer studies have focused on identifying the effects of single nucleotide polymorphisms (SNPs in candidate genes, among which DNA repair genes are increasingly being studied. Genetic variations in DNA repair genes are thought to modulate DNA repair capacity and are suggested to be related to lung cancer risk. We identified a sufficient number of epidemiologic studies on lung cancer to conduct a meta-analysis for genetic polymorphisms in nucleotide excision repair pathway genes, focusing on xeroderma pigmentosum group A (XPA, excision repair cross complementing group 1 (ERCC1, ERCC2/XPD, ERCC4/XPF and ERCC5/XPG. We found an increased risk of lung cancer among subjects carrying the ERCC2 751Gln/Gln genotype (odds ratio (OR = 1.30, 95% confidence interval (CI = 1.14 - 1.49. We found a protective effect of the XPA 23G/G genotype (OR = 0.75, 95% CI = 0.59 - 0.95. Considering the data available, it can be conjectured that if there is any risk association between a single SNP and lung cancer, the risk fluctuation will probably be minimal. Advances in the identification of new polymorphisms and in high-throughput genotyping techniques will facilitate the analysis of multiple genes in multiple DNA repair pathways. Therefore, it is likely that the defining feature of future epidemiologic studies will be the simultaneous analysis of large samples.

  18. Nrf2 facilitates repair of radiation induced DNA damage through homologous recombination repair pathway in a ROS independent manner in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jayakumar, Sundarraj; Pal, Debojyoti; Sandur, Santosh K., E-mail: sskumar@barc.gov.in

    2015-09-15

    Highlights: • Nrf2 inhibition in A549 cells led to attenuated DNA repair and radiosensitization. • Influence of Nrf2 on DNA repair is not linked to its antioxidant function. • Nrf2 influences DNA repair through homologous recombination (HR) repair pathway. • Many genes involved in HR pathway show ARE sequences in their upstream region. - Abstract: Nrf2 is a redox sensitive transcription factor that is involved in the co-ordinated transcription of genes involved in redox homeostasis. But the role of Nrf2 in DNA repair is not investigated in detail. We have employed A549 and MCF7 cells to study the role of Nrf2 on DNA repair by inhibiting Nrf2 using all-trans retinoic acid (ATRA) or by knock down approach prior to radiation exposure (4 Gy). DNA damage and repair analysis was studied by γH2AX foci formation and comet assay. Results suggested that the inhibition of Nrf2 in A549 or MCF7 cells led to significant slowdown in DNA repair as compared to respective radiation controls. The persistence of residual DNA damage even in the presence of free radical scavenger N-acetyl cysteine, suggested that the influence of Nrf2 on DNA repair was not linked to its antioxidant functions. Further, its influence on non-homologous end joining repair pathway was studied by inhibiting both Nrf2 and DNA-PK together. This led to synergistic reduction of survival fraction, indicating that Nrf2 may not be influencing the NHEJ pathway. To investigate the role of homologous recombination repair (HR) pathway, RAD51 foci formation was monitored. There was a significant reduction in the foci formation in cells treated with ATRA or shRNA against Nrf2 as compared to their respective radiation controls. Further, Nrf2 inhibition led to significant reduction in mRNA levels of RAD51. BLAST analysis was also performed on upstream regions of DNA repair genes to identify antioxidant response element and found that many repair genes that are involved in HR pathway may be regulated by Nrf2

  19. DNA double strand breaks repair pathways in mouse male germ cells

    NARCIS (Netherlands)

    Ahmed, E.A.

    2009-01-01

    DNA double strand breaks (DSBs) are induced by ionizing radiation, and during meiotic recombination. DSBs are repaired via two main pathways, homologous recombination (HR) and non homologous end-joining (NHEJ). There are three main types of male germ cells, spermatogonia, spermatocytes and spermatid

  20. Targeting the DNA Repair Pathway in Ewing Sarcoma

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    Elizabeth Stewart

    2014-11-01

    Full Text Available Ewing sarcoma (EWS is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis. PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.

  1. The Use of Extracorporeal Membrane Oxygenation in the Surgical Repair of Bronchial Rupture

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    Ju-Hee Park

    2016-02-01

    Full Text Available Extracorporeal membrane oxygenation (ECMO has been used successfully in critically ill patients with traumatic lung injury and offers an additional treatment modality. ECMO is mainly used as a bridge treatment to delayed surgical management; however, only a few case reports have presented the successful application of ECMO as intraoperative support during the surgical repair of traumatic bronchial injury. A 38-year-old man visited our hospital after a blunt chest trauma. His chest imaging showed hemopneumothorax in the left hemithorax and a finding suspicious for left main bronchus rupture. Bronchoscopy was performed and confirmed a tear in the left main bronchus and a congenital tracheal bronchus. We decided to provide venovenous ECMO support during surgery for bronchial repair. We successfully performed main bronchial repair in this traumatic patient with a congenital tracheal bronchus. We suggest that venovenous ECMO offers a good option for the treatment of bronchial rupture when adequate ventilation is not possible.

  2. The Fanconi anemia pathway and ICL repair: implications for cancer therapy.

    Science.gov (United States)

    Wang, Lily C; Gautier, Jean

    2010-10-01

    Fanconi anemia (FA) is an inherited disease caused by mutations in at least 13 genes and characterized by genomic instability. In addition to displaying strikingly heterogenous clinical phenotypes, FA patients are exquisitely sensitive to treatments with crosslinking agents that create interstrand crosslinks (ICL). In contrast to bacteria and yeast, in which ICLs are repaired through replication-dependent and -independent mechanisms, it is thought that ICLs are repaired primarily during DNA replication in vertebrates. However, recent data indicate that replication-independent ICL repair also operates in vertebrates. While the precise role of the FA pathway in ICL repair remains elusive, increasing evidence suggests that FA proteins function at different steps in the sensing, recognition and processing of ICLs, as well as in signaling from these very toxic lesions, which can be generated by a wide variety of cancer chemotherapeutic drugs. Here, we discuss some of the recent findings that have shed light on the role of the FA pathway in ICL repair, with special emphasis on the implications of these findings for cancer therapy since disruption of FA genes have been associated with cancer predisposition.

  3. The nucleotide excision repair pathway is required for UV-C-induced apoptosis in Caenorhabditis elegans.

    Science.gov (United States)

    Stergiou, L; Doukoumetzidis, K; Sendoel, A; Hengartner, M O

    2007-06-01

    Ultraviolet (UV) radiation is a mutagen of major clinical importance in humans. UV-induced damage activates multiple signaling pathways, which initiate DNA repair, cell cycle arrest and apoptosis. To better understand these pathways, we studied the responses to UV-C light (254 nm) of germ cells in Caenorhabditis elegans. We found that UV activates the same cellular responses in worms as in mammalian cells. Both UV-induced apoptosis and cell cycle arrest were completely dependent on the p53 homolog CEP-1, the checkpoint proteins HUS-1 and CLK-2, and the checkpoint kinases CHK-2 and ATL-1 (the C. elegans homolog of ataxia telangiectasia and Rad3-related); ATM-1 (ataxia telangiectasia mutated-1) was also required, but only at low irradiation doses. Importantly, mutation of genes encoding nucleotide excision repair pathway components severely disrupted both apoptosis and cell cycle arrest, suggesting that these genes not only participate in repair, but also signal the presence of damage to downstream components of the UV response pathway that we delineate here. Our study suggests that whereas DNA damage response pathways are conserved in metazoans in their general outline, there is significant evolution in the relative importance of individual checkpoint genes in the response to specific types of DNA damage.

  4. Perforated schneiderian membrane repair during sinus augmentation in conjunction with immediate implant placement

    Directory of Open Access Journals (Sweden)

    K Hameed Fathima

    2014-01-01

    Full Text Available Dental implant associated rehabilitation of the posterior maxilla poses unique challenge owing to the presence of pneumatized sinuses and atrophied alveolar bone. Sinus augmentation procedure to manage expanded sinuses frequently results in membrane tear resulting in unfavorable stabilization of the graft and associated bone regeneration. Simultaneous implant placement during sinus augmentation procedures frequently requires a minimal alveolar bone height, which when not present forces clinician to defer implant placement resulting in extended treatment duration and multiple surgical appointments. The present case report is about a piezosurgery assisted lateral wall osteotomy approach for sinus augmentation associated with membrane repair with simultaneous implant placement in the posterior maxilla.

  5. Systematic analysis of DNA damage induction and DNA repair pathway activation by continuous wave visible light laser micro-irradiation

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    Britta Muster

    2017-02-01

    Full Text Available Laser micro-irradiation can be used to induce DNA damage with high spatial and temporal resolution, representing a powerful tool to analyze DNA repair in vivo in the context of chromatin. However, most lasers induce a mixture of DNA damage leading to the activation of multiple DNA repair pathways and making it impossible to study individual repair processes. Hence, we aimed to establish and validate micro-irradiation conditions together with inhibition of several key proteins to discriminate different types of DNA damage and repair pathways using lasers commonly available in confocal microscopes. Using time-lapse analysis of cells expressing fluorescently tagged repair proteins and also validation of the DNA damage generated by micro-irradiation using several key damage markers, we show that irradiation with a 405 nm continuous wave laser lead to the activation of all repair pathways even in the absence of exogenous sensitization. In contrast, we found that irradiation with 488 nm laser lead to the selective activation of non-processive short-patch base excision and single strand break repair, which were further validated by PARP inhibition and metoxyamine treatment. We conclude that these low energy conditions discriminated against processive long-patch base excision repair, nucleotide excision repair as well as double strand break repair pathways.

  6. The new base excision repair pathway in mammals mediated by tyrosyl-DNA-phosphodiesterase 1

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    Lavrik O. I.

    2012-06-01

    Full Text Available Human tyrosyl-DNA phosphodiesterase 1 (Tdp1 hydrolyzes the phosphodiester bond at a DNA 3' end linked to a tyrosyl moiety and has been implicated in the repair of Topoisomerase I (TopI-DNA covalent complexes. Tdp1 can also hydrolyze other 3' end DNA alterations including 3' phosphoglycolate and 3' abasic (AP sites, and exhibits the 3' nucleosidase activity indicating that it may function as a general 3' end-processing DNA repair enzyme. Recently we have shown a new Tdp1 activity generating DNA strand break with the 3' phosphate termini from the AP site. AP sites are formed spontaneously and are inevitable intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic, and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair, initiated by DNA glycosylases performing beta, delta-elimination cleavage of the AP sites, has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites that is initiated by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap.

  7. The Fanconi anemia DNA damage repair pathway in the spotlight for germline predisposition to colorectal cancer

    Science.gov (United States)

    Esteban-Jurado, Clara; Franch-Expósito, Sebastià; Muñoz, Jenifer; Ocaña, Teresa; Carballal, Sabela; López-Cerón, Maria; Cuatrecasas, Miriam; Vila-Casadesús, Maria; Lozano, Juan José; Serra, Enric; Beltran, Sergi; Brea-Fernández, Alejandro; Ruiz-Ponte, Clara; Castells, Antoni; Bujanda, Luis; Garre, Pilar; Caldés, Trinidad; Cubiella, Joaquín; Balaguer, Francesc; Castellví-Bel, Sergi

    2016-01-01

    Colorectal cancer (CRC) is one of the most common neoplasms in the world. Fanconi anemia (FA) is a very rare genetic disease causing bone marrow failure, congenital growth abnormalities and cancer predisposition. The comprehensive FA DNA damage repair pathway requires the collaboration of 53 proteins and it is necessary to restore genome integrity by efficiently repairing damaged DNA. A link between FA genes in breast and ovarian cancer germline predisposition has been previously suggested. We selected 74 CRC patients from 40 unrelated Spanish families with strong CRC aggregation compatible with an autosomal dominant pattern of inheritance and without mutations in known hereditary CRC genes and performed germline DNA whole-exome sequencing with the aim of finding new candidate germline predisposition variants. After sequencing and data analysis, variant prioritization selected only those very rare alterations, producing a putative loss of function and located in genes with a role compatible with cancer. We detected an enrichment for variants in FA DNA damage repair pathway genes in our familial CRC cohort as 6 families carried heterozygous, rare, potentially pathogenic variants located in BRCA2/FANCD1, BRIP1/FANCJ, FANCC, FANCE and REV3L/POLZ. In conclusion, the FA DNA damage repair pathway may play an important role in the inherited predisposition to CRC. PMID:27165003

  8. A Novel Repair Technique for the Internal Thermal Control System Dual-Membrane Gas Trap

    Science.gov (United States)

    Leimkuehler, Thomas O.; Patel, Vipul; Reeves, Daniel R.; Holt, James M.

    2005-01-01

    A dual-membrane gas trap is currently used to remove gas bubbles from the Internal Thermal Control System (ITCS) coolant on board the International Space Station (ISS). The gas trap consists of concentric tube membrane pairs, comprised of outer hydrophilic tubes and inner hydrophobic fibers. Liquid coolant passes through the outer hydrophilic membrane, which traps the gas bubbles. The inner hydrophobic fiber allows the trapped gas bubbles to pass through and vent to the ambient atmosphere in the cabin. The gas trap was designed to last for the entire lifetime of the ISS, and therefore was not designed to be repaired. However, repair of these gas traps is now a necessity due to contamination from the on-orbit ITCS fluid and other sources on the ground as well as a limited supply of flight gas traps. This paper describes a novel repair technique that has been developed that will allow the refurbishment of contaminated gas traps and their return to flight use.

  9. DNA double-strand break repair: a tale of pathway choices

    Institute of Scientific and Technical Information of China (English)

    Jing Li; Xingzhi Xu

    2016-01-01

    Deoxyribonucleic acid double-strand breaks (DSBs) are cytotoxic lesions that must be repaired either through homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways.DSB repair is critical for genome integrity,cellular homeostasis and also constitutes the biological foundation for radiotherapy and the majority of chemotherapy.The choice between HR and NHEJ is a complex yet not completely understood process that will entail more future efforts.Herein we review our current understandings about how the choice is made over an antagonizing balance between p53-binding protein 1 and breast cancer 1 in the context of cell cycle stages,downstream effects,and distinct chromosomal histone marks.These exciting areas of research will surely bring more mechanistic insights about DSB repair and be utilized in the clinical settings.

  10. Repair of a Perforated Sinus Membrane with a Subepithelial Palatal Conjunctive Flap: Technique Report and Evaluation

    Directory of Open Access Journals (Sweden)

    S. A. Gehrke

    2012-01-01

    Full Text Available The maxillary sinus grafting procedure has proven to be an acceptable modality for bone augmentation to provide a base for endosseous implants, routinely used for the rehabilitation of posterior maxilla. Perforation of the membrane is the most common complication in this type of procedure. This paper presents a technique for repairing a perforated Schneiderian membrane with a conjunctive connective tissue graft harvested from the palate and shows the histological and radiographic evaluation of the results. Ten consecutives cases with the occurrence of membrane perforation were included in this study. All were repaired with a flap of tissue removed from the palatine portion near to the surgical site. The technique is demonstrated through a clinical case. The results showed successful integration of 88.8% of the implants after 12 months from prosthesis installation. Histological evaluation of the samples showed that the use of nanocrystalized hydroxyapatite showed an adequate stimulation of boné neoformation within 6 months. Radiographic evaluation revealed a small apical implant bone loss, not compromising their anchorages and proservation. Thus, it can be concluded that the use of conjunctive technique with collected palate flap for sealing the perforation of the membrane of the sinus may have predictable result.

  11. The potential of nanoporous anodic aluminium oxide membranes to influence skin wound repair.

    Science.gov (United States)

    Parkinson, Leigh G; Giles, Natalie L; Adcroft, Katharine F; Fear, Mark W; Wood, Fiona M; Poinern, Gerard E

    2009-12-01

    Cells respond to changes in the environment by altering their phenotype. The ability to influence cell behavior by modifying their environment provides an opportunity for therapeutic application, for example, to promote faster wound healing in response to skin injury. Here, we have modified the preparation of an aluminium oxide template to generate large uniform membranes with differing nano-pore sizes. Epidermal cells (keratinocytes) and dermal cells (fibroblasts) readily adhere to these nanoporous membranes. The pore size appears to influence the rate of cell proliferation and migration, important aspects of cell behavior during wound healing. The suitability of the membrane to act as a dressing after a burn injury was assessed in vivo; application of the membrane demonstrated adherence and conformability to the skin surface of a pig, with no observed degradation or detrimental effect on the repair. Our results suggest that keratinocytes are sensitive to changes in topography at the nanoscale level and that this property may be exploited to improve wound repair after tissue injury.

  12. Auxilin facilitates membrane traffic in the early secretory pathway.

    Science.gov (United States)

    Ding, Jingzhen; Segarra, Verónica A; Chen, Shuliang; Cai, Huaqing; Lemmon, Sandra K; Ferro-Novick, Susan

    2016-01-01

    Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER-Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.

  13. Early Steps in the DNA Base Excision Repair Pathway of a Fission Yeast Schizosaccharomyces pombe

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    Kyoichiro Kanamitsu

    2010-01-01

    Full Text Available DNA base excision repair (BER accounts for maintaining genomic integrity by removing damaged bases that are generated endogenously or induced by genotoxic agents. In this paper, we describe the roles of enzymes functioning in the early steps of BER in fission yeast. Although BER is an evolutionarily conserved process, some unique features of the yeast repair pathway were revealed by genetic and biochemical approaches. AP sites generated by monofunctional DNA glycosylases are incised mainly by AP lyase activity of Nth1p, a sole bifunctional glycosylase in yeast, to leave a blocked 3′ end. The major AP endonuclease Apn2p functions predominantly in removing the 3′ block. Finally, a DNA polymerase fills the gap, and a DNA ligase seals the nick (Nth1p-dependent or short patch BER. Apn1p backs up Apn2p. In long patch BER, Rad2p endonuclease removes flap DNA containing a lesion after DNA synthesis. A UV-specific endonuclease Uve1p engages in an alternative pathway by nicking DNA on the 5′ side of oxidative damage. Nucleotide excision repair and homologous recombination are involved in repair of BER intermediates including the AP site and single-strand break with the 3′ block. Other enzymes working in 3′ end processing are also discussed.

  14. Accelerating repaired basement membrane after bevacizumab treatment on alkali-burned mouse cornea

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    Koon-Ja Lee

    2013-04-01

    Full Text Available To understand the corneal regeneration induced by bevacizumab,we investigated the structure changes of stroma andbasement membrane regeneration. A Stick soaked in 0.5 NNaOH onto the mouse cornea and 2.5 mg/ml of bevacizumabwas delivered into an alkali-burned cornea (2 μl by subconjunctivalinjections at 1 hour and 4 days after injury. At 7 daysafter injury, basement membrane regeneration was observedby transmission electron microscope. Uneven and thin epithelialbasement membrane, light density of hemidesmosomes,and edematous collagen fibril bundles are shown in thealkali-burned cornea. Injured epithelial basement membraneand hemidesmosomes and edematous collagen fibril bundlesresulting from alkali-burned mouse cornea was repaired bybevacizumab treatment. This study demonstrates that bevacizumabcan play an important role in wound healing in thecornea by accelerating the reestablishment of basementmembrane integrity that leads to barriers for scar formation.[BMB Reports 2013; 46(4: 195-200

  15. Modeling damage complexity-dependent non-homologous end-joining repair pathway.

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    Yongfeng Li

    Full Text Available Non-homologous end joining (NHEJ is the dominant DNA double strand break (DSB repair pathway and involves several repair proteins such as Ku, DNA-PKcs, and XRCC4. It has been experimentally shown that the choice of NHEJ proteins is determined by the complexity of DSB. In this paper, we built a mathematical model, based on published data, to study how NHEJ depends on the damage complexity. Under an appropriate set of parameters obtained by minimization technique, we can simulate the kinetics of foci track formation in fluorescently tagged mammalian cells, Ku80-EGFP and DNA-PKcs-YFP for simple and complex DSB repair, respectively, in good agreement with the published experimental data, supporting the notion that simple DSB undergo fast repair in a Ku-dependent, DNA-PKcs-independent manner, while complex DSB repair requires additional DNA-PKcs for end processing, resulting in its slow repair, additionally resulting in slower release rate of Ku and the joining rate of complex DNA ends. Based on the numerous experimental descriptions, we investigated several models to describe the kinetics for complex DSB repair. An important prediction of our model is that the rejoining of complex DSBs is through a process of synapsis formation, similar to a second order reaction between ends, rather than first order break filling/joining. The synapsis formation (SF model allows for diffusion of ends before the synapsis formation, which is precluded in the first order model by the rapid coupling of ends. Therefore, the SF model also predicts the higher number of chromosomal aberrations observed with high linear energy transfer (LET radiation due to the higher proportion of complex DSBs compared to low LET radiation, and an increased probability of misrejoin following diffusion before the synapsis is formed, while the first order model does not provide a mechanism for the increased effectiveness in chromosomal aberrations observed.

  16. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  17. MOF Phosphorylation by ATM Regulates 53BP1-Mediated Double-Strand Break Repair Pathway Choice

    Directory of Open Access Journals (Sweden)

    Arun Gupta

    2014-07-01

    Full Text Available Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ or homologous recombination (HR. Here, we report that double-strand breaks (DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase phosphorylation (p-T392-MOF and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.

  18. MOF phosphorylation by ATM regulates 53BP1-mediated DSB repair pathway choice

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R.; Hegdec, Muralidhar L.; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh1, Mayank; Ramnarain, Deepti B.; Hittelman, Walter N.; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K.; Ludwig, Thomas; Pandita, Raj K.; Tyler, Jessica K.; Pandita, Tej K.

    2014-01-01

    Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase. PMID:24953651

  19. Genomically amplified Akt3 activates DNA repair pathway and promotes glioma progression.

    Science.gov (United States)

    Turner, Kristen M; Sun, Youting; Ji, Ping; Granberg, Kirsi J; Bernard, Brady; Hu, Limei; Cogdell, David E; Zhou, Xinhui; Yli-Harja, Olli; Nykter, Matti; Shmulevich, Ilya; Yung, W K Alfred; Fuller, Gregory N; Zhang, Wei

    2015-03-17

    Akt is a robust oncogene that plays key roles in the development and progression of many cancers, including glioma. We evaluated the differential propensities of the Akt isoforms toward progression in the well-characterized RCAS/Ntv-a mouse model of PDGFB-driven low grade glioma. A constitutively active myristoylated form of Akt1 did not induce high-grade glioma (HGG). In stark contrast, Akt2 and Akt3 showed strong progression potential with 78% and 97% of tumors diagnosed as HGG, respectively. We further revealed that significant variations in polarity and hydropathy values among the Akt isoforms in both the pleckstrin homology domain (P domain) and regulatory domain (R domain) were critical in mediating glioma progression. Gene expression profiles from representative Akt-derived tumors indicated dominant and distinct roles for Akt3, consisting primarily of DNA repair pathways. TCGA data from human GBM closely reflected the DNA repair function, as Akt3 was significantly correlated with a 76-gene signature DNA repair panel. Consistently, compared with Akt1 and Akt2 overexpression models, Akt3-expressing human GBM cells had enhanced activation of DNA repair proteins, leading to increased DNA repair and subsequent resistance to radiation and temozolomide. Given the wide range of Akt3-amplified cancers, Akt3 may represent a key resistance factor.

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

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    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.

  1. Modulation of Wound Healing and Scar Formation by MG53 Protein-mediated Cell Membrane Repair*

    Science.gov (United States)

    Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M.; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

    2015-01-01

    Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53−/− mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. PMID:26306047

  2. Signalling pathways that inhibit the capacity of precursor cells for myelin repair.

    Science.gov (United States)

    Sabo, Jennifer K; Cate, Holly S

    2013-01-07

    In demyelinating disorders such as Multiple Sclerosis (MS), targets of injury are myelin and oligodendrocytes, leading to severe neurological dysfunction. Regenerative therapies aimed at promoting oligodendrocyte maturation and remyelination are promising strategies for treatment in demyelinating disorders. Endogenous precursor cells or exogenous transplanted cells are potential sources for remyelinating oligodendrocytes in the central nervous system (CNS). Several signalling pathways have been implicated in regulating the capacity of these cell populations for myelin repair. Here, we review neural precursor cells and oligodendrocyte progenitor cells as potential sources for remyelinating oligodendrocytes and evidence for the functional role of key signalling pathways in inhibiting regeneration from these precursor cell populations.

  3. Signalling Pathways that Inhibit the Capacity of Precursor Cells for Myelin Repair

    Directory of Open Access Journals (Sweden)

    Jennifer K. Sabo

    2013-01-01

    Full Text Available In demyelinating disorders such as Multiple Sclerosis (MS, targets of injury are myelin and oligodendrocytes, leading to severe neurological dysfunction. Regenerative therapies aimed at promoting oligodendrocyte maturation and remyelination are promising strategies for treatment in demyelinating disorders. Endogenous precursor cells or exogenous transplanted cells are potential sources for remyelinating oligodendrocytes in the central nervous system (CNS. Several signalling pathways have been implicated in regulating the capacity of these cell populations for myelin repair. Here, we review neural precursor cells and oligodendrocyte progenitor cells as potential sources for remyelinating oligodendrocytes and evidence for the functional role of key signalling pathways in inhibiting regeneration from these precursor cell populations.

  4. Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase.

    Science.gov (United States)

    Lee, Kyung-Jong; Saha, Janapriya; Sun, Jingxin; Fattah, Kazi R; Wang, Shu-Chi; Jakob, Burkhard; Chi, Linfeng; Wang, Shih-Ya; Taucher-Scholz, Gisela; Davis, Anthony J; Chen, David J

    2016-02-29

    Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.

  5. A Review of Recent Experiments on Step-to-Step “Hand-off” of the DNA Intermediates in Mammalian Base Excision Repair Pathways1

    OpenAIRE

    Prasad, R.; Beard, W A; Batra, V. K.; Liu, Y.; Shock, D. D.; Wilson, S H

    2011-01-01

    The current “working model” for mammalian base excision repair involves two sub-pathways termed single-nucleotide base excision repair and long patch base excision repair that are distinguished by their repair patch sizes and the enzymes/co-factors involved. These base excision repair sub-pathways are designed to sequester the various DNA intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apo...

  6. Calpains, cleaved mini-dysferlinC72, and L-type channels underpin calcium-dependent muscle membrane repair.

    Science.gov (United States)

    Lek, Angela; Evesson, Frances J; Lemckert, Frances A; Redpath, Gregory M I; Lueders, Ann-Katrin; Turnbull, Lynne; Whitchurch, Cynthia B; North, Kathryn N; Cooper, Sandra T

    2013-03-20

    Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlinC72. Mini-dysferlinC72-rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlinC72 and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein- or phospholipid-binding role for muscle membrane repair.

  7. Repair of Nerve Cell Membrane Damage by Calcium-Dependent, Membrane-Binding Proteins (Revised)

    Science.gov (United States)

    2012-09-01

    Alzheimer disease amyloid beta protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum , Proc Natl Acad Sci U S A...Calcium signaling and amyloid toxicity in Alzheimer disease, J Biol Chem 285 (2010) 12463-12468. [14] H.A. Lashuel, P.T. Lansbury, Are amyloid

  8. Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Muralidhar L Hegde; Tapas K Hazra; Sankar Mitra

    2008-01-01

    Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3' OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APEl, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E.coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3' phosphate ter-mini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APEl. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

  9. RAG2’s Acidic Hinge Restricts Repair-Pathway Choice and Promotes Genomic Stability

    Directory of Open Access Journals (Sweden)

    Marc A. Coussens

    2013-09-01

    Full Text Available V(DJ recombination-associated DNA double-strand breaks (DSBs are normally repaired by the high-fidelity classical nonhomologous end-joining (cNHEJ machinery. Previous studies implicated the recombination-activating gene (RAG/DNA postcleavage complex (PCC in regulating pathway choice by preventing access to inappropriate repair mechanisms such as homologous recombination (HR and alternative NHEJ (aNHEJ. Here, we report that RAG2’s “acidic hinge,” previously of unknown function, is critical for several key steps. Mutations that reduce the hinge’s negative charge destabilize the PCC, disrupt pathway choice, permit repair of RAG-mediated DSBs by the translocation-prone aNHEJ machinery, and reduce genomic stability in developing lymphocytes. Structural predictions and experimental results support our hypothesis that reduced flexibility of the hinge underlies these outcomes. Furthermore, sequence variants present in the human population reduce the hinge’s negative charge, permit aNHEJ, and diminish genomic integrity.

  10. Biodegradable polymer nanofiber membrane for the repair of cutaneous wounds in dogs - two case reports

    Directory of Open Access Journals (Sweden)

    Lívia Gomes Amaral

    2016-12-01

    Full Text Available The study of wound healing and its treatment is extremely important in veterinary medicine due to the high frequency of wounds and the difficulty in treating wounds by second intention. Thus, the objective of this study was to evaluate the use of a nanofiber membrane made of biodegradable polymers as a method of wound treatment in dogs. This study comprised two dogs with bite wounds. Debridement and cleaning was performed followed by the application of the membrane. In one dog, the wound was in the left proximal calcaneal region with clinical signs of infection, necrotic tissue, and muscle and the gastrocnemius tendon were exposed. The wound displayed rapid formation of granulation tissue which became excessive, so it was necessary to debride several times. However, with the suspension of the use of the membrane, formation of this tissue was not observed, and the wound evolved to epithelialization and fast contraction. In the second dog, there was a deep wound on the medial aspect of the proximal right hind limb, with clinical signs of infection, with muscle exposure. Once the membrane was placed, granulation tissue formed, and the membrane was used until the level of this tissue reached the skin. The wound underwent rapid epithelialization and contraction, without developing exuberant granulation tissue. Efficient wound repair was observed and the dogs exhibited greater comfort during application and use of the membrane. More studies should be conducted in dogs focusing on the application of this membrane until the appearance of healthy granulation tissue, as continued use seems to stimulate the formation of exuberant granulation tissue.

  11. CtIP-BRCA1 modulates the choice of DNA double-strand break repair pathway throughout the cell cycle

    OpenAIRE

    Yun, Maximina H.; Hiom, Kevin

    2009-01-01

    The repair of DNA double-strand breaks (DSB) is tightly regulated during the cell cycle. In G1 phase, the absence of a sister chromatid means that repair of DSB occurs through non-homologous end-joining (NHEJ) or microhomology-mediated end-joining (MMEJ)1. These pathways often involve loss of DNA sequences at the break site and are therefore error-prone. In late S and G2 phases, even though DNA end-joining pathways remain functional2, there is an increase in repair of DSB by homologous recomb...

  12. Helicobacter pylori Disrupts Host Cell Membranes, Initiating a Repair Response and Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Hsueh-Fen Juan

    2012-08-01

    Full Text Available Helicobacter pylori (H. pylori, the human stomach pathogen, lives on the inner surface of the stomach and causes chronic gastritis, peptic ulcer, and gastric cancer. Plasma membrane repair response is a matter of life and death for human cells against physical and biological damage. We here test the hypothesis that H. pylori also causes plasma membrane disruption injury, and that not only a membrane repair response but also a cell proliferation response are thereby activated. Vacuolating cytotoxin A (VacA and cytotoxin-associated gene A (CagA have been considered to be major H. pylori virulence factors. Gastric cancer cells were infected with H. pylori wild type (vacA+/cagA+, single mutant (ΔvacA or ΔcagA or double mutant (ΔvacA/ΔcagA strains and plasma membrane disruption events and consequent activation of membrane repair components monitored. H. pylori disrupts the host cell plasma membrane, allowing localized dye and extracellular Ca2+ influx. Ca2+-triggered members of the annexin family, A1 and A4, translocate, in response to injury, to the plasma membrane, and cell surface expression of an exocytotic maker of repair, LAMP-2, increases. Additional forms of plasma membrane disruption, unrelated to H. pylori exposure, also promote host cell proliferation. We propose that H. pylori activation of a plasma membrane repair is pro-proliferative. This study might therefore provide new insight into potential mechanisms of H. pylori-induced gastric carcinogenesis.

  13. DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases

    Science.gov (United States)

    Bettencourt, Conceição; Hensman‐Moss, Davina; Flower, Michael; Wiethoff, Sarah; Brice, Alexis; Goizet, Cyril; Stevanin, Giovanni; Koutsis, Georgios; Karadima, Georgia; Panas, Marios; Yescas‐Gómez, Petra; García‐Velázquez, Lizbeth Esmeralda; Alonso‐Vilatela, María Elisa; Lima, Manuela; Raposo, Mafalda; Traynor, Bryan; Sweeney, Mary; Wood, Nicholas; Giunti, Paola; Durr, Alexandra; Holmans, Peter; Houlden, Henry; Tabrizi, Sarah J.

    2016-01-01

    Objective The polyglutamine diseases, including Huntington's disease (HD) and multiple spinocerebellar ataxias (SCAs), are among the commonest hereditary neurodegenerative diseases. They are caused by expanded CAG tracts, encoding glutamine, in different genes. Longer CAG repeat tracts are associated with earlier ages at onset, but this does not account for all of the difference, and the existence of additional genetic modifying factors has been suggested in these diseases. A recent genome‐wide association study (GWAS) in HD found association between age at onset and genetic variants in DNA repair pathways, and we therefore tested whether the modifying effects of variants in DNA repair genes have wider effects in the polyglutamine diseases. Methods We assembled an independent cohort of 1,462 subjects with HD and polyglutamine SCAs, and genotyped single‐nucleotide polymorphisms (SNPs) selected from the most significant hits in the HD study. Results In the analysis of DNA repair genes as a group, we found the most significant association with age at onset when grouping all polyglutamine diseases (HD+SCAs; p = 1.43 × 10–5). In individual SNP analysis, we found significant associations for rs3512 in FAN1 with HD+SCAs (p = 1.52 × 10–5) and all SCAs (p = 2.22 × 10–4) and rs1805323 in PMS2 with HD+SCAs (p = 3.14 × 10–5), all in the same direction as in the HD GWAS. Interpretation We show that DNA repair genes significantly modify age at onset in HD and SCAs, suggesting a common pathogenic mechanism, which could operate through the observed somatic expansion of repeats that can be modulated by genetic manipulation of DNA repair in disease models. This offers novel therapeutic opportunities in multiple diseases. Ann Neurol 2016;79:983–990 PMID:27044000

  14. Modulation of wound healing and scar formation by MG53 protein-mediated cell membrane repair.

    Science.gov (United States)

    Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

    2015-10-02

    Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53(-/-) mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Enhanced Critical Size Defect Repair in Rabbit Mandible by Electrospun Gelatin/β-TCP Composite Nanofibrous Membranes

    Directory of Open Access Journals (Sweden)

    Mingming Xu

    2015-01-01

    Full Text Available The design and fabrication of biodegradable barrier membranes with satisfactory structure and composition remain a considerable challenge for periodontal tissue regeneration. We have developed a biomimetic nanofibrous membrane made from a composite of gelatin and β-tricalcium phosphate (β-TCP. We previously confirmed the in vitro biological performance of the membrane material, but the efficacy of the membranes in promoting bone repair in situ has not yet been examined. Gelatin/β-TCP composite nanofibers were fabricated by incorporation of 20 wt.% β-TCP nanoparticles into electrospun gelatin nanofibers. Electron microscopy showed that the composite membranes presented a nonwoven structure with an interconnected porous network and had a rough surface due to the β-TCP nanoparticles, which were distributed widely and uniformly throughout the gelatin-fiber matrix. The repair efficacy of rabbit mandible defects implanted with bone substitute (Bio-Oss and covered with the gelatin/β-TCP composite nanofibrous membrane was evaluated in comparison with pure gelatin nanofibrous membrane. Gross observation, histological examination, and immunohistochemical analysis showed that new bone formation and defect closure were significantly enhanced by the composite membranes compared to the pure gelatin ones. From these results, we conclude that nanofibrous gelatin/β-TCP composite membranes could serve as effective barrier membranes for guided tissue regeneration.

  16. Perichondrium/cartilage composite graft for repairing large tympanic membrane perforations and hearing improvement

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiao-wei; YANG Hua; GAO Ru-zhen; YU Rong; GAO Zhi-qiang

    2010-01-01

    Background The main risk factors for postoperative failure in tympanoplasties are large perforations that are difficult to repair, annular perforations, and a tympanic membrane (TM) with extensive granular myringitis that require middle ear exploration and mastoidectomy. The aim of this study was to investigate a novel technique of perichondrium/cartilage composite graft for repairing the large TM perforation in the patient of otitis media.Methods Retrospective chart reviews were conducted for 102 patients with large tympanic membrane perforations, who had undergone tympanoplasty from August 2005 to August 2008. Tympanoplasty or tympanomastoidectomy using a perichondrium/cartilage composite graft was analyzed. The tragal or conchal perichondrium/cartilage was used to replace the tympanic membrane in patients.Results Patients aged from 13 to 67 years were followed up in average for 24 months (10-36 months). Seventy-four ears (72.61%) were used the tragal perichondrium/cartilage as graft material and 27 ears (27.39%) were used the conchal perichondrium/cartilage. Graft take was successful in all patients. Postoperative complications such as wound infection, hematoma, or sensorineural hearing loss were not identified. Nine patients (8.82%) had the partial ossicular replacement prosthesis, 14 patients (13.72%) using the autologous curved incus and 79 patients (77.45%) without prosthesis. Successful closure occurred in 92% of the ears. A total of 85.8% patients achieved a postoperative hearing improvement.Conclusions The graft underlay tympanoplasty using perichonddum/cartilage composite is effective for the majority of patients with large perforation. The hearing was improved even if the mastoidectomy was required in the patients with otitis media with extensive granulation.

  17. Electrochemical performance of a localized repair. Effect of applying a primer and a bonding membrane

    Directory of Open Access Journals (Sweden)

    Castro-Borges, P.

    2003-12-01

    Full Text Available It is customary, in the constructions at the North of Yucatán, to employ primers to the rebars as a repair method in reinforced concrete, it has been observed in the literature that using primers alone presents an electrochemical behaviour still under discussion. Under these circumstances, several investigations have focused on the evaluation of the factor against or in favour of using primers as a repair method in a tropical marine environment. In this paper, the effects of applying a bonding membrane and the galvanic effect generated by the repair are also discussed. Small reinforced concrete columns were prepared and exposed under accelerated degradation cycles. After the activation of the rebars, the columns were repaired applying three types of primers, leaving one bar as control. After curing, the columns were exposed again to the same environment and their electrochemical behaviour was monitored. Corrosion potentials, polarization resistance, carbonation and chloride content were measured as a function of time. Conclusions suggest that the bonding membrane reduces the corrosion rates for the conditions and exposure time studied. The galvanic effect was observed after repairing the columns with and without bonding membrane, but with a tendency to reduce with time. The primers applied helped to reduce the galvanic effect of the repair, and to increase the polarization resistance.

    En las construcciones del norte de Yucatán se emplean, por costumbre, las imprimaciones al refuerzo como método de reparación al hormigón armado. Se ha observado en la literatura que la utilización sólo de imprimaciones presenta un comportamiento electroquímico, motivo éste, aún, de amplia discusión. Ante estas situaciones se llevaron a cabo varias investigaciones que tienen como objetivo la evaluación de las situaciones que favorezcan o no la utilización de las imprimaciones como método de reparación en un ambiente tropical marino. En este

  18. XLF/Cernunnos: An important but puzzling participant in the nonhomologous end joining DNA repair pathway.

    Science.gov (United States)

    Menon, Vijay; Povirk, Lawrence F

    2017-10-01

    DNA double strand breaks (DSBs) are one of the most deleterious DNA lesions that promote cell death, genomic instability and carcinogenesis. The two major cellular mechanisms that repair DSBs are Nonhomologous End-Joining (NHEJ) and Homologous Recombination Repair (HRR). NHEJ is the predominant pathway, in which XLF (also called Cernunnos) is a key player. Patients with XLF mutation exhibit microcephaly, lymphopenia, and growth retardation, and are immunodeficient and radiosensitive. During NHEJ, XLF interacts with XRCC4-Ligase IV, stimulates its ligase activity, and forms DNA-binding filaments of alternating XLF and XRCC4 dimers that may serve to align broken DNA and promote ligation of noncomplementary ends. Despite its central role in NHEJ, the effects of XLF deficiency are surprisingly variable in different biological contexts, and different individual cell lines. This review summarizes the role of XLF in NHEJ, and the unexpected complexity of its interplay with other repair factors in supporting radiosurvival and V(D)J recombination. Copyright © 2017. Published by Elsevier B.V.

  19. Impaired nucleotide excision repair pathway as a possible factor in pathogenesis of head and neck cancer

    Energy Technology Data Exchange (ETDEWEB)

    Sliwinski, T. [Department of Molecular Genetics, University of Lodz, Lodz (Poland); Markiewicz, L. [Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Lodz (Poland); Rusin, P. [Department of Molecular Genetics, University of Lodz, Lodz (Poland); Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Lodz (Poland); Kabzinski, J. [Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Lodz (Poland); Dziki, L. [Department of General and Colorectal Surgery, Medical University of Lodz, Lodz (Poland); Milonski, J.; Olszewski, J. [Department of Otolaryngology and Oncology, Medical University of Lodz, Lodz (Poland); Blaszczyk, J. [Department of Human Physiology, Medical University of Lodz, Lodz (Poland); Szemraj, J. [Department of Medical Biochemistry, Medical University of Lodz, Lodz (Poland); Majsterek, I., E-mail: ireneusz.majsterek@umed.lodz.pl [Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, Lodz (Poland)

    2011-11-01

    Tobacco smoking is one of the major risk factors in pathogenesis of head and neck squamous cell carcinomas (HNSCC). Many of the chemical compounds present in tobacco are well-known carcinogens which form adducts with DNA. Cells remove these adducts mainly by the nucleotide excision repair pathway (NER). NER also eliminates a broad spectrum of pyrimidine dimers (CPD) and photo-products (6-4PP) induced by UV-radiation or DNA cross-links after cisplatin anti-cancer treatment. In this study DNA damage and repair was examined in peripheral blood lymphocytes obtained from 20 HNSCC patients and 20 healthy controls as well as HTB-43 larynx and SSC-25 tongue cancer cell lines. DNA repair kinetics in the examined cells after cisplatin or UV-radiation treatment were investigated using alkaline comet assay during 240 min of post-treatment incubation. MTT assay was used to analyse cell viability and the Annexin V-FITC kit specific for kinase-3 was employed to determine apoptosis after treating the cells with UV-radiation at dose range from 0.5 to 60 J/m{sup 2}. NER capability was assessed in vitro with cell extracts by the use of a bacterial plasmid irradiated with UV-light as a substrate for the repair. The results show that lymphocytes from HNSCC patients and HTB-43 or SSC-25 cancer cells were more sensitive to genotoxic treatment with UV-radiation and displayed impaired DNA repair. Also evidenced was a higher rate of apoptosis induction after UV-radiation treatment of lymphocytes from the HNSCC patients and the HTB-43 cancer cells than after treatment of those from healthy donors. Finally, our results showed that there was a significant decrease in NER capacity in HTB-43 or SSC-25 cancer cells as well as in peripheral blood lymphocytes of HNSCC patients compared to controls. In conclusion, we suggest that the impaired NER pathway might be a critical factor in pathogenesis of head and neck cancer.

  20. Highly stable and self-repairing membrane-mimetic 2D nanomaterials assembled from lipid-like peptoids

    Science.gov (United States)

    Jin, Haibao; Jiao, Fang; Daily, Michael D.; Chen, Yulin; Yan, Feng; Ding, Yan-Huai; Zhang, Xin; Robertson, Ellen J.; Baer, Marcel D.; Chen, Chun-Long

    2016-07-01

    An ability to develop sequence-defined synthetic polymers that both mimic lipid amphiphilicity for self-assembly of highly stable membrane-mimetic 2D nanomaterials and exhibit protein-like functionality would revolutionize the development of biomimetic membranes. Here we report the assembly of lipid-like peptoids into highly stable, crystalline, free-standing and self-repairing membrane-mimetic 2D nanomaterials through a facile crystallization process. Both experimental and molecular dynamics simulation results show that peptoids assemble into membranes through an anisotropic formation process. We further demonstrated the use of peptoid membranes as a robust platform to incorporate and pattern functional objects through large side-chain diversity and/or co-crystallization approaches. Similar to lipid membranes, peptoid membranes exhibit changes in thickness upon exposure to external stimuli; they can coat surfaces in single layers and self-repair. We anticipate that this new class of membrane-mimetic 2D nanomaterials will provide a robust matrix for development of biomimetic membranes tailored to specific applications.

  1. Genetic variation in DNA repair pathways and risk of non-Hodgkin's lymphoma.

    Directory of Open Access Journals (Sweden)

    Justin Rendleman

    Full Text Available Molecular and genetic evidence suggests that DNA repair pathways may contribute to lymphoma susceptibility. Several studies have examined the association of DNA repair genes with lymphoma risk, but the findings from these reports have been inconsistent. Here we provide the results of a focused analysis of genetic variation in DNA repair genes and their association with the risk of non-Hodgkin's lymphoma (NHL. With a population of 1,297 NHL cases and 1,946 controls, we have performed a two-stage case/control association analysis of 446 single nucleotide polymorphisms (SNPs tagging the genetic variation in 81 DNA repair genes. We found the most significant association with NHL risk in the ATM locus for rs227060 (OR = 1.27, 95% CI: 1.13-1.43, p = 6.77×10(-5, which remained significant after adjustment for multiple testing. In a subtype-specific analysis, associations were also observed for the ATM locus among both diffuse large B-cell lymphomas (DLBCL and small lymphocytic lymphomas (SLL, however there was no association observed among follicular lymphomas (FL. In addition, our study provides suggestive evidence of an interaction between SNPs in MRE11A and NBS1 associated with NHL risk (OR = 0.51, 95% CI: 0.34-0.77, p = 0.0002. Finally, an imputation analysis using the 1,000 Genomes Project data combined with a functional prediction analysis revealed the presence of biologically relevant variants that correlate with the observed association signals. While the findings generated here warrant independent validation, the results of our large study suggest that ATM may be a novel locus associated with the risk of multiple subtypes of NHL.

  2. The Fanconi anemia pathway: Repairing the link between DNA damage and squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Romick-Rosendale, Lindsey E. [Division of Oncology, Cancer and Blood Diseases Institute, Cincinnati Children' s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229 (United States); Lui, Vivian W.Y.; Grandis, Jennifer R. [Department of Otolaryngology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Wells, Susanne I., E-mail: Susanne.Wells@cchmc.org [Division of Oncology, Cancer and Blood Diseases Institute, Cincinnati Children' s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229 (United States)

    2013-03-15

    Fanconi anemia (FA) is a rare inherited recessive disease caused by mutations in one of fifteen genes known to encode FA pathway components. In response to DNA damage, nuclear FA proteins associate into high molecular weight complexes through a cascade of post-translational modifications and physical interactions, followed by the repair of damaged DNA. Hematopoietic cells are particularly sensitive to the loss of these interactions, and bone marrow failure occurs almost universally in FA patients. FA as a disease is further characterized by cancer susceptibility, which highlights the importance of the FA pathway in tumor suppression, and will be the focus of this review. Acute myeloid leukemia is the most common cancer type, often subsequent to bone marrow failure. However, FA patients are also at an extreme risk of squamous cell carcinoma (SCC) of the head and neck and gynecological tract, with an even greater incidence in those individuals who have received a bone marrow transplant and recovered from hematopoietic disease. FA tumor suppression in hematopoietic versus epithelial compartments could be mechanistically similar or distinct. Definition of compartment specific FA activities is now critical to assess the effects of today's bone marrow failure treatments on tomorrow's solid tumor development. It is our hope that current therapies can then be optimized to decrease the risk of malignant transformation in both hematopoietic and epithelial cells. Here we review our current understanding of the mechanisms of action of the Fanconi anemia pathway as it contributes to stress responses, DNA repair and squamous cell carcinoma susceptibility.

  3. Homologous recombination is a primary pathway to repair DNA double-strand breaks generated during DNA rereplication.

    Science.gov (United States)

    Truong, Lan N; Li, Yongjiang; Sun, Emily; Ang, Katrina; Hwang, Patty Yi-Hwa; Wu, Xiaohua

    2014-10-17

    Re-initiation of DNA replication at origins within a given cell cycle would result in DNA rereplication, which can lead to genome instability and tumorigenesis. DNA rereplication can be induced by loss of licensing control at cellular replication origins, or by viral protein-driven multiple rounds of replication initiation at viral origins. DNA double-strand breaks (DSBs) are generated during rereplication, but the mechanisms of how these DSBs are repaired to maintain genome stability and cell viability are poorly understood in mammalian cells. We generated novel EGFP-based DSB repair substrates, which specifically monitor the repair of rereplication-associated DSBs. We demonstrated that homologous recombination (HR) is an important mechanism to repair rereplication-associated DSBs, and sister chromatids are used as templates for such HR-mediated DSB repair. Micro-homology-mediated non-homologous end joining (MMEJ) can also be used but to a lesser extent compared to HR, whereas Ku-dependent classical non-homologous end joining (C-NHEJ) has a minimal role to repair rereplication-associated DSBs. In addition, loss of HR activity leads to severe cell death when rereplication is induced. Therefore, our studies identify HR, the most conservative repair pathway, as the primary mechanism to repair DSBs upon rereplication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways.

    Science.gov (United States)

    Arnal, Suzzette M; Holub, Abigail J; Salus, Sandra S; Roth, David B

    2010-05-01

    V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC's ability to restrict repair pathway choice.

  5. Repairing Fetal Membranes with a Self-adhesive Ultrathin Polymeric Film: Evaluation in Mid-gestational Rabbit Model.

    Science.gov (United States)

    Pensabene, Virginia; Patel, Premal P; Williams, Phillip; Cooper, Trisha L; Kirkbride, Kellye C; Giorgio, Todd D; Tulipan, Noel B

    2015-08-01

    Preterm premature rupture of membranes causes 40% of all preterm births, affecting 150000 women each year in the United States. Prenatal diagnostic procedures and surgical interventions increase incidence of adverse events, leading to iatrogenic membrane rupture after a fetoscopic procedure in 45% of cases. We propose an ultrathin, self-adherent, poly-L-lactic acid patch ("nanofilm") as a reparative wound closure after endoscopic/fetoscopic procedures. These nanofilms are compatible with application in wet conditions and with minimally invasive instrumentation. Ex vivo studies to evaluate the nanofilm were conducted using human chorion-amnion (CA) membranes. A custom-built inflation device was used for mechanical characterization of CA membranes and for assessment of nanofilm adhesion and sealing of membrane defects up to 3 mm in size. These ex vivo tests demonstrated the ability of the nanofilm to seal human CA defects ranging in size from 1 to 3 mm in diameter. In vivo survival studies were conducted in 25 mid-gestational rabbits, defects were created by perforating the uterus and the CA membranes and subsequently using the nanofilm to seal these wounds. These in vivo studies confirmed the successful sealing of defects smaller than 3 mm observed ex vivo. Histological analysis of whole harvested uteri 7 days after surgery showed intact uterine walls in 59% of the nanofilm repaired fetuses, along with increased uterine size and intrauterine development in 63% of the cases. In summary, we have developed an ultrathin, self-adhesive nanofilm for repair of uterine membrane defects.

  6. Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Joo, Woo; Xu, Guozhou; Persky, Nicole S.; Smogorzewska, Agata; Rudge, Derek G.; Buzovetsky, Olga; Elledge, Stephen J.; Pavletich, Nikola P. (Harvard-Med); (Cornell); (MSKCC)

    2011-08-29

    Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

  7. Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway

    Energy Technology Data Exchange (ETDEWEB)

    W Joo; G Xu; n Persky; A Smogorzewska; D Rudge; O Buzovetsky; S Elledge; N Pavletich

    2011-12-31

    Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

  8. Self-Repairing Membranes for Inflatable Structures Inspired by a Rapid Wound Sealing Process of Climbing Plants

    Institute of Scientific and Technical Information of China (English)

    Markus Rampf; Olga Speck; Thomas Speck; Rolf H. Luchsinger

    2011-01-01

    A new self-repairing membrane for inflatable light weight structures such as rubber boats or Tensairity(R) constructions is presented. Inspired by rapid self-sealing processes in plants, a thin soft cellular polyurethane foam coating is applied on the inside of a fabric substrate, which closes the fissure if the membrane is punctured with a spike. Experimental tests are carried out with a purpose built setup by measuring the air mass flow through a leak in a damaged membrane sample. It is shown that the weight per unit area of the self-repairing foam as well as the curing of the two component PU-foam under an overpressure influence the repair efficiency. Curing the foam under overpressure affects the relative density as well as the microstructure of the foam coatings. Maximal median repair efficiencies of 0.999 have been obtained with 0.16 g·cm-2 foam cured at 1 bar overpressure. These results suggest that the bio-inspired technique has the potential to extend the functional integrity of injured inflatable structures dramatically.

  9. Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells

    OpenAIRE

    Johnson, Roger D.; Jasin, Maria

    2000-01-01

    In mammalian cells, repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. By definition, homologous recombination requires a template with sufficient sequence identity to the damaged molecule in order to direct repair. We now show that the sister chromatid acts as a repair template in a substantial proportion of DSB repair events. The outcome of sister chromatid repair is primarily gene conversion unassociated with reciprocal exchange. This contras...

  10. Preventing damage limitation: targeting DNA-PKcs and DNA double strand break repair pathways for ovarian cancer therapy

    Directory of Open Access Journals (Sweden)

    Daniela A Dungl

    2015-10-01

    Full Text Available Platinum-based chemotherapy is the cornerstone of ovarian cancer treatment, and its efficacy is dependent on the generation of DNA damage, with subsequent induction of apoptosis. Inappropriate or aberrant activation of the DNA damage response network is are associated with resistance to platinum, and defects in DNA repair pathways play critical roles in determining patient response to chemotherapy. In ovarian cancer, tumour cell defects in homologous recombination - a repair pathway activated in response to DNA double strand breaks (DSB - are most commonly associated with platinum sensitive disease. However, despite initial sensitivity, the emergence of resistance is frequent. Here, we review strategies for directly interfering with DNA repair pathways, with particular focus on direct inhibition of non-homologous end joining (NHEJ, another DSB repair pathway. DNA-PKcs is a core component of NHEJ and it has shown considerable promise as a chemosensitization target in numerous cancer types, including ovarian cancer where it functions to promote platinum-induced survival signalling, via AKT activation. The development of pharmacological inhibitors of DNA-PKcs is on-going, and clinic-ready agents offer real hope to patients with chemoresistant disease.

  11. Endoscopic observation of different repair patterns in human traumatic tympanic membrane perforations.

    Science.gov (United States)

    Huang, Peng; Zhang, Shujun; Gong, Xinhong; Wang, Xuesong; Lou, Zi-Han

    2017-08-03

    In the last decade, there has been an increasing use of biomaterial patches in the regeneration of traumatic tympanic membrane perforations. The major advantages of biomaterial patches are to provisionally restore the physiological function of the middle ear, thereby immediately improving ear symptoms, and act as a scaffold for epithelium migration. However, whether there are additional biological effects on eardrum regeneration is unclear for biological material patching in the clinic. This study evaluated the healing response for different repair patterns in human traumatic tympanic membrane perforations by endoscopic observation. In total, 114 patients with traumatic tympanic membrane perforations were allocated sequentially to two groups: the spontaneous healing group (n=57) and Gelfoam patch-treated group (n=57). The closure rate, closure time, and rate of otorrhea were compared between the groups at 3 months. Ultimately, 107 patients were analyzed in the two groups (52 patients in the spontaneous healing group vs. 55 patients in the Gelfoam patch-treated group). The overall closure rate at the end of the 3 month follow-up period was 90.4% in the spontaneous healing group and 94.5% in the Gelfoam patch-treated group; the difference was not statistically significant (p>0.05). However, the total average closure time was significantly different between the two groups (26.8±9.1 days in the spontaneous healing group vs. 14.7±9.1 days in the Gelfoam patch-treated group, p<0.01). In addition, the closure rate was not significantly different between the spontaneous healing group and Gelfoam patch-treated group regardless of the perforation size. The closure time in the Gelfoam patch-treated group was significantly shorter than that in the spontaneous healing group regardless of the perforation size (small perforations: 7.1±1.6 days vs. 12.6±3.9, medium-sized perforations: 13.3±2.2 days vs. 21.8±4.2 days, and large perforations: 21.2±4.7 days vs. 38.4±5.7 days

  12. 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.

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

    Science.gov (United States)

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

    2015-10-01

    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 T4endonuclease 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 T4endonuclease 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, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 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.

  14. 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)

  15. Elementary Energy Transfer Pathways in Allochromatium vinosum Photosynthetic Membranes

    Energy Technology Data Exchange (ETDEWEB)

    Lüer, Larry; Carey, Anne-Marie; Henry, Sarah; Maiuri, Margherita; Hacking, Kirsty; Polli, Dario; Cerullo, Giulio; Cogdell, Richard J.

    2015-11-01

    Allochromatium vinosum (formerly Chromatium vinosum) purple bacteria are known to adapt their light-harvesting strategy during growth according to environmental factors such as temperature and average light intensity. Under low light illumination or low ambient temperature conditions, most of the LH2 complexes in the photosynthetic membranes form a B820 exciton with reduced spectral overlap with LH1. To elucidate the reason for this light and temperature adaptation of the LH2 electronic structure, we performed broadband femtosecond transient absorption spectroscopy as a function of excitation wavelength in A. vinosum membranes. A target analysis of the acquired data yielded individual rate constants for all relevant elementary energy transfer (ET) processes. We found that the ET dynamics in high-light-grown membranes was well described by a homogeneous model, with forward and backward rate constants independent of the pump wavelength. Thus, the overall B800→B850→B890→ Reaction Center ET cascade is well described by simple triexponential kinetics. In the low-light-grown membranes, we found that the elementary backward transfer rate constant from B890 to B820 was strongly reduced compared with the corresponding constant from B890 to B850 in high-light-grown samples. The ET dynamics of low-light-grown membranes was strongly dependent on the pump wavelength, clearly showing that the excitation memory is not lost throughout the exciton lifetime. The observed pump energy dependence of the forward and backward ET rate constants suggests exciton diffusion via B850→ B850 transfer steps, making the overall ET dynamics nonexponential. Our results show that disorder plays a crucial role in our understanding of low-light adaptation in A. vinosum.

  16. Elementary Energy Transfer Pathways in Allochromatium vinosum Photosynthetic Membranes.

    Science.gov (United States)

    Lüer, Larry; Carey, Anne-Marie; Henry, Sarah; Maiuri, Margherita; Hacking, Kirsty; Polli, Dario; Cerullo, Giulio; Cogdell, Richard J

    2015-11-01

    Allochromatium vinosum (formerly Chromatium vinosum) purple bacteria are known to adapt their light-harvesting strategy during growth according to environmental factors such as temperature and average light intensity. Under low light illumination or low ambient temperature conditions, most of the LH2 complexes in the photosynthetic membranes form a B820 exciton with reduced spectral overlap with LH1. To elucidate the reason for this light and temperature adaptation of the LH2 electronic structure, we performed broadband femtosecond transient absorption spectroscopy as a function of excitation wavelength in A. vinosum membranes. A target analysis of the acquired data yielded individual rate constants for all relevant elementary energy transfer (ET) processes. We found that the ET dynamics in high-light-grown membranes was well described by a homogeneous model, with forward and backward rate constants independent of the pump wavelength. Thus, the overall B800→B850→B890→ Reaction Center ET cascade is well described by simple triexponential kinetics. In the low-light-grown membranes, we found that the elementary backward transfer rate constant from B890 to B820 was strongly reduced compared with the corresponding constant from B890 to B850 in high-light-grown samples. The ET dynamics of low-light-grown membranes was strongly dependent on the pump wavelength, clearly showing that the excitation memory is not lost throughout the exciton lifetime. The observed pump energy dependence of the forward and backward ET rate constants suggests exciton diffusion via B850→ B850 transfer steps, making the overall ET dynamics nonexponential. Our results show that disorder plays a crucial role in our understanding of low-light adaptation in A. vinosum.

  17. Canonical DNA Repair Pathways Influence R-Loop-Driven Genome Instability.

    Science.gov (United States)

    Stirling, Peter C; Hieter, Philip

    2016-07-22

    DNA repair defects create cancer predisposition in humans by fostering a higher rate of mutations. While DNA repair is quite well characterized, recent studies have identified previously unrecognized relationships between DNA repair and R-loop-mediated genome instability. R-loops are three-stranded nucleic acid structures in which RNA binds to genomic DNA to displace a loop of single-stranded DNA. Mutations in homologous recombination, nucleotide excision repair, crosslink repair, and DNA damage checkpoints have all now been linked to formation and function of transcription-coupled R-loops. This perspective will summarize recent literature linking DNA repair to R-loop-mediated genomic instability and discuss how R-loops may contribute to mutagenesis in DNA-repair-deficient cancers.

  18. Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

    Energy Technology Data Exchange (ETDEWEB)

    Henrique Barreta, Marcos [Universidade Federal de Santa Catarina, Campus Universitario de Curitibanos, Curitibanos, SC (Brazil); Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Garziera Gasperin, Bernardo; Braga Rissi, Vitor; Cesaro, Matheus Pedrotti de [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Ferreira, Rogerio [Centro de Educacao Superior do Oeste-Universidade do Estado de Santa Catarina, Chapeco, SC (Brazil); Oliveira, Joao Francisco de; Goncalves, Paulo Bayard Dias [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Bordignon, Vilceu, E-mail: vilceu.bordignon@mcgill.ca [Department of Animal Science, McGill University, Ste-Anne-De-Bellevue, QC (Canada)

    2012-10-01

    This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

  19. Inflammatory and repair pathways induced in human bronchoalveolar lavage cells with ozone inhalation.

    Directory of Open Access Journals (Sweden)

    Pascale Leroy

    Full Text Available Inhalation of ambient levels of ozone causes airway inflammation and epithelial injury.To examine the responses of airway cells to ozone-induced oxidative injury, 19 subjects (7 with asthma were exposed to clean air (0ppb, medium (100ppb, and high (200ppb ambient levels of ozone for 4h on three separate occasions in a climate-controlled chamber followed by bronchoscopy with bronchoalveolar lavage (BAL 24h later. BAL cell mRNA expression was examined using Affymetrix GeneChip Microarray. The role of a differentially expressed gene (DEG in epithelial injury was evaluated in an in vitro model of injury [16HBE14o- cell line scratch assay].Ozone exposure caused a dose-dependent up-regulation of several biologic pathways involved in inflammation and repair including chemokine and cytokine secretion, activity, and receptor binding; metalloproteinase and endopeptidase activity; adhesion, locomotion, and migration; and cell growth and tumorigenesis regulation. Asthmatic subjects had 1.7- to 3.8-fold higher expression of many DEGs suggestive of increased proinflammatory and matrix degradation and remodeling signals. The most highly up-regulated gene was osteopontin, the protein level of which in BAL fluid increased in a dose-dependent manner after ozone exposure. Asthmatic subjects had a disproportionate increase in non-polymerized osteopontin with increasing exposure to ozone. Treatment with polymeric, but not monomeric, osteopontin enhanced the migration of epithelial cells and wound closure in an α9β1 integrin-dependent manner.Expression profiling of BAL cells after ozone exposure reveals potential regulatory genes and pathways activated by oxidative stress. One DEG, osteopontin, promotes epithelial wound healing in an in vitro model of injury.

  20. Modulation of DNA Damage and Repair Pathways by Human Tumour Viruses

    Directory of Open Access Journals (Sweden)

    Robert Hollingworth

    2015-05-01

    Full Text Available With between 10% and 15% of human cancers attributable to viral infection, there is great interest, from both a scientific and clinical viewpoint, as to how these pathogens modulate host cell functions. Seven human tumour viruses have been identified as being involved in the development of specific malignancies. It has long been known that the introduction of chromosomal aberrations is a common feature of viral infections. Intensive research over the past two decades has subsequently revealed that viruses specifically interact with cellular mechanisms responsible for the recognition and repair of DNA lesions, collectively known as the DNA damage response (DDR. These interactions can involve activation and deactivation of individual DDR pathways as well as the recruitment of specific proteins to sites of viral replication. Since the DDR has evolved to protect the genome from the accumulation of deleterious mutations, deregulation is inevitably associated with an increased risk of tumour formation. This review summarises the current literature regarding the complex relationship between known human tumour viruses and the DDR and aims to shed light on how these interactions can contribute to genomic instability and ultimately the development of human cancers.

  1. In vivo and in vitro evaluation of an Acetobacter xylinum synthesized microbial cellulose membrane intended for guided tissue repair

    Directory of Open Access Journals (Sweden)

    de Lima-Neto João

    2009-03-01

    Full Text Available Abstract Background Barrier materials as cellulose membranes are used for guided tissue repair. However, it is essential that the surrounding tissues accept the device. The present study histologically evaluated tissue reaction to a microbial cellulose membrane after subcutaneous implantation in mice. Furthermore, the interaction between mesenchymal stem cells and the biomaterial was studied in vitro to evaluate its ability to act as cellular scaffold for tissue engineering. Methods Twenty-five Swiss Albino mice were used. A 10 × 10 mm cellulose membrane obtained through biosynthesis using Acetobacter xylinum bacteria was implanted into the lumbar subcutaneous tissue of each mouse. The mice were euthanatized at seven, 15, 30, 60, and 90 days, and the membrane and surrounding tissues were collected and examined by histology. Results A mild inflammatory response without foreign body reaction was observed until 30 days post-surgery around the implanted membrane. Polarized microscopy revealed that the membrane remained intact at all evaluation points. Scanning electron microscopy of the cellulose membrane surface showed absence of pores. The in vitro evaluation of the interaction between cells and biomaterial was performed through viability staining analysis of the cells over the biomaterial, which showed that 95% of the mesenchymal stem cells aggregating to the cellulose membrane were alive and that 5% were necrotic. Scanning electron microscopy showed mesenchymal stem cells with normal morphology and attached to the cellulose membrane surface. Conclusion The microbial cellulose membrane evaluated was found to be nonresorbable, induced a mild inflammatory response and may prove useful as a scaffold for mesenchymal stem cells.

  2. Highly stable and self-repairing membrane-mimetic 2D nanomaterials assembled from lipid-like peptoids

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Haibao; Jiao, Fang; Daily, Michael D.; Chen, Yulin; Yan, Feng; Ding, Yanhuai; Zhang, Xin; Robertson, Ellen J.; Baer, Marcel D.; Chen, Chunlong

    2016-07-12

    Two-dimensional (2D) materials with molecular-scale thickness have attracted increasing interest for separation, electronic, catalytic, optical, energy and biomedical applications.1-4 Although extensive research on 2D materials, such as graphene and graphene oxide, has been performed in recent years, progress is limited on self-assembly of 2D materials from sequence-specific macromolecules,5-7 especially from synthetic sequences that could exhibit lipid-like self-assembly of bilayer sheets and mimic membrane proteins for functions. The creation of such new class of materials could enable development of highly stable biomimetic membranes that exhibit cell-membrane-like molecular transport with exceptional selectively and high transport rates.8,9 Here we demonstrate self-assembly of lipid-like 12-mer peptoids into extremely stable, crystalline, flexible and free-standing 2D membrane materials. As with cell membranes, upon exposure to external stimuli, these materials exhibit changes in thickness,10 varying from 3.5 nm to 5.6 nm. We find that self-assembly occurs through a facile crystallization process, in which inter-peptoid hydrogen bonds and enhanced hydrophobic interactions drive the formation of a highly-ordered structure. Molecular simulation confirms this is the energetically favored structure. Displaying functional groups at arbitrary locations of membrane-forming peptoids produces membranes with similar structures. This research further shows that single-layer membranes can be coated onto substrate surfaces. Moreover, membranes with mechanically-induced defects can self-repair. Given that peptoids are sequence-specific and exhibit protein-like molecular recognition with enhanced stability,11 we anticipate our membranes to be a robust platform tailored to specific applications.

  3. The Hypoxia-Inducible Factor Pathway, Prolyl Hydroxylase Domain Protein Inhibitors, and Their Roles in Bone Repair and Regeneration

    Directory of Open Access Journals (Sweden)

    Lihong Fan

    2014-01-01

    Full Text Available Hypoxia-inducible factors (HIFs are oxygen-dependent transcriptional activators that play crucial roles in angiogenesis, erythropoiesis, energy metabolism, and cell fate decisions. The group of enzymes that can catalyse the hydroxylation reaction of HIF-1 is prolyl hydroxylase domain proteins (PHDs. PHD inhibitors (PHIs activate the HIF pathway by preventing degradation of HIF-α via inhibiting PHDs. Osteogenesis and angiogenesis are tightly coupled during bone repair and regeneration. Numerous studies suggest that HIFs and their target gene, vascular endothelial growth factor (VEGF, are critical regulators of angiogenic-osteogenic coupling. In this brief perspective, we review current studies about the HIF pathway and its role in bone repair and regeneration, as well as the cellular and molecular mechanisms involved. Additionally, we briefly discuss the therapeutic manipulation of HIFs and VEGF in bone repair and bone tumours. This review will expand our knowledge of biology of HIFs, PHDs, PHD inhibitors, and bone regeneration, and it may also aid the design of novel therapies for accelerating bone repair and regeneration or inhibiting bone tumours.

  4. Synergistic interactions between RAD5, RAD16, and RAD54, three partially homologous yeast DNA repair genes each in a different repair pathway

    Energy Technology Data Exchange (ETDEWEB)

    Glassner, B.J. [Univ. of California, Berkeley, CA (United States); Mortimer, R.K. [Univ. of California, Berkeley, CA (United States)]|[Lawrence Berkeley Laboratory, Berkeley, CA (United States)

    1994-07-01

    Considerable homology has recently been noted between the proteins encoded by the RAD5, RAD16 and RAD54 genes of Saccharomyces cerevisiae. These genes are members of the RAD6, RAD3 and RAD50 epistasis groups, respectively, which correspond to the three major DNA repair pathways in yeast. These proteins also share homology with other eucaryotic proteins, including those encoded by SNF2 and MO1 of yeast, brahma and lodestar of Drosophila and the human ERCC6 gene. The homology shares features with known helicases, suggesting a newly identified helicase subfamily. We have constructed a series of congenic single-, double- and triple-deletion mutants involving RAD5, RAD16 and RAD54 to examine the interactions between these genes. Each deletion mutation alone has only a moderate effect on survival after exposure to UV radiation. Each pairwise-double mutant exhibits marked synergism. The triple-deletion mutant displays further synergism. These results confirm the assignment of the RAD54 gene to the RAD50 epistasis group and suggest that the RAD16 gene plays a larger role in DNA repair after exposure to UV radiation than has been suggested previously. Additionally, the proteins encoded by RAD5, RAD16, and RAD54 may compete for the same substrate after damage induced by UV radiation, possibly at an early step in their respective pathways. 49 refs., 6 figs., 2 tabs.

  5. Inhibitory effect of membrane-binding drugs on excision repair of DNA damage in UV-irradiated Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Todo, T.; Yonei, S. (Kyoto Univ. (Japan). Lab. of Radiation Biology)

    1983-04-01

    The effects of procaine and lidocaine on DNA-repair processes were investigated in UV-irradiated cells of E. coli with different DNA-repair capacities. The cells were irradiated with various doses of UV and then incubated at 37/sup 0/C in M9 buffer (liquid-holding) or in EM9 medium in the presence or absence of membrane-binding drugs. The results obtained are as follows. (1) In strains H/r30 (wild-type for DNA repair) and NG30 (recA/sup -/), the increase in survival with increase in time of liquid-holding was almost completely inhibited by the addition of procaine and lidocaine. The same trends were observable under conditions of post-irradiation incubation in EM9 medium, more efficiently in recA/sup -/ strain than in the wild-type strain. (2) The addition of these drugs gave an apparent enhancement of the frequency of UV-induced mutation to arginine prototrophy, corresponding to a decrease in survival. (3) There were negligible effects of the drugs on survival and mutation in the excision-repair-defective strain, Hs30 (uvrB/sup -/). (4) The removal of thymine dimers from DNA was actually reduced by the addition of procaine.

  6. Vascular basement membranes as pathways for the passage of fluid into and out of the brain.

    Science.gov (United States)

    Morris, Alan W J; Sharp, Matthew MacGregor; Albargothy, Nazira J; Fernandes, Rute; Hawkes, Cheryl A; Verma, Ajay; Weller, Roy O; Carare, Roxana O

    2016-05-01

    In the absence of conventional lymphatics, drainage of interstitial fluid and solutes from the brain parenchyma to cervical lymph nodes is along basement membranes in the walls of cerebral capillaries and tunica media of arteries. Perivascular pathways are also involved in the entry of CSF into the brain by the convective influx/glymphatic system. The objective of this study is to differentiate the cerebral vascular basement membrane pathways by which fluid passes out of the brain from the pathway by which CSF enters the brain. Experiment 1: 0.5 µl of soluble biotinylated or fluorescent Aβ, or 1 µl 15 nm gold nanoparticles was injected into the mouse hippocampus and their distributions determined at 5 min by transmission electron microscopy. Aβ was distributed within the extracellular spaces of the hippocampus and within basement membranes of capillaries and tunica media of arteries. Nanoparticles did not enter capillary basement membranes from the extracellular spaces. Experiment 2: 2 µl of 15 nm nanoparticles were injected into mouse CSF. Within 5 min, groups of nanoparticles were present in the pial-glial basement membrane on the outer aspect of cortical arteries between the investing layer of pia mater and the glia limitans. The results of this study and previous research suggest that cerebral vascular basement membranes form the pathways by which fluid passes into and out of the brain but that different basement membrane layers are involved. The significance of these findings for neuroimmunology, Alzheimer's disease, drug delivery to the brain and the concept of the Virchow-Robin space are discussed.

  7. DNA repair pathways in radiation induced cellular damage: a molecular approach

    NARCIS (Netherlands)

    L.R. van Veelen (Lieneke)

    2005-01-01

    markdownabstract__Abstract__ DNA damage, especially double-strand breaks, can be induced by endogenous or exogenous darnaging agents, such as ionizing radiation. Repair of DNA damage is very important in maintaining genomic stability. Incorrect repair may lead to chromosomal aberrations,

  8. DNA repair pathways in radiation induced cellular damage: a molecular approach

    NARCIS (Netherlands)

    L.R. van Veelen (Lieneke)

    2005-01-01

    markdownabstract__Abstract__ DNA damage, especially double-strand breaks, can be induced by endogenous or exogenous darnaging agents, such as ionizing radiation. Repair of DNA damage is very important in maintaining genomic stability. Incorrect repair may lead to chromosomal aberrations, translocat

  9. Base excision repair pathway: PARP1 genotypes as modulators of therapy response in cervical cancer patients.

    Science.gov (United States)

    Nogueira, Augusto; Assis, Joana; Faustino, Ilda; Pereira, Deolinda; Catarino, Raquel; Medeiros, Rui

    2017-02-01

    Genetic polymorphisms in genes of the base excision repair (BER) pathway appear to modulate the therapy response of cancer patients. PARP1 protein recognizes the DNA strand damage and facilitates the subsequent recruitment of BER proteins. Few studies have reported an association between PARP1 Val762Ala polymorphism (rs1136410) and cancer therapy response. The purpose of our study was to determine whether PARP1 Val762Ala polymorphism have prognostic value in patients with cervical cancer. Two hundred and sixty adult patients, with histologically confirmed cervical cancer, at FIGO-stages IB2-IVA, primarily treated with concurrent chemotherapy (cisplatin) and radiotherapy. Overall survival (OS) and disease-free survival (DFS) were the primary end points of the analysis. The PARP1 Val762Ala genetic variants were analyzed by allelic discrimination by real-time PCR. We observed that peri- and postmenopausal women carrying the C-allele present a statistically significant lower OS and DFS (log-rank test, p = 0.008 and p = 0.006, respectively) among those with early stage cervical cancer. Cox regression analysis confirmed these results, after adjustment for other prognostic factors (for OS: HR, 3.70; 95%CI, 1.32-10.38; p = 0.013 and for DFS: HR, 3.97; 95%CI, 1.59-9.93; p = 0.003). This is the first study evaluating the effect of PARP1 Val762Ala polymorphism in treatment response in cervical cancer patients. PARP1 genotypes may contribute as an independent prognostic factor in cervical cancer, being useful in predicting the clinical outcome.

  10. Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents

    Science.gov (United States)

    Klapacz, Joanna; Pottenger, Lynn H.; Engelward, Bevin P.; Heinen, Christopher D.; Johnson, George E.; Clewell, Rebecca A.; Carmichael, Paul L.; Adeleye, Yeyejide; Andersen, Melvin E.

    2016-01-01

    From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. PMID:27036068

  11. The role of C/EBPβ phosphorylation in modulating membrane phospholipids repairing in LPS-induced human lung/bronchial epithelial cells.

    Science.gov (United States)

    Shu, Shiyu; Xu, Yan; Xie, Ling; Ouyang, Yufang

    2017-09-20

    study of underlying mechanism show that the activity of C/EBP β depends on its phosphorylation:LPS stimulation reduced C/EBP β phosphorylation and suppressed the transcription of CCSP1 in BEAS-2B cells, which resulted in enhanced PLA2 and the consequent membrane damage. And further study shows that overexpression of CDK2(Cyclindependent kinase 2), promoted the phosphorylation of C/EBP β and inhibited PLA2 through the C/EBP β/CCSP1/PLA2 pathway, so as to attenuate membrane damage. The significance of this study lies in that artificial C/EBP β phosphorylation regulation may ease the membrane damage in ALI and improve membrane repair. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Membrane-displayed peptide ligand activates the pheromone response pathway in Saccharomyces cerevisiae.

    Science.gov (United States)

    Hara, Keisuke; Ono, Takuya; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2012-05-01

    The budding yeast, Saccharomyces cerevisiae, is an attractive host for studying G protein-coupled receptors (GPCRs). We developed a system in which a peptide ligand specific for GPCR is displayed on yeast plasma membrane. The model system described here is based on yeast plasma membrane display of an analogue of α-factor, which is a peptide ligand for Ste2p, the GPCR that activates the yeast pheromone response pathway. α-Factor analogues, containing linkers of varying lengths and produced in yeast cells, became attached to the cell plasma membrane by linking to the glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein Yps1p. We were able to demonstrate that an optimized α-factor analogue activated the pheromone response pathway in S. cerevisiae, as assessed by a fluorescent reporter assay. Furthermore, it was shown that linker length strongly influenced signalling pathway activation. To our knowledge, this is the first report documenting functional signalling by a plasma membrane-displayed ligand in S. cerevisiae.

  13. Ndrg3 gene regulates DSB repair during meiosis through modulation the ERK signal pathway in the male germ cells

    Science.gov (United States)

    Pan, Hongjie; Zhang, Xuan; Jiang, Hanwei; Jiang, Xiaohua; Wang, Liu; Qi, Qi; Bi, Yuan; Wang, Jian; Shi, Qinghua; Li, Runsheng

    2017-01-01

    The N-myc downstream regulated gene (NDRG) family consists of 4 members, NDRG-1, -2, -3, -4. Physiologically, we found Ndrg3, a critical gene which led to homologous lethality in the early embryo development, regulated the male meiosis in mouse. The expression of Ndrg3 was enhanced specifically in germ cells, and reached its peak level in the pachytene stage spermatocyte. Haplo-insufficiency of Ndrg3 gene led to sub-infertility during the male early maturation. In the Ndrg3+/− germ cells, some meiosis events such as DSB repair and synaptonemal complex formation were impaired. Disturbances on meiotic prophase progression and spermatogenesis were observed. In mechanism, the attenuation of pERK1/2 signaling was detected in the heterozygous testis. With our primary spermatocyte culture system, we found that lactate promoted DSB repair via ERK1/2 signaling in the male mouse germ cells in vitro. Deficiency of Ndrg3 gene attenuated the activation of ERK which further led to the aberrancy of DSB repair in the male germ cells in mouse. Taken together, we reported that Ndrg3 gene modulated the lactate induced ERK pathway to facilitate DSB repair in male germ cells, which further regulated meiosis and subsequently fertility in male mouse. PMID:28290521

  14. Ndrg3 gene regulates DSB repair during meiosis through modulation the ERK signal pathway in the male germ cells.

    Science.gov (United States)

    Pan, Hongjie; Zhang, Xuan; Jiang, Hanwei; Jiang, Xiaohua; Wang, Liu; Qi, Qi; Bi, Yuan; Wang, Jian; Shi, Qinghua; Li, Runsheng

    2017-03-14

    The N-myc downstream regulated gene (NDRG) family consists of 4 members, NDRG-1, -2, -3, -4. Physiologically, we found Ndrg3, a critical gene which led to homologous lethality in the early embryo development, regulated the male meiosis in mouse. The expression of Ndrg3 was enhanced specifically in germ cells, and reached its peak level in the pachytene stage spermatocyte. Haplo-insufficiency of Ndrg3 gene led to sub-infertility during the male early maturation. In the Ndrg3(+/-) germ cells, some meiosis events such as DSB repair and synaptonemal complex formation were impaired. Disturbances on meiotic prophase progression and spermatogenesis were observed. In mechanism, the attenuation of pERK1/2 signaling was detected in the heterozygous testis. With our primary spermatocyte culture system, we found that lactate promoted DSB repair via ERK1/2 signaling in the male mouse germ cells in vitro. Deficiency of Ndrg3 gene attenuated the activation of ERK which further led to the aberrancy of DSB repair in the male germ cells in mouse. Taken together, we reported that Ndrg3 gene modulated the lactate induced ERK pathway to facilitate DSB repair in male germ cells, which further regulated meiosis and subsequently fertility in male mouse.

  15. Importance of the cell cycle phase for the choice of the appropriate DSB repair pathway, for genome stability maintenance: the trans-S double-strand break repair model.

    Science.gov (United States)

    Delacôte, Fabien; Lopez, Bernard S

    2008-01-01

    A DNA double-strand break (DSB) is a highly harmful lesion that can lead to genome rearrangements. Two main pathways compete for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). Depending on the cell cycle phase, the choice of one DSB repair pathway over the other will secure genome stability maintenance or in contrast will increase the risk of genetic instability. HR with the sister chromatid is an efficient way to maintain genome stability, for damage occurring at a post-replication stage. However, in G(1) checkpoint-defective cells, DSBs produced in the G(1) phase and not repaired by NHEJ, can progress through S phase and be processed by HR in late S/G(2) phase. We propose the "trans-S DSB repair" model to account for these data. In this situation HR cannot use the sister chromatid (which is also broken at the same locus) and is thus forced to use ectopic homologous sequences dispersed through the genome, increasing the risk of genetic instability. This shows that the two DSB repair pathways can compete through the cell cycle and underlines the importance of the association between the cell cycle checkpoint and the appropriate DNA repair pathway for genome stability maintenance.

  16. GENETIC AND MOLECULAR ANALYSIS OF DNA DAMAGE REPAIR AND TOLERANCE PATHWAYS.

    Energy Technology Data Exchange (ETDEWEB)

    SUTHERLAND, B.M.

    2001-07-26

    Radiation can damage cellular components, including DNA. Organisms have developed a panoply of means of dealing with DNA damage. Some repair paths have rather narrow substrate specificity (e.g. photolyases), which act on specific pyrimidine photoproducts in a specific type (e.g., DNA) and conformation (double-stranded B conformation) of nucleic acid. Others, for example, nucleotide excision repair, deal with larger classes of damages, in this case bulky adducts in DNA. A detailed discussion of DNA repair mechanisms is beyond the scope of this article, but one can be found in the excellent book of Friedberg et al. [1] for further detail. However, some DNA damages and paths for repair of those damages important for photobiology will be outlined below as a basis for the specific examples of genetic and molecular analysis that will be presented below.

  17. Classical and alternative end-joining pathways for repair of lymphocyte-specific and general DNA double-strand breaks.

    Science.gov (United States)

    Boboila, Cristian; Alt, Frederick W; Schwer, Bjoern

    2012-01-01

    Classical nonhomologous end joining (C-NHEJ) is one of the two major known pathways for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Our understanding of C-NHEJ has been derived, in significant part, through studies of programmed physiologic DNA DSBs formed during V(D)J recombination in the developing immune system. Studies of immunoglobulin heavy-chain (IgH) class-switch recombination (CSR) also have revealed that there is an "alternative" end-joining process (A-EJ) that can function, relatively robustly, in the repair of DSBs in activated mature B lymphocytes. This A-EJ process has also been implicated in the formation of oncogenic translocations found in lymphoid tumors. In this review, we discuss our current understanding of C-NHEJ and A-EJ in the context of V(D)J recombination, CSR, and the formation of chromosomal translocations. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. PTH1–34 Blocks Radiation-induced Osteoblast Apoptosis by Enhancing DNA Repair through Canonical Wnt Pathway*

    Science.gov (United States)

    Chandra, Abhishek; Lin, Tiao; Zhu, Ji; Tong, Wei; Huo, Yanying; Jia, Haoruo; Zhang, Yejia; Liu, X. Sherry; Cengel, Keith; Xia, Bing; Qin, Ling

    2015-01-01

    Focal radiotherapy for cancer patients has detrimental effects on bones within the radiation field and the primary clinical signs of bone damage include the loss of functional osteoblasts. We reported previously that daily injection of parathyroid hormone (PTH, 1–34) alleviates radiation-induced osteopenia in a preclinical radiotherapy model by improving osteoblast survival. To elucidate the molecular mechanisms, we irradiated osteoblastic UMR 106-01 cells and calvarial organ culture and demonstrated an anti-apoptosis effect of PTH1–34 on these cultures. Inhibitor assay indicated that PTH exerts its radioprotective action mainly through protein kinase A/β-catenin pathway. γ-H2AX foci staining and comet assay revealed that PTH efficiently promotes the repair of DNA double strand breaks (DSBs) in irradiated osteoblasts via activating the β-catenin pathway. Interestingly, Wnt3a alone also blocked cell death and accelerated DNA repair in primary osteoprogenitors, osteoblastic and osteocytic cells after radiation through the canonical signaling. Further investigations revealed that both Wnt3a and PTH increase the amount of Ku70, a core protein for initiating the assembly of DSB repair machinery, in osteoblasts after radiation. Moreover, down-regulation of Ku70 by siRNA abrogated the prosurvival effect of PTH and Wnt3a on irradiated osteoblasts. In summary, our results identify a novel role of PTH and canonical Wnt signaling in regulating DSB repair machinery and apoptosis in osteoblasts and shed light on using PTH1–34 or Wnt agonist as possible therapy for radiation-induced osteoporosis. PMID:25336648

  19. Ku regulates the non-homologous end joining pathway choice of DNA double-strand break repair in human somatic cells.

    Directory of Open Access Journals (Sweden)

    Farjana Fattah

    2010-02-01

    Full Text Available The repair of DNA double-strand breaks (DSBs is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR and non-homologous end joining (NHEJ. In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways-the main Ku heterodimer-dependent or "classic" NHEJ (C-NHEJ pathway and an "alternative" NHEJ (A-NHEJ pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PK(cs, XLF, and LIGIV, and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PK(cs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PK(cs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice.

  20. Ku regulates the non-homologous end joining pathway choice of DNA double-strand break repair in human somatic cells.

    Directory of Open Access Journals (Sweden)

    Farjana Fattah

    2010-02-01

    Full Text Available The repair of DNA double-strand breaks (DSBs is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR and non-homologous end joining (NHEJ. In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways-the main Ku heterodimer-dependent or "classic" NHEJ (C-NHEJ pathway and an "alternative" NHEJ (A-NHEJ pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PK(cs, XLF, and LIGIV, and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PK(cs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PK(cs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice.

  1. Alternative end-joining and classical nonhomologous end-joining pathways repair different types of double-strand breaks during class-switch recombination.

    Science.gov (United States)

    Cortizas, Elena M; Zahn, Astrid; Hajjar, Maurice E; Patenaude, Anne-Marie; Di Noia, Javier M; Verdun, Ramiro E

    2013-12-01

    Classical nonhomologous end-joining (C-NHEJ) and alternative end-joining (A-EJ) are the main DNA double-strand break (DSB) repair pathways when a sister chromatid is not available. However, it is not clear how one pathway is chosen over the other to process a given DSB. To address this question, we studied in mouse splenic B cells and CH12F3 cells how C-NHEJ and A-EJ repair DSBs initiated by the activation-induced deaminase during IgH (Igh) class-switch recombination (CSR). We show in this study that lowering the deamination density at the Igh locus increases DSB resolution by microhomology-mediated repair while decreasing C-NHEJ activity. This process occurs without affecting 53BP1 and γH2AX levels during CSR. Mechanistically, lowering deamination density increases exonuclease I recruitment and single-stranded DNA at the Igh locus and promotes C-terminal binding protein interacting protein and MSH2-dependent DSB repair during CSR. Indeed, reducing activation-induced deaminase levels increases CSR efficiency in C-NHEJ-defective cells, suggesting enhanced use of an A-EJ pathway. Our results establish a mechanism by which C-NHEJ and this C-terminal binding protein interacting protein/MSH2-dependent pathway that relies on microhomology can act concurrently but independently to repair different types of DSBs and reveal that the density of DNA lesions influences the choice of DSB repair pathway during CSR.

  2. Promotion of Dental Pulp Cell Migration and Pulp Repair by a Bioceramic Putty Involving FGFR-mediated Signaling Pathways.

    Science.gov (United States)

    Zhang, J; Zhu, L X; Cheng, X; Lin, Y; Yan, P; Peng, B

    2015-06-01

    Mineral trioxide aggregate is the currently recommended material of choice for clinical pulp repair despite several disadvantages, including handling inconvenience. Little is known about the signaling mechanisms involved in bioceramic-mediated dental pulp repair-particularly, dental pulp cell (DPC) migration. This study evaluated the effects of iRoot BP Plus, a novel ready-to-use nanoparticulate bioceramic putty, on DPC migration in vitro and pulp repair in vivo, focusing on possible involvement of fibroblast growth factor receptor (FGFR)-related signaling, including mitogen-activated protein kinase and Akt pathways. Treatment with iRoot BP Plus extracts enhanced horizontal and vertical migration of DPCs, which was comparable with the effects induced by mineral trioxide aggregate extracts. The DPCs exposed to iRoot BP Plus extracts demonstrated no evident apoptosis. Importantly, treatment with iRoot BP Plus extracts resulted in rapid activation of FGFR, p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK), and Akt signaling in DPCs. Confocal immunofluorescence staining revealed that iRoot BP Plus stimulated focal adhesion formation and stress fiber assembly in DPCs, in addition to upregulating the expression of focal adhesion molecules, including p-focal adhesion kinase, p-paxillin, and vinculin. Moreover, activation of FGFR, ERK, JNK, and Akt were found to mediate the upregulated expression of focal adhesion molecules, stress fiber assembly, and enhanced DPC migration induced by iRoot BP Plus. Consistent with the in vitro results, we observed induction of homogeneous dentin bridge formation and expression of p-focal adhesion kinase, p-FGFR, p-ERK 1/2, p-JNK, and p-Akt near injury sites by iRoot BP Plus in an in vivo pulp repair model. These data demonstrate that iRoot BP Plus can promote DPC migration and pulp repair involving the FGFR-mediated ERK 1/2, JNK, and Akt pathways. These findings provide

  3. A trans-well-based cellular model for the rapid pre-evaluation of tympanic membrane repair materials.

    Science.gov (United States)

    Hung, Shih-Han; Su, Chin-Hui; Tseng, How

    2016-08-01

    It is important to have a standardized tympanic membrane (TM) perforation platform to evaluate the various myringoplasty materials that have been studied and developed extensively during recent years. However, currently there are no cellular models specifically designed for this purpose, and animal models remain unsatisfactory. The purpose of this study is to propose an inexpensive, readily available, well-controlled, and easy-to-create cellular model as a substitute for use in the evaluation of TM repairing materials. A trans-well model was created using a cell culture insert with a round hole created at the center of the polycarbonate membrane. HaCaT cells were cultured on the fenestrated culture insert, and the desired myringoplasty graft was placed at the center of the window for one week and observed by fluorescent microscopy under vital staining. Under this cellular model, there was notable migration of HaCaT cells onto the positive control graft (rabbit fascia), while only a few cell clusters were observed on the negative control graft (paper). Model validation showed that the cell migration ratio for the PLLA + 1% hyaluronic acid (HA) graft is significantly higher than using myringoplasty paper, poly L-lactide (PLLA), or PLLA + 0.5% HA (p model might be a useful pre-evaluation platform for the evaluation of TM repairing materials. The model is inexpensive, readily available, easy to create, and standardized for use.

  4. Toxicity and mutagenicity of plumbagin and the induction of a possible new DNA repair pathway in Escherichia coli.

    Science.gov (United States)

    Farr, S B; Natvig, D O; Kogoma, T

    1985-12-01

    Actively growing Escherichia coli cells exposed to plumbagin, a redox cycling quinone that increases the flux of O2- radicals in the cell, were mutagenized or killed by this treatment. The toxicity of plumbagin was not found to be mediated by membrane damage. Cells pretreated with plumbagin could partially reactivate lambda phage damaged by exposure to riboflavin plus light, a treatment that produces active oxygen species. The result suggested the induction of a DNA repair response. Lambda phage damaged by H2O2 treatment were not reactivated in plumbagin-pretreated cells, nor did H2O2-pretreated cells reactivate lambda damaged by treatment with riboflavin plus light. Plumbagin treatment did not induce lambda phage in a lysogen, nor did it cause an increase in beta-galactosidase production in a dinD::Mu d(lac Ap) promoter fusion strain. Cells pretreated with nonlethal doses of plumbagin showed enhanced survival upon exposure to high concentrations of plumbagin, but were unchanged in their susceptibility to far-UV irradiation. polA and recA mutants were not significantly more sensitive than wild type to killing by plumbagin. However, xth-1 mutants were partially resistant to plumbagin toxicity. It is proposed that E. coli has an inducible DNA repair response specific for the type of oxidative damage generated during incubation with plumbagin. Furthermore, this response appears to be qualitatively distinct from the SOS response and the repair response induced by H2O2.

  5. Interplay between Target Sequences and Repair Pathways Determines Distinct Outcomes of AID-Initiated Lesions.

    Science.gov (United States)

    Chen, Zhangguo; Eder, Maxwell D; Elos, Mihret T; Viboolsittiseri, Sawanee S; Chen, Xiaomi; Wang, Jing H

    2016-03-01

    Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of Ab gene diversification. However, precise mechanisms regulating AID deamination frequency remain incompletely understood. Moreover, it is not known whether different sequence contexts influence the preferential access of mismatch repair or uracil glycosylase (UNG) to AID-initiated U:G mismatches. In this study, we employed two knock-in models to directly compare the mutability of core Sμ and VDJ exon sequences and their ability to regulate AID deamination and subsequent repair process. We find that the switch (S) region is a much more efficient AID deamination target than the V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by mismatch repair deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating Ab gene diversity and AID targeting specificity.

  6. Pentavalent antimony uptake pathway through erythrocyte membranes: molecular and atomic fluorescence approaches.

    Science.gov (United States)

    Barrera, Camila; López, Silvana; Aguilar, Luis; Mercado, Luis; Bravo, Manuel; Quiroz, Waldo

    2016-04-01

    Previous studies by our group have shown that Sb(V) is able to enter red blood cells in a dynamic process and is reduced to Sb(III) by glutathione. The present study aims to investigate a possible entry pathway for Sb(V) through the erythrocyte membrane. Applying fluorescence spectroscopy studies with Laurdan and diphenylhexatriene (DPH) probes, it was found that there was no interaction between Sb(V) and membrane lipids. By comparing the Sb(V) entry percentages through lipid vesicles and sealed erythrocyte membranes, it was found that Sb(V) required protein channels to pass through the membrane. The competitive inhibition results using HCO3 (-) and Cl(-) showed that the Sb(V) uptake rate through the membrane fell approximately 50-70 % until full inhibition was reached, which was possibly due to the inhibition of the anion exchanger 1 (AE1) channel. Finally, the fluorescence measurements with the 5-iodoacetamidofluorescein (5-IAF) probe showed that Sb(V) interacted with membrane protein SH groups during this process.

  7. A Cross-Cancer Genetic Association Analysis of the DNA repair and DNA Damage Signaling Pathways for Lung, Ovary, Prostate, Breast and Colorectal Cancer

    Science.gov (United States)

    Scarbrough, Peter M.; Weber, Rachel Palmieri; Iversen, Edwin S.; Brhane, Yonathan; Amos, Christopher I.; Kraft, Peter; Hung, Rayjean J.; Sellers, Thomas A.; Witte, John S.; Pharoah, Paul; Henderson, Brian E.; Gruber, Stephen B.; Hunter, David J.; Garber, Judy E.; Joshi, Amit D.; McDonnell, Kevin; Easton, Doug F.; Eeles, Ros; Kote-Jarai, Zsofia; Muir, Kenneth; Doherty, Jennifer A.; Schildkraut, Joellen M.

    2015-01-01

    Background DNA damage is an established mediator of carcinogenesis, though GWAS have identified few significant loci. This cross-cancer site, pooled analysis was performed to increase the power to detect common variants of DNA repair genes associated with cancer susceptibility. Methods We conducted a cross-cancer analysis of 60,297 SNPs, at 229 DNA repair gene regions, using data from the NCI Genetic Associations and Mechanisms in Oncology (GAME-ON) Network. Our analysis included data from 32 GWAS and 48,734 controls and 51,537 cases across five cancer sites (breast, colon, lung, ovary, and prostate). Because of the unavailability of individual data, data were analyzed at the aggregate level. Meta-analysis was performed using the Association analysis for SubSETs (ASSET) software. To test for genetic associations that might escape individual variant testing due to small effect sizes, pathway analysis of eight DNA repair pathways was performed using hierarchical modeling. Results We identified three susceptibility DNA repair genes, RAD51B (p < 5.09 × 10−6), MSH5 (p < 5.09 × 10−6) and BRCA2 (p = 5.70 × 10−6). Hierarchical modeling identified several pleiotropic associations with cancer risk in the base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination pathways. Conclusions Only three susceptibility loci were identified which had all been previously reported. In contrast, hierarchical modeling identified several pleiotropic cancer risk associations in key DNA repair pathways. Impact Results suggest that many common variants in DNA repair genes are likely associated with cancer susceptibility through small effect sizes that do not meet stringent significance testing criteria. PMID:26637267

  8. Urothelial endocytic vesicle recycling and lysosomal degradative pathway regulated by lipid membrane composition.

    Science.gov (United States)

    Grasso, E J; Calderón, R O

    2013-02-01

    The urothelium, a specialized epithelium that covers the mucosa cell surface of the urinary bladder, undergoes dramatic morphological changes during the micturition cycle that involve a membrane apical traffic. This traffic was first described as a lysosomal pathway, in addition to the known endocytosis/exocytosis membrane recycling. In an attempt to understand the role of membrane lipid composition in those effects, we previously described the lipid-dependent leakage of the endocytosed vesicle content. In this work, we demonstrated clear differences in the traffic of both the fluid probe and the membrane-bound probe in urothelial umbrella cells by using spectrofluorometry and/or confocal and epifluorescence microscopy. Different membrane lipid compositions were established by using three diet formulae enriched in oleic acid, linoleic acid and a commercial formula. Between three and five animals for each dietary treatment were used for each analysis. The decreased endocytosis of both fluid and membrane-bound probes (approximately 32 and 49 % lower, respectively) in oleic acid-derived umbrella cells was concomitant with an increased recycling (approximately 4.0 and 3.7 times, respectively) and diminished sorting to the lysosome (approximately 23 and 37 %, respectively) when compared with the control umbrella cells. The higher intravesicular pH and the impairment of the lysosomal pathway of oleic acid diet-derived vesicles compared to linoleic acid diet-derived vesicles and control diet-derived vesicles correlate with our findings of a lower V-ATPase activity previously reported. We integrated the results obtained in the present and previous work to determine the sorting of endocytosed material (fluid and membrane-bound probes) into the different cell compartments. Finally, the weighted average effect of the individual alterations on the intracellular distribution was evaluated. The results shown in this work add evidences for the modulatory role of the membrane

  9. Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Noda, Taichi [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Takahashi, Akihisa [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Kondo, Natsuko [Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Mori, Eiichiro [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Okamoto, Noritomo [Department of Otorhinolaryngology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Nakagawa, Yosuke [Department of Oral and Maxillofacial Surgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Ohnishi, Ken [Department of Biology, Ibaraki Prefectual University of Health Sciences, 4669-2 Ami, Ami-mati, Inasiki-gun, Ibaraki 300-0394 (Japan); Zdzienicka, Malgorzata Z. [Department of Molecular Cell Genetics, Collegium Medicum in Bydgoszcz, Nicolaus-Copernicus-University in Torun, ul. Sklodowskiej-Curie 9, 85-094 Bydgoszcz (Poland); Thompson, Larry H. [Biosciences and Biotechnology Division, L452, Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551-0808 (United States); Helleday, Thomas [Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ (United Kingdom); Department of Genetics, Microbiology and Toxicology Stockholm University, SE-106 91 Stockholm (Sweden); Asada, Hideo [Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); and others

    2011-01-07

    The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA{sup -/-}, FANCC{sup -/-}, FANCA{sup -/-}C{sup -/-}, FANCD2{sup -/-} and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical {gamma}H2AX-staining assay. Although the sensitivity of FANCA{sup -/-}, FANCC{sup -/-} and FANCA{sup -/-}C{sup -/-} cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2{sup -/-} cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, {gamma}H2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex. -- Research highlights: {yields} We examined to clarify the repair pathways of formaldehyde-induced DNA damage. Formaldehyde induces DNA double strand breaks (DSBs). {yields} DSBs are repaired through the Fanconi anemia (FA) repair pathway. {yields} This pathway is independent of the FA nuclear core complex. {yields} We also found that homologous recombination repair was induced by formaldehyde.

  10. TGFβ1 protects cells from γ-IR by enhancing the activity of the NHEJ repair pathway.

    Science.gov (United States)

    Kim, Mi-Ra; Lee, Jeeyong; An, You Sun; Jin, Yeung Bae; Park, In-Chul; Chung, Eunkyung; Shin, Incheol; Barcellos-Hoff, Mary Helen; Yi, Jae Youn

    2015-02-01

    Several groups have reported that TGFβ1 regulates cellular responses to γ-irradiation; however, the exact mechanism has not been fully elucidated. In the current study, the role of TGFβ1 in cellular responses to γ-irradiation was investigated in detail. The data indicate that TGFβ1 pretreatment decreased the aftermath of ionizing radiation (IR)-induced DNA damage in a SMAD-dependent manner. To determine the underlying mechanism for these effects, the extent of IR-induced DNA repair activity in the presence or absence of TGFβ1 was examined. Studies reveal that TGFβ1 upregulated DNA ligase IV (Lig4), augmented IR-induced nuclear retention of the DNA ligase, and enhanced nonhomologous end-joining (NHEJ) repair activity. In addition, knockdown of Lig4 reduced the TGFβ1-induced protection against IR. Overall, these data indicate that TGFβ1 facilitates the NHEJ repair process upon γ-irradiation and thereby enhances long-term survival. These findings provide new insight and a possible approach to controlling genotoxic stress by the TGFβ signaling pathway. ©2014 American Association for Cancer Research.

  11. RAG2 mutants alter DSB repair pathway choice in vivo and illuminate the nature of 'alternative NHEJ'.

    Science.gov (United States)

    Gigi, Vered; Lewis, Susanna; Shestova, Olga; Mijušković, Martina; Deriano, Ludovic; Meng, Wenzhao; Luning Prak, Eline T; Roth, David B

    2014-06-01

    DNA double-stranded breaks (DSBs) can be repaired by several mechanisms, including classical NHEJ (c-NHEJ) and a poorly defined, error-prone process termed alternative NHEJ (a-NHEJ). How cells choose between these alternatives to join physiologic DSBs remains unknown. Here, we show that deletion of RAG2's C-terminus allows a-NHEJ to repair RAG-mediated DSBs in developing lymphocytes from both c-NHEJ-proficient and c-NHEJ-deficient mice, demonstrating that the V(D)J recombinase influences repair pathway choice in vivo. Analysis of V(D)J junctions revealed that, contrary to expectation, junctional characteristics alone do not reliably distinguish between a-NHEJ and c-NHEJ. These data suggest that a-NHEJ is not necessarily mutagenic, and may be more prevalent than previously appreciated. Whole genome sequencing of a lymphoma arising in a p53(-/-) mouse bearing a C-terminal RAG2 truncation reveals evidence of a-NHEJ and also of aberrant recognition of DNA sequences resembling RAG recognition sites.

  12. Electroacupuncture in the repair of spinal cord injury: inhibiting the Notch signaling pathway and promoting neural stem cell proliferation

    Directory of Open Access Journals (Sweden)

    Xin Geng

    2015-01-01

    Full Text Available Electroacupuncture for the treatment of spinal cord injury has a good clinical curative effect, but the underlying mechanism is unclear. In our experiments, the spinal cord of adult Sprague-Dawley rats was clamped for 60 seconds. Dazhui (GV14 and Mingmen (GV4 acupoints of rats were subjected to electroacupuncture. Enzyme-linked immunosorbent assay revealed that the expression of serum inflammatory factors was apparently downregulated in rat models of spinal cord injury after electroacupuncture. Hematoxylin-eosin staining and immunohistochemistry results demonstrated that electroacupuncture contributed to the proliferation of neural stem cells in rat injured spinal cord, and suppressed their differentiation into astrocytes. Real-time quantitative PCR and western blot assays showed that electroacupuncture inhibited activation of the Notch signaling pathway induced by spinal cord injury. These findings indicate that electroacupuncture repaired the injured spinal cord by suppressing the Notch signaling pathway and promoting the proliferation of endogenous neural stem cells.

  13. Electroacupuncture in the repair of spinal cord injury:inhibiting the Notch signaling pathway and promoting neural stem cell proliferation

    Institute of Scientific and Technical Information of China (English)

    Xin Geng; Tao Sun; Jing-hui Li; Ning Zhao; Yong Wang; Hua-lin Yu

    2015-01-01

    Electroacupuncture for the treatment of spinal cord injury has a good clinical curative effect, but the underlying mechanism is unclear. In our experiments, the spinal cord of adult Sprague-Daw-ley rats was clamped for 60 seconds.Dazhui (GV14) andMingmen (GV4) acupoints of rats were subjected to electroacupuncture. Enzyme-linked immunosorbent assay revealed that the expres-sion of serum inlfammatory factors was apparently downregulated in rat models of spinal cord injury after electroacupuncture. Hematoxylin-eosin staining and immunohistochemistry results demonstrated that electroacupuncture contributed to the proliferation of neural stem cells in rat injured spinal cord, and suppressed their differentiation into astrocytes. Real-time quantitative PCR and western blot assays showed that electroacupuncture inhibited activation of the Notch signaling pathway induced by spinal cord injury. These ifndings indicate that electroacupuncture repaired the injured spinal cord by suppressing the Notch signaling pathway and promoting the proliferation of endogenous neural stem cells.

  14. New discoveries linking transcription to DNA repair and damage tolerance pathways.

    Science.gov (United States)

    Cohen, Susan E; Walker, Graham C

    2011-01-01

    In Escherichia coli, the transcription elongation factor NusA is associated with all elongating RNA polymerases where it functions in transcription termination and antitermination. Here, we review our recent results implicating NusA in the recruitment of DNA repair and damage tolerance mechanisms to sites of stalled transcription complexes.

  15. DNA Double-Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities

    NARCIS (Netherlands)

    A. Shibata (Atsushi); D. Moiani (Davide); A.S. Arvai (Andrew); J. Perry (Jefferson); S.M. Harding (Shane); M.-M. Genois (Marie-Michelle); R. Maity (Ranjan); S.E. van Rossum-Fikkert (Sari); A. Kertokalio (Aryandi); F. Romoli (Filippo); A. Ismail (Amani); E. Ismalaj (Ermal); E. Petricci (Elena); M.J. Neale (Matthew); R.G. Bristow (Robert); J.-Y. Masson (Jean-Yves); C. Wyman (Claire); P.A. Jeggo (Penny); J.A. Tainer (John)

    2014-01-01

    textabstractMRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employ

  16. DNA Double-Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities

    NARCIS (Netherlands)

    A. Shibata (Atsushi); D. Moiani (Davide); A.S. Arvai (Andrew); J. Perry (Jefferson); S.M. Harding (Shane); M.-M. Genois (Marie-Michelle); R. Maity (Ranjan); S.E. van Rossum-Fikkert (Sari); A. Kertokalio (Aryandi); F. Romoli (Filippo); A. Ismail (Amani); E. Ismalaj (Ermal); E. Petricci (Elena); M.J. Neale (Matthew); R.G. Bristow (Robert); J.-Y. Masson (Jean-Yves); C. Wyman (Claire); P.A. Jeggo (Penny); J.A. Tainer (John)

    2014-01-01

    textabstractMRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we

  17. Human umbilical cord mesenchymal stem cell-loaded amniotic membrane for the repair of radial nerve injury

    Institute of Scientific and Technical Information of China (English)

    Zhi Li; Hanjiao Qin; Zishan Feng; Wei Liu; Ye Zhou; Lifeng Yang; Wei Zhao; Youjun Li

    2013-01-01

    In this study, we loaded human umbilical cord mesenchymal stem cells onto human amniotic membrane with epithelial cells to prepare nerve conduits, i.e., a relatively closed nerve regeneration chamber. After neurolysis, the injured radial nerve was enwrapped with the prepared nerve conduit, which was fixed to the epineurium by sutures, with the cellon the inner surface of the conduit. Simultaneously, a 1.0 mL aliquot of human umbilical cord mesenchymal stem cellsuspension was injected into the distal and proximal ends of the injured radial nerve with 1.0 cm intervals. A total of 1.75 × 107 cells were seeded on the amniotic membrane. In the control group, patients received only neurolysis. At 12 weeks after celltransplantation, more than 80%of patients exhibited obvious improvements in muscular strength, and touch and pain sensations. In contrast, these improve-ments were observed only in 55-65% of control patients. At 8 and 12 weeks, muscular electro-physiological function in the region dominated by the injured radial nerve was significantly better in the transplantation group than the control group. After celltransplantation, no immunological rejec-tions were observed. These findings suggest that human umbilical cord mesenchymal stem cel-loaded amniotic membrane can be used for the repair of radial nerve injury.

  18. Laser induced wounding of the plasma membrane and methods to study the repair process.

    Science.gov (United States)

    Jimenez, Ana J; Maiuri, Paolo; Lafaurie-Janvore, Julie; Perez, Franck; Piel, Matthieu

    2015-01-01

    Cells are constantly exposed to agents that can trigger the perforation of their plasma membrane. This damage occurs naturally, and the frequency and intensity depends on how much cells are exposed to damaging threats. The following protocol is a simple and powerful method to damage the plasma membrane using laser ablation. It allows the induction of a single and localized wound at the plasma membrane of cultured cells, which can be followed with fast time-lapse imaging. The first part of the protocol describes simple cell culture techniques and the material ideal to make the experiments. A second part of the protocol gives advice about the procedures to make effective wounds in cells while ensuring a good survival rate. We also propose different ways to follow the opening and closure of the plasma membrane. Finally, we describe the procedure to efficiently analyze the data acquired after single cell photodamage to characterize the wounding process.

  19. Redox regulation of genome stability by effects on gene expression, epigenetic pathways and DNA damage/repair.

    Science.gov (United States)

    Mikhed, Yuliya; Görlach, Agnes; Knaus, Ulla G; Daiber, Andreas

    2015-08-01

    Reactive oxygen and nitrogen species (e.g. H2O2, nitric oxide) confer redox regulation of essential cellular signaling pathways such as cell differentiation, proliferation, migration and apoptosis. In addition, classical regulation of gene expression or activity, including gene transcription to RNA followed by translation to the protein level, by transcription factors (e.g. NF-κB, HIF-1α) and mRNA binding proteins (e.g. GAPDH, HuR) is subject to redox regulation. This review will give an update of recent discoveries in this field, and specifically highlight the impact of reactive oxygen and nitrogen species on DNA repair systems that contribute to genomic stability. Emphasis will be placed on the emerging role of redox mechanisms regulating epigenetic pathways (e.g. miRNA, DNA methylation and histone modifications). By providing clinical correlations we discuss how oxidative stress can impact on gene regulation/activity and vise versa, how epigenetic processes, other gene regulatory mechanisms and DNA repair can influence the cellular redox state and contribute or prevent development or progression of disease.

  20. Genistein sensitizes sarcoma cells in vitro and in vivo by enhancing apoptosis and by inhibiting DSB repair pathways.

    Science.gov (United States)

    Liu, X X; Sun, C; Jin, X D; Li, P; Zheng, X G; Zhao, T; Li, Q

    2016-06-01

    The aim of this work was to investigate the radiosensitization effects of genistein on mice sarcoma cells and the corresponding biological mechanisms in vitro and in vivo Using the non-toxic dosage of 10 μM genistein, the sensitizer enhancement ratios after exposure to X-rays at 50% cell survival (IC50) was 1.45 for S180 cells. For mice cotreated with genistein and X-rays, the excised tumor tissues had reduced blood vessels and decreased size and volume compared with the control and irradiation-only groups. Moreover, a significant increase in apoptosis was accompanied by upregulation of Bax and downregulation of Bcl-2 in the mitochondria, and lots of cytochrome c being transferred to the cytoplasm. Furthermore, X-rays combined with genistein inhibited the activity of DNA-PKcs, so DNA-injured sites were dominated by Ku70/80, leading to incompleteness of homologous recombination (HR) and non-homologous end-joining (NHEJ) repairs and the eventual occurrence of cell apoptosis. Our study, for the first time, demonstrated that genistein sensitized sarcoma cells to X-rays and that this radiosensitizing effect depended on induction of the mitochondrial apoptosis pathway and inhibition of the double-strand break (DSB) repair pathways.

  1. Redox regulation of genome stability by effects on gene expression, epigenetic pathways and DNA damage/repair

    Directory of Open Access Journals (Sweden)

    Yuliya Mikhed

    2015-08-01

    Full Text Available Reactive oxygen and nitrogen species (e.g. H2O2, nitric oxide confer redox regulation of essential cellular signaling pathways such as cell differentiation, proliferation, migration and apoptosis. In addition, classical regulation of gene expression or activity, including gene transcription to RNA followed by translation to the protein level, by transcription factors (e.g. NF-κB, HIF-1α and mRNA binding proteins (e.g. GAPDH, HuR is subject to redox regulation. This review will give an update of recent discoveries in this field, and specifically highlight the impact of reactive oxygen and nitrogen species on DNA repair systems that contribute to genomic stability. Emphasis will be placed on the emerging role of redox mechanisms regulating epigenetic pathways (e.g. miRNA, DNA methylation and histone modifications. By providing clinical correlations we discuss how oxidative stress can impact on gene regulation/activity and vise versa, how epigenetic processes, other gene regulatory mechanisms and DNA repair can influence the cellular redox state and contribute or prevent development or progression of disease.

  2. The Rate and Spectrum of Spontaneous Mutations in Mycobacterium smegmatis, a Bacterium Naturally Devoid of the Postreplicative Mismatch Repair Pathway

    Directory of Open Access Journals (Sweden)

    Sibel Kucukyildirim

    2016-07-01

    Full Text Available Mycobacterium smegmatis is a bacterium that is naturally devoid of known postreplicative DNA mismatch repair (MMR homologs, mutS and mutL, providing an opportunity to investigate how the mutation rate and spectrum has evolved in the absence of a highly conserved primary repair pathway. Mutation accumulation experiments of M. smegmatis yielded a base-substitution mutation rate of 5.27 × 10−10 per site per generation, or 0.0036 per genome per generation, which is surprisingly similar to the mutation rate in MMR-functional unicellular organisms. Transitions were found more frequently than transversions, with the A:T→G:C transition rate significantly higher than the G:C→A:T transition rate, opposite to what is observed in most studied bacteria. We also found that the transition-mutation rate of M. smegmatis is significantly lower than that of other naturally MMR-devoid or MMR-knockout organisms. Two possible candidates that could be responsible for maintaining high DNA fidelity in this MMR-deficient organism are the ancestral-like DNA polymerase DnaE1, which contains a highly efficient DNA proofreading histidinol phosphatase (PHP domain, and/or the existence of a uracil-DNA glycosylase B (UdgB homolog that might protect the GC-rich M. smegmatis genome against DNA damage arising from oxidation or deamination. Our results suggest that M. smegmatis has a noncanonical Dam (DNA adenine methylase methylation system, with target motifs differing from those previously reported. The mutation features of M. smegmatis provide further evidence that genomes harbor alternative routes for improving replication fidelity, even in the absence of major repair pathways.

  3. Triple-helix formation induces recombination in mammalian cells via a nucleotide excision repair-dependent pathway.

    Science.gov (United States)

    Faruqi, A F; Datta, H J; Carroll, D; Seidman, M M; Glazer, P M

    2000-02-01

    The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.

  4. A new possibility for repairing the anal dysfunction by promoting regeneration of the reflex pathways in the enteric nervous system.

    Science.gov (United States)

    Katsui, Renta; Kojima, Yu; Kuniyasu, Hiroki; Shimizu, Juichiro; Koyama, Fumikazu; Fujii, Hisao; Nakajima, Yoshiyuki; Takaki, Miyako

    2008-04-01

    Moderate rectal distension elicits recto-rectal reflex contractions and simultaneous recto-internal anal sphincter reflex relaxations that together comprise the defecation reflex. Both reflexes are controlled by 1) pelvic nerves, 2) lumbar colonic nerves, and 3) enteric nervous system. The aim of the present study was to explore a novel approach to repairing the defecation reflex dysfunction by using the plasticity of enteric nervous pathways. Experiments were performed in anesthetized guinea pigs with ethyl carbamate. The rectum 30 mm oral from the anal verge was transected without damage to extrinsic nerves, and subsequent end-to-end one-layer anastomosis was performed. Recovery of the defecation reflex and associated reflex pathways were evaluated. Eight weeks after sectioning of intrinsic reflex nerve pathways in the rectum, the defecation reflex recovered to the control level, accompanied with regeneration of reflex pathways. The 5-HT(4)-receptor agonist mosapride (0.5 and 1.0 mg/kg) significantly (P nervous system with local application of BDNF.

  5. Heterozygous Vangl2Looptail mice reveal novel roles for the planar cell polarity pathway in adult lung homeostasis and repair

    Science.gov (United States)

    Poobalasingam, Thanushiyan; Yates, Laura L.; Walker, Simone A.; Pereira, Miguel; Gross, Nina Y.; Ali, Akmol; Kolatsi-Joannou, Maria; Jarvelin, Marjo-Riitta; Pekkanen, Juha; Papakrivopoulou, Eugenia; Long, David A.; Griffiths, Mark; Wagner, Darcy; Königshoff, Melanie; Hind, Matthew; Minelli, Cosetta; Lloyd, Clare M.

    2017-01-01

    ABSTRACT Lung diseases impose a huge economic and health burden worldwide. A key aspect of several adult lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), including emphysema, is aberrant tissue repair, which leads to an accumulation of damage and impaired respiratory function. Currently, there are few effective treatments available for these diseases and their incidence is rising. The planar cell polarity (PCP) pathway is critical for the embryonic development of many organs, including kidney and lung. We have previously shown that perturbation of the PCP pathway impairs tissue morphogenesis, which disrupts the number and shape of epithelial tubes formed within these organs during embryogenesis. However, very little is known about the role of the PCP pathway beyond birth, partly because of the perinatal lethality of many PCP mouse mutant lines. Here, we investigate heterozygous Looptail (Lp) mice, in which a single copy of the core PCP gene, Vangl2, is disrupted. We show that these mice are viable but display severe airspace enlargement and impaired adult lung function. Underlying these defects, we find that Vangl2Lp/+ lungs exhibit altered distribution of actin microfilaments and abnormal regulation of the actin-modifying protein cofilin. In addition, we show that Vangl2Lp/+ lungs exhibit many of the hallmarks of tissue damage, including an altered macrophage population, abnormal elastin deposition and elevated levels of the elastin-modifying enzyme, Mmp12, all of which are observed in emphysema. In vitro, disruption of VANGL2 impairs directed cell migration and reduces the rate of repair following scratch wounding of human alveolar epithelial cells. Moreover, using population data from a birth cohort of young adults, all aged 31, we found evidence of an interactive effect between VANGL2 and smoking on lung function. Finally, we show that PCP genes VANGL2 and SCRIB are significantly downregulated in lung

  6. DNA repair and damage pathway related cancer suppressor genes in low-dose-rate irradiated AKR/J an IR mice

    Energy Technology Data Exchange (ETDEWEB)

    Bang, Hyun Soon; Bong, Jin Jong; Kang, Yumi; Choi, Moo Hyun; Lee, Hae Un; Yoo, Jae Young; Choi, Seung Jin; Kim, Hee Sun [Radiation Health Research Institute, Korea Hydro and Nuclear Power Co., Ltd, Gyeongju (Korea, Republic of); Lee, Kyung Mi [Global Research Lab, BAERI Institute, Dept. of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of)

    2012-11-15

    It has been reported that low-dose-rate radiation stimulates the immune response, prolongs life span and inhibits carcinogenesis. The high dose-rate radiation influences the expression of DNA repair and damage-related genes. In contrast, DNA repair and damage signaling triggered by low-dose-rate irradiation remain unclear. In the present study, we investigated the differential expression of DNA repair and damage pathway related genes in the thymus of AKR/J and ICR mice after 100th day low-dose-rate irradiation. Our findings demonstrated that low-dose-rate γ -radiation suppressed tumorigenesis.

  7. Electron Pathways through Erythrocyte Plasma Membrane in Human Physiology and Pathology: Potential Redox Biomarker?

    Directory of Open Access Journals (Sweden)

    Elena Matteucci

    2007-01-01

    Full Text Available Erythrocytes are involved in the transport of oxygen and carbon dioxide in the body. Since pH is the influential factor in the Bohr-Haldane effect, pHi is actively maintained via secondary active transports Na+/H+ exchange and HC3 -/Cl- anion exchanger. Because of the redox properties of the iron, hemoglobin generates reactive oxygen species and thus, the human erythrocyte is constantly exposed to oxidative damage. Although the adult erythrocyte lacks protein synthesis and cannot restore damaged proteins, it is equipped with high activity of protective enzymes. Redox changes in the cell initiate various signalling pathways. Plasma membrane oxido-reductases (PMORs are transmembrane electron transport systems that have been found in the membranes of all cells and have been extensively characterized in the human erythrocyte. Erythrocyte PMORs transfer reducing equivalents from intracellular reductants to extracellular oxidants, thus their most important role seems to be to enable the cell respond to changes in intra- and extra-cellular redox environments.So far the activity of erythrocyte PMORs in disease states has not been systematically investigated. This review summarizes present knowledge on erythrocyte electron transfer activity in humans (health, type 1 diabetes, diabetic nephropathy, and chronic uremia and hypothesizes an integrated model of the functional organization of erythrocyte plasma membrane where electron pathways work in parallel with transport metabolons to maintain redox homeostasis.

  8. Electron Pathways through Erythrocyte Plasma Membrane in Human Physiology and Pathology: Potential Redox Biomarker?

    Science.gov (United States)

    Matteucci, Elena; Giampietro, Ottavio

    2007-09-17

    Erythrocytes are involved in the transport of oxygen and carbon dioxide in the body. Since pH is the influential factor in the Bohr-Haldane effect, pHi is actively maintained via secondary active transports Na(+)/H(+) exchange and HC(3) (-)/Cl(-) anion exchanger. Because of the redox properties of the iron, hemoglobin generates reactive oxygen species and thus, the human erythrocyte is constantly exposed to oxidative damage. Although the adult erythrocyte lacks protein synthesis and cannot restore damaged proteins, it is equipped with high activity of protective enzymes. Redox changes in the cell initiate various signalling pathways. Plasma membrane oxido-reductases (PMORs) are transmembrane electron transport systems that have been found in the membranes of all cells and have been extensively characterized in the human erythrocyte. Erythrocyte PMORs transfer reducing equivalents from intracellular reductants to extracellular oxidants, thus their most important role seems to be to enable the cell respond to changes in intra- and extra-cellular redox environments.So far the activity of erythrocyte PMORs in disease states has not been systematically investigated. This review summarizes present knowledge on erythrocyte electron transfer activity in humans (health, type 1 diabetes, diabetic nephropathy, and chronic uremia) and hypothesizes an integrated model of the functional organization of erythrocyte plasma membrane where electron pathways work in parallel with transport metabolons to maintain redox homeostasis.

  9. An alternative membrane transport pathway for phosphate and adenine nucleotides in mitochondria and its possible function.

    Science.gov (United States)

    Reynafarje, B; Lehninger, A L

    1978-10-01

    This paper describes the properties and a possible biological role of a transport process across the inner membrane of rat liver mitochondria resulting in the exchange of ATP(4-) (out) for ADP(3-) (in) + 0.5 phosphate(2-) (in). This transmembrane exchange reaction, designated as the ATP-ADP-phosphate exchange, is specific for the ligands shown, electroneutral, insensitive to N-ethylmaleimide or mersalyl, inhibited by atractyloside, and appears to occur only in the direction as written. It is thus distinct from the well-known phosphate-hydroxide and phosphate-dicarboxylate exchange systems, which are inhibited by mersalyl, and from the ATP-ADP exchanger, which does not transport phosphate. During ATP hydrolysis by mitochondria, half of the phosphate formed from ATP passes from the matrix to the medium by the mersalyl-insensitive ATP-ADP-phosphate exchange and the other half by the well-known mersalyl-sensitive phosphate-hydroxide exchange. These and other considerations have led to a hypothesis for the pathway and stoichiometry of ATP-dependent reverse electron transport, characterized by a requirement of 1.33 molecules of ATP per pair of electrons reversed and by the utilization of a different membrane transport pathway for phosphate and adenine nucleotides than is taken in forward electron flow and oxidative phosphorylation. The possible occurrence of independent pathways for ATP-forming forward electron flow and ATP-consuming reverse electron flow is consonant with the fact that the opposing degradative and synthetic pathways in the central routes of cell metabolism generally have different pathways that are independently regulated.

  10. Saturated fatty acids alter the late secretory pathway by modulating membrane properties.

    Science.gov (United States)

    Payet, Laurie-Anne; Pineau, Ludovic; Snyder, Ellen C R; Colas, Jenny; Moussa, Ahmed; Vannier, Brigitte; Bigay, Joelle; Clarhaut, Jonathan; Becq, Frédéric; Berjeaud, Jean-Marc; Vandebrouck, Clarisse; Ferreira, Thierry

    2013-12-01

    Saturated fatty acids (SFA) have been reported to alter organelle integrity and function in many cell types, including muscle and pancreatic β-cells, adipocytes, hepatocytes and cardiomyocytes. SFA accumulation results in increased amounts of ceramides/sphingolipids and saturated phospholipids (PL). In this study, using a yeast-based model that recapitulates most of the trademarks of SFA-induced lipotoxicity in mammalian cells, we demonstrate that these lipid species act at different levels of the secretory pathway. Ceramides mostly appear to modulate the induction of the unfolded protein response and the transcription of nutrient transporters destined to the cell surface. On the other hand, saturated PL, by altering membrane properties, directly impact vesicular budding at later steps in the secretory pathway, i.e. at the trans-Golgi Network level. They appear to do so by increasing lipid order within intracellular membranes which, in turn, alters the recruitment of loose lipid packing-sensing proteins, required for optimal budding, to nascent vesicles. We propose that this latter general mechanism could account for the well-documented deleterious impacts of fatty acids on the last steps of the secretory pathway in several cell types.

  11. Simulation and analysis of cellular internalization pathways and membrane perturbation for graphene nanosheets.

    Science.gov (United States)

    Mao, Jian; Guo, Ruohai; Yan, Li-Tang

    2014-07-01

    Clarifying the mechanisms of cellular interactions of graphene family nanomaterials is an urgent issue to the development of guidelines for safer biomedical applications and to the evaluation of health and environment impacts. By combining large-scale computer simulations, theoretical analysis, and experimental discussions, here we present a systematic study on the interactions of graphene nanosheets having various oxidization degrees with a model lipid bilayer membrane. In the mesoscopic simulations, we investigate the detailed translocation pathways of these materials across a 56 × 56 nm(2) membrane patch which allows us to fully consider the role of membrane perturbation during this process. A phase diagram regarding the transmembrane translocation mechanisms of graphene nanosheets is thereby obtained in the space of oxidization degree and particle size. Then, we propose a theoretical approach to analyze the effects of various initial equilibrium states of graphene nanosheets with membrane on their following cellular uptake process. Finally, we demonstrate that the simulation and theoretical results reproduce some important experimental findings towards the mechanisms of cytotoxicity and antibacterial activity of graphene materials. These results not only provide new insight into the cellular internalization mechanism of graphene-based nanomaterials but also offer fundamental understanding on their physicochemical properties which can be precisely tailored for safer biomedical and environment applications.

  12. Identification of a residue in the translocation pathway of a membrane carrier.

    Science.gov (United States)

    Yan, R T; Maloney, P C

    1993-10-08

    Preliminary work using directed mutagenesis proved that cysteine is not required for operation of UhpT, the anion exchange protein responsible for glucose 6-phosphate transport by E. coli. We then made a detailed study of C143 and C265, because these cysteines impart sensitivity to p-chloromercuribenzosulfonate (PCMBS), a sulfhydral agent resembling glucose 6-phosphate in size, shape, and charge. We showed that C143 was exposed to the cytoplasm, as expected from hydropathy analysis, but we found no sidedness for C265. Rather, C265 was accessible to PCMBS from both membrane surfaces. And since the attack at C265 was blocked by glucose 6-phosphate, position 265 must lie directly on the pathway taken by the substrate as it moves through this membrane carrier.

  13. The type and yield of ionising radiation induced chromosomal aberrations depend on the efficiency of different DSB repair pathways in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Natarajan, Adayapalam T.; Berni, Andrea; Marimuthu, Kodumudi M. [Department of Agrobiology and Agrochemistry, University of Tuscia, Via San Camillo de Lellis, 01100 Viterbo (Italy); Palitti, Fabrizio [Department of Agrobiology and Agrochemistry, University of Tuscia, Via San Camillo de Lellis, 01100 Viterbo (Italy)], E-mail: palitti@unitus.it

    2008-07-03

    In order to evaluate the relative role of two major DNA double strand break repair pathways, i.e., non-homologous end joining (NHEJ) and homologous recombination repair (HRR), CHO mutants deficient in these two pathways and the parental cells (AA8) were X-irradiated with various doses. The cells were harvested at different times after irradiation, representing G{sub 2}, S and G{sub 1} phase at the time of irradiation, The mutant cell lines used were V33 (NHEJ deficient), Irs1SF, 51-D1 (HRR deficient). In addition to parental cell line (AA8), a revertant of V33, namely V33-155 was employed. Both types of mutant cells responded with increased frequencies of chromosomal aberrations at all recovery times in comparison to the parental and revertant cells. Mutant cells deficient in NHEJ were more sensitive in all cell stages in comparison to HRR deficient mutant cells, indicating NHEJ is the major repair pathway for DSB repair through out the cell cycle. Both chromosome and chromatid types of exchange aberrations were observed following G{sub 1} irradiation (16 and 24 h recovery). Interestingly, configurations involving both chromosome (dicentrics) and chromatid exchanges were encountered in G{sub 1} irradiated V33 cells. This may indicate that unrepaired DSBs accumulate in G{sub 1} in these mutant cells and carried over to S phase, where they are repaired by HRR or other pathways such as B-NHEJ (back up NHEJ), which appear to be highly error prone. Both NHEJ and HRR, which share some of the same proteins in their pathways, are involved in the repair of DSBs leading to chromosomal aberrations, but with a major role of NHEJ in all stages of cell cycle.

  14. Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

    Directory of Open Access Journals (Sweden)

    Scheuring David

    2012-09-01

    Full Text Available Abstract Background In yeast and mammals, many plasma membrane (PM proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. Results Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. Conclusions Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route

  15. RIP4 is a target of multiple signal transduction pathways in keratinocytes: Implications for epidermal differentiation and cutaneous wound repair

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Stephanie [Charite, University Medicine Berlin, Institute of Physiology, Arnimallee 22, D-14195 Berlin (Germany); Munz, Barbara, E-mail: barbara.munz@charite.de [Charite, University Medicine Berlin, Institute of Physiology, Arnimallee 22, D-14195 Berlin (Germany)

    2010-01-01

    Receptor interacting protein 4 (RIP4) is an important regulator of epidermal morphogenesis during embryonic development. We could previously show that expression of the rip4 gene is strongly downregulated in cutaneous wound repair, which might be initiated by a broad variety of growth factors and cytokines. Here, we demonstrate that in keratinocytes, rip4 expression is controlled by a multitude of different signal transduction pathways, such as the p38 mitogen-activated protein kinase (MAPK) and the nuclear factor kappa B (NF-{kappa}B) cascade, in a unique and specific manner. Furthermore, we show that the steroid dexamethasone abolishes the physiological rip4 downregulation after injury and might thus contribute to the phenotype of reduced and delayed wound reepithelialization seen in glucocorticoid-treated patients. As a whole, our data indicate that rip4 expression is regulated in a complex manner, which might have therapeutic implications.

  16. Meta-analyses identify 13 novel loci associated with age at menopause and highlights DNA repair and immune pathways

    Science.gov (United States)

    Stolk, Lisette; Perry, John RB; Chasman, Daniel I; He, Chunyan; Mangino, Massimo; Sulem, Patrick; Barbalic, Maja; Broer, Linda; Byrne, Enda M; Ernst, Florian; Esko, Tõnu; Franceschini, Nora; Gudbjartsson, Daniel F; Hottenga, Jouke-Jan; Kraft, Peter; McArdle, Patick F; Porcu, Eleonora; Shin, So-Youn; Smith, Albert V; van Wingerden, Sophie; Zhai, Guangju; Zhuang, Wei V; Albrecht, Eva; Alizadeh, Behrooz Z; Aspelund, Thor; Bandinelli, Stefania; Lauc, Lovorka Barac; Beckmann, Jacques S; Boban, Mladen; Boerwinkle, Eric; Broekmans, Frank J; Burri, Andrea; Campbell, Harry; Chanock, Stephen J; Chen, Constance; Cornelis, Marilyn C; Corre, Tanguy; Coviello, Andrea D; d’Adamo, Pio; Davies, Gail; de Faire, Ulf; de Geus, Eco JC; Deary, Ian J; Dedoussis, George VZ; Deloukas, Panagiotis; Ebrahim, Shah; Eiriksdottir, Gudny; Emilsson, Valur; Eriksson, Johan G; Fauser, Bart CJM; Ferreli, Liana; Ferrucci, Luigi; Fischer, Krista; Folsom, Aaron R; Garcia, Melissa E; Gasparini, Paolo; Gieger, Christian; Glazer, Nicole; Grobbee, Diederick E; Hall, Per; Haller, Toomas; Hankinson, Susan E; Hass, Merli; Hayward, Caroline; Heath, Andrew C; Hofman, Albert; Ingelsson, Erik; Janssens, A Cecile JW; Johnson, Andrew D; Karasik, David; Kardia, Sharon LR; Keyzer, Jules; Kiel, Douglas P; Kolcic, Ivana; Kutalik, Zoltán; Lahti, Jari; Lai, Sandra; Laisk, Triin; Laven, Joop SE; Lawlor, Debbie A; Liu, Jianjun; Lopez, Lorna M; Louwers, Yvonne V; Magnusson, Patrik KE; Marongiu, Mara; Martin, Nicholas G; Klaric, Irena Martinovic; Masciullo, Corrado; McKnight, Barbara; Medland, Sarah E; Melzer, David; Mooser, Vincent; Navarro, Pau; Newman, Anne B; Nyholt, Dale R; Onland-Moret, N. Charlotte; Palotie, Aarno; Paré, Guillaume; Parker, Alex N; Pedersen, Nancy L; Peeters, Petra HM; Pistis, Giorgio; Plump, Andrew S; Polasek, Ozren; Pop, Victor JM; Psaty, Bruce M; Räikkönen, Katri; Rehnberg, Emil; Rotter, Jerome I; Rudan, Igor; Sala, Cinzia; Salumets, Andres; Scuteri, Angelo; Singleton, Andrew; Smith, Jennifer A; Snieder, Harold; Soranzo, Nicole; Stacey, Simon N; Starr, John M; Stathopoulou, Maria G; Stirrups, Kathleen; Stolk, Ronald P; Styrkarsdottir, Unnur; Sun, Yan V; Tenesa, Albert; Thorand, Barbara; Toniolo, Daniela; Tryggvadottir, Laufey; Tsui, Kim; Ulivi, Sheila; van Dam, Rob M; van der Schouw, Yvonne T; van Gils, Carla H; van Nierop, Peter; Vink, Jacqueline M; Visscher, Peter M; Voorhuis, Marlies; Waeber, Gérard; Wallaschofski, Henri; Wichmann, H Erich; Widen, Elisabeth; Gent, Colette JM Wijnands-van; Willemsen, Gonneke; Wilson, James F; Wolffenbuttel, Bruce HR; Wright, Alan F; Yerges-Armstrong, Laura M; Zemunik, Tatijana; Zgaga, Lina; Zillikens, M. Carola; Zygmunt, Marek; Arnold, Alice M; Boomsma, Dorret I; Buring, Julie E.; Crisponi, Laura; Demerath, Ellen W; Gudnason, Vilmundur; Harris, Tamara B; Hu, Frank B; Hunter, David J; Launer, Lenore J; Metspalu, Andres; Montgomery, Grant W; Oostra, Ben A; Ridker, Paul M; Sanna, Serena; Schlessinger, David; Spector, Tim D; Stefansson, Kari; Streeten, Elizabeth A; Thorsteinsdottir, Unnur; Uda, Manuela; Uitterlinden, André G; van Duijn, Cornelia M; Völzke, Henry; Murray, Anna; Murabito, Joanne M; Visser, Jenny A; Lunetta, Kathryn L

    2011-01-01

    To identify novel loci for age at natural menopause, we performed a meta-analysis of 22 genome-wide association studies in 38,968 women of European descent, with replication in up to 14,435 women. In addition to four known loci, we identified 13 new age at natural menopause loci (P < 5 × 10−8). The new loci included genes implicated in DNA repair (EXO1, HELQ, UIMC1, FAM175A, FANCI, TLK1, POLG, PRIM1) and immune function (IL11, NLRP11, BAT2). Gene-set enrichment pathway analyses using the full GWAS dataset identified exodeoxyribonuclease, NFκB signalling and mitochondrial dysfunction as biological processes related to timing of menopause. PMID:22267201

  17. Repair of Nerve Cell Membrance Damage by Calcium-Dependent, Membrane-Binding Proteins

    Science.gov (United States)

    2013-09-01

    signaling and amyloid toxicity in Alzheimer disease, J Biol Chem 285 (2010) 12463-12468. [14] H.A. Lashuel, P.T. Lansbury, Are amyloid diseases caused by...protein aggregates that mimic bacterial pore-forming toxins?, Q Rev Biophys 39 (2006) 167-201. [15] N. Arispe, E. Rojas, H.B. Pollard, Alzheimer ...disease amyloid beta protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum , Proc Natl Acad Sci U S A 90 (1993) 567

  18. Coordination of altered DNA repair and damage pathways in arsenite-exposed keratinocytes.

    Science.gov (United States)

    Hamadeh, Hisham K; Trouba, Kevin J; Amin, Rupesh P; Afshari, Cynthia A; Germolec, Dori

    2002-10-01

    Human exposure to arsenic, a ubiquitous and toxic environmental pollutant, is associated with an increased incidence of skin cancer. However, the mechanism(s) associated with AsIII-mediated toxicity and carcinogenesis at low levels of exposure remains elusive. Aberrations in cell proliferation, oxidative damage, and DNA-repair fidelity have been implicated in sodium arsenite (AsIII)-mediated carcinogenicity and toxicity, but these events have been examined in isolation in the majority of biological models of arsenic exposure. We hypothesized that the simultaneous interaction of these effects may be important in arsenic-mediated neoplasia in the skin. To evaluate this, normal human epidermal keratinocytes (NHEK) were exposed to nontoxic doses (0.005-5 micro M) of AsIII and monitored for several physiological endpoints at the times when cells were harvested for gene expression measurements (1-24 h). Two-fluor cDNA microarray analyses indicated that AsIII treatment decreased the expression of genes associated with DNA repair (e.g., p53 and Damage-specific DNA-binding protein 2) and increased the expression of genes indicative of the cellular response to oxidative stress (e.g., Superoxide dismutase 1, NAD(P)H quinone oxidoreductase, and Serine/threonine kinase 25). AsIII also modulated the expression of certain transcripts associated with increased cell proliferation (e.g., Cyclin G1, Protein kinase C delta), oncogenes, and genes associated with cellular transformation (e.g., Gro-1 and V-yes). These observations correlated with measurements of cell proliferation and mitotic measurements as AsIII treatment resulted in a dose-dependent increase in cellular mitoses at 24 h and an increase in cell proliferation at 48 h of exposure. Data in this manuscript demonstrates that AsIII exposure simultaneously modulates DNA repair, cell proliferation, and redox-related gene expression in nontransformed, normal NHEK. It is anticipated that data in this report will serve as a

  19. Plasma membrane calcium ATPase proteins as novel regulators of signal transduction pathways

    Institute of Scientific and Technical Information of China (English)

    Mary; Louisa; Holton; Michael; Emerson; Ludwig; Neyses; Angel; L; Armesilla

    2010-01-01

    Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular freecalcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulindependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.

  20. SELECTIVE-INHIBITION OF REPAIR OF ACTIVE GENES BY HYPERTHERMIA IS DUE TO INHIBITION OF GLOBAL AND TRANSCRIPTION COUPLED REPAIR PATHWAYS

    NARCIS (Netherlands)

    SAKKERS, RJ; FILON, AR; BRUNSTING, JF; KAMPINGA, HH; KONINGS, AWT; MULLENDERS, LHF

    1995-01-01

    Hyperthermia specifically inhibits the repair of UV-induced DNA photolesions in transcriptionally active genes, To define more precisely which mechanisms underlie the heat-induced inhibition of repair of active genes, removal of cyclobutane pyrimidine dimers (CPDs) was studied in human fibroblasts w

  1. Metformin enhances radiosensitivity via inhibition of DNA repair pathway in colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Youn Kyoung; Kim, Mi Sook; Lee, Ji Young; Song, Kyung Hee; Choi, Kyul; Kim, Eun Ho; Ha, Hun Joo [Ewha Womans University, Seoul (Korea, Republic of)

    2014-04-15

    In this study, we provide a scientific rationale for the clinical application of metformin as a radiosensitizer in colorectal cancer. Colorectal cancer (CRC) is the third most common cancer in men and the second most common cancer in women worldwide. Currently, it is one of the commonest chemoradiotherapy worked better than the radiotherapy or chemotherapy in colorectal cancer. To enhance radiosensitivity of tumor cells for chemoradiotherapy, it is to use potential anticancer agents that act as radiosensitizers. Metformin, one of the most widely used antidiabetic drugs, has recently been associated with potential antitumorigenic effects. Our data shows that metformin combined with radiation enhances the efficacy of radiotherapy and down-regulates DNA repair proteins. Therefore, we provides a scientific rationale for the clinical application of metformin as a radiosensitizer in colorectal cancer.

  2. The Role of Fanconi/BRCA DNA Repair Pathway in Epithelial Ovarian Carcinogenesis

    Science.gov (United States)

    2015-01-01

    Mutagen 35:206-221. Wu JH, Jones NJ (2012): Assessment of DNA interstrand crosslinks using the modified alkaline comet assay. Methods Mol Biol 817...transferred to nitrocellulose membranes, which were probed for FANCD2 (1:100) or nucleoporin (1:200) (Santa Cruz Biotechnology; Santa Cruz, CA), as an...10 µg of total protein from each fraction •Protein separation by SDS/PAGE and transferred to nitrocellulose •Probe for FANCD2 •Loading controls

  3. Folding outer membrane proteins independently of the β-barrel assembly machinery: an assembly pathway for multimeric complexes?

    Science.gov (United States)

    Huysmans, Gerard H M

    2016-06-15

    Since the discovery of the essential role of the β-barrel assembly machinery (BAM) for the membrane insertion of outer membrane proteins (OMPs) that are unrelated in sequence, members of this universally conserved family dominate discussions on OMP assembly in bacteria, mitochondria and chloroplasts. However, several multimeric bacterial OMPs assemble independently of the catalyzing BAM-component BamA. Recent progress on this alternative pathway is reviewed here, and a model for BAM-independent assembly for multimeric OMPs is proposed in which monomer delivery to the membrane and stable prepore formation are key steps towards productive membrane insertion.

  4. A Role for BLM in Double-Strand Break Repair Pathway Choice: Prevention of CtIP/Mre11-Mediated Alternative Nonhomologous End-Joining

    DEFF Research Database (Denmark)

    Grabarz, Anastazja; Guirouilh-Barbat, Josée; Barascu, Aurelia

    2013-01-01

    The choice of the appropriate double-strand break (DSB) repair pathway is essential for the maintenance of genomic stability. Here, we show that the Bloom syndrome gene product, BLM, counteracts CtIP/MRE11-dependent long-range deletions (>200 bp) generated by alternative end-joining (A-EJ). BLM r...

  5. Contribution of membrane mucins to tumor progression through modulation of cellular growth signaling pathways.

    Science.gov (United States)

    Carraway, Kermit L; Funes, Melanie; Workman, Heather C; Sweeney, Colleen

    2007-01-01

    Mucins are large, heavily O-glycosylated proteins expressed by epithelial tissues. The canonical function of membrane mucins is to provide protection to vulnerable epithelia by forming a steric barrier against assault, and by contributing to the formation of protective extracellular mucin gels. The aberrant overexpression of mucins is thought to contribute to tumor progression by allowing tumor cells to evade immune recognition, and by aiding in the breakdown of cell-cell and cell-matrix contacts to facilitate migration and metastasis. Recent evidence suggests that we should now modify our thinking about mucin function by considering their roles in signaling pathways leading to cellular growth control. Here we review the markedly divergent mechanisms by which membrane mucins, specifically MUC1 and MUC4, influence pathways contributing to cellular proliferation and survival. The cytoplasmic domain of MUC1 serves as a scaffold for the assembly of a variety of signaling proteins, while MUC4 influences the trafficking and localization of growth factor receptors, and hence their responses to external stimuli. We also discuss how tumor cells exploit these mechanisms to promote their own growth and metastasis.

  6. Ion-conduction pathways in self-organised ureidoarene-heteropolysiloxane hybrid membranes.

    Science.gov (United States)

    Michau, Mathieu; Barboiu, Mihail; Caraballo, Rémi; Arnal-Hérault, Carole; Perriat, Pascal; van der Lee, Arie; Pasc, A

    2008-01-01

    This paper reports on hybrid organic-inorganic dense membrane materials in which protons and ions are envisioned to diffuse along the hydrophilic pathways. The hierarchical generation of functional hybrid materials was realised in two steps. First, the self-assembling properties of 3-(ureidoarene)propyltriethoxysilane compounds 1-5 in aprotic solvents were determined, revealing the formation of supramolecular oligomers. Compounds 1-5 generate organogels in chloroform or in acetone, leading in a second sol-gel transcription step to hybrid membrane materials on a nanoscopic scale. The crystal structures of 1-5 indicate that the arrangement is mainly defined by periodic parallel sheets, resulting from the alignment of hydrophobic organic and inorganic silica layers. Hybrid materials MB 1-MB 4, with a similar lamellar structure, define particularly attractive functional transport devices; they are oriented along the organic layers and sandwiched between the two siloxane layers. These systems have been employed successfully to design solid dense membranes and illustrate how the self-organised hybrid materials perform interesting and potentially useful functions.

  7. Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis.

    Science.gov (United States)

    Maassen, Nicole; Freese, Stefan; Schruff, Barbara; Passoth, Volkmar; Klinner, Ulrich

    2008-08-01

    Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.

  8. Ferlins: regulators of vesicle fusion for auditory neurotransmission, receptor trafficking and membrane repair.

    Science.gov (United States)

    Lek, Angela; Evesson, Frances J; Sutton, R Bryan; North, Kathryn N; Cooper, Sandra T

    2012-02-01

    Ferlins are a family of multiple C2 domain proteins with emerging roles in vesicle fusion and membrane trafficking. Ferlin mutations are associated with muscular dystrophy (dysferlin) and deafness (otoferlin) in humans, and infertility in Caenorhabditis elegans (Fer-1) and Drosophila (misfire), demonstrating their importance for normal cellular functioning. Ferlins show ancient origins in eukaryotic evolution and are detected in all eukaryotic kingdoms, including unicellular eukaryotes and apicomplexian protists, suggesting origins in a common ancestor predating eukaryotic evolutionary branching. The characteristic feature of the ferlin family is their multiple tandem cytosolic C2 domains (five to seven C2 domains), the most of any protein family, and an extremely rare feature amongst eukaryotic proteins. Ferlins also bear a unique nested DysF domain and small conserved 60-70 residue ferlin-specific sequences (Fer domains). Ferlins segregate into two subtypes based on the presence (type I ferlin) or absence (type II ferlin) of the DysF and FerA domains. Ferlins have diverse tissue-specific and developmental expression patterns, with ferlin animal models united by pathologies arising from defects in vesicle fusion. Consistent with their proposed role in vesicle trafficking, ferlin interaction partners include cytoskeletal motors, other vesicle-associated trafficking proteins and transmembrane receptors or channels. Herein we summarize the research history of the ferlins, an intriguing family of structurally conserved proteins with a preserved ancestral function as regulators of vesicle fusion and receptor trafficking.

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

    Science.gov (United States)

    Redrejo-Rodríguez, Modesto; Saint-Pierre, Christine; Couve, Sophie; Mazouzi, Abdelghani; Ishchenko, Alexander A; Gasparutto, Didier; Saparbaev, Murat

    2011-01-01

    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. 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. This study demonstrates that both BER and NIR pathways can compete and/or back-up each other to remove hydantoin DNA lesions in vivo.

  10. 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.

  11. Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks

    OpenAIRE

    Yang, Yun-Gui; Saidi, Amal; Frappart, Pierre-Olivier; Min, WooKee; Barrucand, Christelle; Dumon-Jones, Valérie; Michelon, Jocelyne; Herceg, Zdenko; Wang, Zhao-Qi

    2006-01-01

    NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting...

  12. A rapid, membrane-dependent pathway directs furrow formation through RalA in the early Drosophila embryo.

    Science.gov (United States)

    Holly, Ryan M; Mavor, Lauren M; Zuo, Zhongyuan; Blankenship, J Todd

    2015-07-01

    Plasma membrane furrow formation is crucial in cell division and cytokinesis. Furrow formation in early syncytial Drosophila embryos is exceptionally rapid, with furrows forming in as little as 3.75 min. Here, we use 4D imaging to identify furrow formation, stabilization, and regression periods, and identify a rapid, membrane-dependent pathway that is essential for plasma membrane furrow formation in vivo. Myosin II function is thought to provide the ingression force for cytokinetic furrows, but the role of membrane trafficking pathways in guiding furrow formation is less clear. We demonstrate that a membrane trafficking pathway centered on Ras-like protein A (RalA) is required for fast furrow ingression in the early fly embryo. RalA function is absolutely required for furrow formation and initiation. In the absence of RalA and furrow function, chromosomal segregation is aberrant and polyploid nuclei are observed. RalA localizes to syncytial furrows, and mediates the movement of exocytic vesicles to the plasma membrane. Sec5, which is an exocyst complex subunit and localizes to ingressing furrows in wild-type embryos, becomes punctate and loses its cortical association in the absence of RalA function. Rab8 also fails to traffic to the plasma membrane and accumulates aberrantly in the cytoplasm in RalA disrupted embryos. RalA localization precedes F-actin recruitment to the furrow tip, suggesting that membrane trafficking might function upstream of cytoskeletal remodeling. These studies identify a pathway, which stretches from Rab8 to RalA and the exocyst complex, that mediates rapid furrow formation in early Drosophila embryos.

  13. Effects of collagen membranes enriched with in vitro-differentiated N1E-115 cells on rat sciatic nerve regeneration after end-to-end repair

    Directory of Open Access Journals (Sweden)

    Fornaro Michele

    2010-02-01

    Full Text Available Abstract Peripheral nerves possess the capacity of self-regeneration after traumatic injury but the extent of regeneration is often poor and may benefit from exogenous factors that enhance growth. The use of cellular systems is a rational approach for delivering neurotrophic factors at the nerve lesion site, and in the present study we investigated the effects of enwrapping the site of end-to-end rat sciatic nerve repair with an equine type III collagen membrane enriched or not with N1E-115 pre-differentiated neural cells. After neurotmesis, the sciatic nerve was repaired by end-to-end suture (End-to-End group, end-to-end suture enwrapped with an equine collagen type III membrane (End-to-EndMemb group; and end-to-end suture enwrapped with an equine collagen type III membrane previously covered with neural cells pre-differentiated in vitro from N1E-115 cells (End-to-EndMembCell group. Along the postoperative, motor and sensory functional recovery was evaluated using extensor postural thrust (EPT, withdrawal reflex latency (WRL and ankle kinematics. After 20 weeks animals were sacrificed and the repaired sciatic nerves were processed for histological and stereological analysis. Results showed that enwrapment of the rapair site with a collagen membrane, with or without neural cell enrichment, did not lead to any significant improvement in most of functional and stereological predictors of nerve regeneration that we have assessed, with the exception of EPT which recovered significantly better after neural cell enriched membrane employment. It can thus be concluded that this particular type of nerve tissue engineering approach has very limited effects on nerve regeneration after sciatic end-to-end nerve reconstruction in the rat.

  14. Calmodulin Mediates DNA Repair Pathways Involving H2AX in Response to Low-Dose Radiation Exposure of RAW 264.7 Macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Smallwood, Heather S.; Lopez Ferrer, Daniel; Eberlein, P. Elis; Watson, David J.; Squier, Thomas C.

    2009-02-05

    Understanding the molecular mechanisms that modulate macrophage radioresistance is necessary for the development of effective radiation therapies, as tumor-associated macrophages promote both angiogenesis and matrix remodeling that, in turn, enhance metastasis. In this respect, we have identified a dose-dependent increase in the abundance of the calcium regulatory protein calmodulin (CaM) in RAW 264.7 macrophages upon irradiation. CaM overexpression results in increased macrophage survival following radiation exposure, acting to diminish the sensitivity to low-dose exposures. Increases in CaM abundance also result in an increase in the number of phosphorylated histone H2AX protein complexes associated with DNA repair following macrophage irradiation, with no change in the extent of double-stranded DNA damage. In comparison, when NFκB-dependent pathways are inhibited, through the expression of a dominant-negative IκB construct, there is no significant increase in phosphorylated H2AX upon irradiation. These results indicate that the molecular basis for the up-regulation of histone H2AX mediated DNA-repair pathways is not the result of nonspecific NFκB-dependent pathways or a specific threshold of DNA damage. Rather, increases in CaM abundance act to minimize the low-dose hypersensitivity to radiation to enhance macrophage radioresistance through processes that include the upregulation of DNA repair pathways involving histone protein H2AX phosphorylation.

  15. Rupturing the hemi-fission intermediate in membrane fission under tension: Reaction coordinates, kinetic pathways, and free-energy barriers.

    Science.gov (United States)

    Zhang, Guojie; Müller, Marcus

    2017-08-14

    Membrane fission is a fundamental process in cells, involved inter alia in endocytosis, intracellular trafficking, and virus infection. Its underlying molecular mechanism, however, is only incompletely understood. Recently, experiments and computer simulation studies have revealed that dynamin-mediated membrane fission is a two-step process that proceeds via a metastable hemi-fission intermediate (or wormlike micelle) formed by dynamin's constriction. Importantly, this hemi-fission intermediate is remarkably metastable, i.e., its subsequent rupture that completes the fission process does not occur spontaneously but requires additional, external effects, e.g., dynamin's (unknown) conformational changes or membrane tension. Using simulations of a coarse-grained, implicit-solvent model of lipid membranes, we investigate the molecular mechanism of rupturing the hemi-fission intermediate, such as its pathway, the concomitant transition states, and barriers, as well as the role of membrane tension. The membrane tension is controlled by the chemical potential of the lipids, and the free-energy landscape as a function of two reaction coordinates is obtained by grand canonical Wang-Landau sampling. Our results show that, in the course of rupturing, the hemi-fission intermediate undergoes a "thinning → local pinching → rupture/fission" pathway, with a bottle-neck-shaped cylindrical micelle as a transition state. Although an increase of membrane tension facilitates the fission process by reducing the corresponding free-energy barrier, for biologically relevant tensions, the free-energy barriers still significantly exceed the thermal energy scale kBT.

  16. Ubiquitin-Proteasome-Collagen (CUP Pathway in Preterm Premature Rupture of Fetal Membranes

    Directory of Open Access Journals (Sweden)

    Xinliang Zhao

    2017-06-01

    Full Text Available Spontaneous preterm birth (sPTB occurs before 37 gestational weeks, with preterm premature rupture of the membranes (PPROM and spontaneous preterm labor (sPTL as the predominant adverse outcomes. Previously, we identified altered expression of long non-coding RNAs (lncRNAs and message RNAs (mRNAs related to the ubiquitin proteasome system (UPS in human placentas following pregnancy loss and PTB. We therefore hypothesized that similar mechanisms might underlie PPROM. In the current study, nine pairs of ubiquitin-proteasome-collagen (CUP pathway–related mRNAs and associated lncRNAs were found to be differentially expressed in PPROM and sPTL. Pathway analysis showed that the functions of their protein products were inter-connected by ring finger protein. Twenty variants including five mutations were identified in CUP-related genes in sPTL samples. Copy number variations were found in COL19A1, COL28A1, COL5A1, and UBAP2 of sPTL samples. The results reinforced our previous findings and indicated the association of the CUP pathway with the development of sPTL and PPROM. This association was due not only to the genetic variation, but also to the epigenetic regulatory function of lncRNAs. Furthermore, the findings suggested that the loss of collagen content in PPROM could result from degradation and/or suppressed expression of collagens.

  17. Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Martini

    2011-09-01

    Full Text Available Meiotic DNA double-strand breaks (DSBs initiate crossover (CO recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs. Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs. First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

  18. CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes

    DEFF Research Database (Denmark)

    Nielsen, C H; Marquart, H V; Prodinger, W M;

    2001-01-01

    Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane-bound C3 convertase. We have investigated whether this might lead to the generation of a C5...... convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4.4 mM MgCl2/20 mM EGTA, which abrogates the classical pathway of complement without affecting...... the alternative pathway. Blockade of the CR2 ligand-binding site with the monoclonal antibody FE8 resulted in 56 +/- 13% and 71 +/- 9% inhibition of the C3-fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade...

  19. Removal and Degradation Pathways of Sulfamethoxazole Present in Synthetic Municipal Wastewater via an Anaerobic Membrane Bioreactor

    KAUST Repository

    Sanchez Huerta, Claudia

    2016-05-01

    The current global water crisis in addition to continues contamination of natural water bodies with harmful organic micropollutants (OMPs) have driven the development of new water treatment technologies that allow the efficient removal of such compounds. Among a long list of OMPs, antibiotics are considered as top priority pollutants to be treated due to their great resistance to biological treatments and their potential to develop bacterial resistance. Different approaches, such as membrane-based and advance oxidation processes have been proposed to alleviate or minimize antibiotics discharge into aquatic environments. However most of these processes are costly and generate either matrices with high concentration of OMPs or intermediate products with potentially greater toxicity or persistence. Therefore, this thesis proposes the study of an anaerobic membrane bioreactor (AnMBR) for the treatment of synthetic municipal wastewater containing sulfamethoxazole (SMX), a world widely used antibiotic. Besides the general evaluation of AnMBR performance in the COD removal and biogas production, this research mainly focuses on the SMX removal and its degradation pathway. Thus 5 SMX quantification was performed through solid phase extraction-liquid chromatography/mass spectrometry and the identification of its transformation products (TPs) was assessed by gas chromatography/mass spectrometry technique. The results achieved showed that, working under optimal conditions (35°C, pH 7 and ORP around -380 to -420 mV) and after a biomass adaptation period (maintaining 0.85 VSS/TSS ratio), the AnMBR process provided over 95% COD removal and 95-98% SMX removal, while allowing stable biogas composition and methane production (≈130 mL CH4/g CODremoved). Kinetic analysis through a batch test showed that after 24 h of biological reaction, AnMBR process achieved around 94% SMX removal, indicating a first order kinetic reaction with K= 0.119, which highlights the high degradation

  20. A robust network of double-strand break repair pathways governs genome integrity during C. elegans development.

    NARCIS (Netherlands)

    Pontier, D.B.; Tijsterman, M.

    2009-01-01

    To preserve genomic integrity, various mechanisms have evolved to repair DNA double-strand breaks (DSBs). Depending on cell type or cell cycle phase, DSBs can be repaired error-free, by homologous recombination, or with concomitant loss of sequence information, via nonhomologous end-joining (NHEJ) o

  1. A robust network of double-strand break repair pathways governs genome integrity during C. elegans development.

    NARCIS (Netherlands)

    Pontier, D.B.; Tijsterman, M.

    2009-01-01

    To preserve genomic integrity, various mechanisms have evolved to repair DNA double-strand breaks (DSBs). Depending on cell type or cell cycle phase, DSBs can be repaired error-free, by homologous recombination, or with concomitant loss of sequence information, via nonhomologous end-joining (NHEJ)

  2. Comparative analysis of meiotic progression in female mice bearing mutations in genes of the DNA mismatch repair pathway.

    Science.gov (United States)

    Kan, Rui; Sun, Xianfei; Kolas, Nadine K; Avdievich, Elena; Kneitz, Burkhard; Edelmann, Winfried; Cohen, Paula E

    2008-03-01

    The DNA mismatch repair (MMR) family functions in a variety of contexts to preserve genome integrity in most eukaryotes. In particular, members of the MMR family are involved in the process of meiotic recombination in germ cells. MMR gene mutations in mice result in meiotic disruption during prophase I, but the extent of this disruption often differs between male and female meiocytes. To address the role of MMR proteins specifically in female meiosis, we explored the progression of oocytes through prophase I and the meiotic divisions in mice harboring deletions in members of the MMR pathway (Mlh1, Mlh3, Exo1, and an ATPase-deficient variant of Mlh1, Mlh1(G67R)). The colocalization of MLH1 and MLH3, key proteins involved in stabilization of nascent crossovers, was dependent on intact heterodimer formation and was highly correlated with the ability of oocytes to progress through to metaphase II. The exception was Exo1(-/-) oocytes, in which normal MLH1/MLH3 localization was observed followed by failure to proceed to metaphase II. All mutant oocytes were able to resume meiosis after dictyate arrest, but they showed a dramatic decline in chiasmata (to less than 25% of normal), accompanied by varied progression through metaphase I. Taken together, these results demonstrate that MMR function is required for the formation and stabilization of crossovers in mammalian oocytes and that, in the absence of a functional MMR system, the failure to maintain chiasmata results in a reduced ability to proceed normally through the first and second meiotic divisions, despite near-normal levels of meiotic resumption after dictyate arrest.

  3. Assessing SNP-SNP interactions among DNA repair, modification and metabolism related pathway genes in breast cancer susceptibility.

    Directory of Open Access Journals (Sweden)

    Yadav Sapkota

    Full Text Available Genome-wide association studies (GWASs have identified low-penetrance common variants (i.e., single nucleotide polymorphisms, SNPs associated with breast cancer susceptibility. Although GWASs are primarily focused on single-locus effects, gene-gene interactions (i.e., epistasis are also assumed to contribute to the genetic risks for complex diseases including breast cancer. While it has been hypothesized that moderately ranked (P value based weak single-locus effects in GWASs could potentially harbor valuable information for evaluating epistasis, we lack systematic efforts to investigate SNPs showing consistent associations with weak statistical significance across independent discovery and replication stages. The objectives of this study were i to select SNPs showing single-locus effects with weak statistical significance for breast cancer in a GWAS and/or candidate-gene studies; ii to replicate these SNPs in an independent set of breast cancer cases and controls; and iii to explore their potential SNP-SNP interactions contributing to breast cancer susceptibility. A total of 17 SNPs related to DNA repair, modification and metabolism pathway genes were selected since these pathways offer a priori knowledge for potential epistatic interactions and an overall role in breast carcinogenesis. The study design included predominantly Caucasian women (2,795 cases and 4,505 controls from Alberta, Canada. We observed two two-way SNP-SNP interactions (APEX1-rs1130409 and RPAP1-rs2297381; MLH1-rs1799977 and MDM2-rs769412 in logistic regression that conferred elevated risks for breast cancer (P(interaction<7.3 × 10(-3. Logic regression identified an interaction involving four SNPs (MBD2-rs4041245, MLH1-rs1799977, MDM2-rs769412, BRCA2-rs1799943 (P(permutation = 2.4 × 10(-3. SNPs involved in SNP-SNP interactions also showed single-locus effects with weak statistical significance, while BRCA2-rs1799943 showed stronger statistical significance (P

  4. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  5. Significant accumulation of persistent organic pollutants and dysregulation in multiple DNA damage repair pathways in the electronic-waste-exposed populations

    Energy Technology Data Exchange (ETDEWEB)

    He, Xiaobo; Jing, Yaqing; Wang, Jianhai; Li, Keqiu [Basic Medical College, Tianjin Medical University, Tianjin 300070 (China); Yang, Qiaoyun [Department of Occupational and Environmental Health, School of Public Health, Tianjin Medical University, Tianjin 300070 (China); Zhao, Yuxia [Basic Medical College, Tianjin Medical University, Tianjin 300070 (China); Li, Ran [State Key Joint Laboratory for Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering and Center for Environment and Health, Peking University, Beijing 100871 (China); Ge, Jie [Department of Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060 (China); Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060 (China); Qiu, Xinghua, E-mail: xhqiu@pku.edu.cn [State Key Joint Laboratory for Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering and Center for Environment and Health, Peking University, Beijing 100871 (China); Li, Guang, E-mail: lig@tijmu.edu.cn [Basic Medical College, Tianjin Medical University, Tianjin 300070 (China)

    2015-02-15

    Electronic waste (e-waste) has created a worldwide environmental and health problem, by generating a diverse group of hazardous compounds such as persistent organic pollutants (POPs). Our previous studies demonstrated that populations from e-waste exposed region have a significantly higher level of chromosomal aberrancy and incidence of DNA damage. In this study, we further demonstrated that various POPs persisted at a significantly higher concentration in the exposed group than those in the unexposed group. The level of reactive oxygen species and micronucleus rate were also significantly elevated in the exposed group. RNA sequencing analysis revealed 31 genes in DNA damage responses and repair pathways that were differentially expressed between the two groups (Log 2 ratio >1 or <−1). Our data demonstrated that both females and males of the exposed group have activated a series of DNA damage response genes; however many important DNA repair pathways have been dysregulated. Expressions of NEIL1/3 and RPA3, which are critical in initiating base pair and nucleotide excision repairs respectively, have been downregulated in both females and males of the exposed group. In contrast, expression of RNF8, an E3 ligase involved in an error prone non-homologous end joining repair for DNA double strand break, was upregulated in both genders of the exposed group. The other genes appeared to be differentially expressed only when the males or females of the two groups were compared respectively. Importantly, the expression of cell cycle regulatory gene CDC25A that has been implicated in multiple kinds of malignant transformation was significantly upregulated among the exposed males while downregulated among the exposed females. In conclusion, our studies have demonstrated significant correlations between e-waste disposing and POPs accumulation, DNA lesions and dysregulation of multiple DNA damage repair mechanisms in the residents of the e-waste exposed region. - Highlights:

  6. Plants Possess a Cyclic Mitochondrial Metabolic Pathway similar to the Mammalian Metabolic Repair Mechanism Involving Malate Dehydrogenase and l-2-Hydroxyglutarate Dehydrogenase.

    Science.gov (United States)

    Hüdig, Meike; Maier, Alexander; Scherrers, Isabell; Seidel, Laura; Jansen, Erwin E W; Mettler-Altmann, Tabea; Engqvist, Martin K M; Maurino, Veronica G

    2015-09-01

    Enzymatic side reactions can give rise to the formation of wasteful and toxic products that are removed by metabolite repair pathways. In this work, we identify and characterize a mitochondrial metabolic repair mechanism in Arabidopsis thaliana involving malate dehydrogenase (mMDH) and l-2-hydroxyglutarate dehydrogenase (l-2HGDH). We analyze the kinetic properties of both A. thaliana mMDH isoforms, and show that they produce l-2-hydroxyglutarate (l-2HG) from 2-ketoglutarate (2-KG) at low rates in side reactions. We identify A. thaliana l-2HGDH as a mitochondrial FAD-containing oxidase that converts l-2HG back to 2-KG. Using loss-of-function mutants, we show that the electrons produced in the l-2HGDH reaction are transferred to the mitochondrial electron transport chain through the electron transfer protein (ETF). Thus, plants possess the biochemical components of an l-2HG metabolic repair system identical to that found in mammals. While deficiencies in the metabolism of l-2HG result in fatal disorders in mammals, accumulation of l-2HG in plants does not adversely affect their development under a range of tested conditions. However, orthologs of l-2HGDH are found in all examined genomes of viridiplantae, indicating that the repair reaction we identified makes an essential contribution to plant fitness in as yet unidentified conditions in the wild.

  7. The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway

    Energy Technology Data Exchange (ETDEWEB)

    Schiestl, R.H.; Prakash, S.; Prakash, L. (Univ. of Rochester School of Medicine, NY (USA))

    1990-04-01

    rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, the authors have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6{Delta}) mutations and show that they also suppress the {gamma}-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of {gamma}-ray sensitivity. The six suppressor mutations they isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. They show that suppression of rad6{Delta} is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6{Delta} SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.

  8. Non-homologous end joining is the responsible pathway for the repair of fludarabine-induced DNA double strand breaks in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Campos-Nebel, Marcelo de [Departamento de Genetica, Instituto de Investigaciones Hematologicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires (Argentina)], E-mail: mnebel@hematologia.anm.edu.ar; Larripa, Irene; Gonzalez-Cid, Marcela [Departamento de Genetica, Instituto de Investigaciones Hematologicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires (Argentina)

    2008-11-10

    Fludarabine (FLU), an analogue of adenosine, interferes with DNA synthesis and inhibits the chain elongation leading to replication arrest and DNA double strand break (DSB) formation. Mammalian cells use two main pathways of DSB repair to maintain genomic stability: homologous recombination (HR) and non-homologous end joining (NHEJ). The aim of the present work was to evaluate the repair pathways employed in the restoration of DSB formed following replication arrest induced by FLU in mammalian cells. Replication inhibition was induced in human lymphocytes and fibroblasts by FLU. DSB occurred in a dose-dependent manner on early/middle S-phase cells, as detected by {gamma}H2AX foci formation. To test whether conservative HR participates in FLU-induced DSB repair, we measured the kinetics of Rad51 nuclear foci formation in human fibroblasts. There was no significant induction of Rad51 foci after FLU treatment. To further confirm these results, we analyzed the frequency of sister chromatid exchanges (SCE) in both human cells. We did not find increased frequencies of SCE after FLU treatment. To assess the participation of NHEJ pathway in the repair of FLU-induced damage, we used two chemical inhibitors of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), vanillin and wortmannin. Human fibroblasts pretreated with DNA-PKcs inhibitors showed increased levels of chromosome breakages and became more sensitive to cell death. An active role of NHEJ pathway was also suggested from the analysis of Chinese hamster cell lines. XR-C1 (DNA-PKcs-deficient) and XR-V15B (Ku80-deficient) cells showed hypersensitivity to FLU as evidenced by the increased frequency of chromosome aberrations, decreased mitotic index and impaired survival rates. In contrast, CL-V4B (Rad51C-deficient) and V-C8 (Brca2-deficient) cell lines displayed a FLU-resistant phenotype. Together, our results suggest a major role for NHEJ repair in the preservation of genome integrity against FLU

  9. Successful management of severe unilateral re-expansion pulmonary edema after mitral valve repair with mini-thoracotomy using extracorporeal membrane oxygenation.

    Science.gov (United States)

    Kitahara, Hiroto; Okamoto, Kazuma; Kudo, Mikihiko; Yoshitake, Akihiro; Hayashi, Kanako; Inaba, Yu; Ai, Kimiaki; Suzuki, Takeshi; Morisaki, Hiroshi; Shimizu, Hideyuki

    2017-03-01

    A 60-year-old man received mitral valve repair via right mini-thoracotomy, which was followed by unilateral re-expansion pulmonary edema on the right side and severe hemoptysis just after the surgery. Despite differential lung ventilation with unilateral high positive end expiratory pressure was initiated for the affected right lung, respiratory function did not improved and hemodynamics was collapsed in the next day. Veno-venous extracorporeal membrane oxygenation was initiated by cannulation of the right jugular and the left femoral vein. After pulmonary function recovered gradually, veno-venous extracorporeal membrane oxygenation was terminated on the fifth postoperative day. He was discharged in ambulatory condition on postoperative day 52.

  10. Significant accumulation of persistent organic pollutants and dysregulation in multiple DNA damage repair pathways in the electronic-waste-exposed populations.

    Science.gov (United States)

    He, Xiaobo; Jing, Yaqing; Wang, Jianhai; Li, Keqiu; Yang, Qiaoyun; Zhao, Yuxia; Li, Ran; Ge, Jie; Qiu, Xinghua; Li, Guang

    2015-02-01

    Electronic waste (e-waste) has created a worldwide environmental and health problem, by generating a diverse group of hazardous compounds such as persistent organic pollutants (POPs). Our previous studies demonstrated that populations from e-waste exposed region have a significantly higher level of chromosomal aberrancy and incidence of DNA damage. In this study, we further demonstrated that various POPs persisted at a significantly higher concentration in the exposed group than those in the unexposed group. The level of reactive oxygen species and micronucleus rate were also significantly elevated in the exposed group. RNA sequencing analysis revealed 31 genes in DNA damage responses and repair pathways that were differentially expressed between the two groups (Log2 ratio >1 or waste disposing and POPs accumulation, DNA lesions and dysregulation of multiple DNA damage repair mechanisms in the residents of the e-waste exposed region.

  11. Alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations.

    Science.gov (United States)

    Iliakis, George; Murmann, Tamara; Soni, Aashish

    2015-11-01

    DNA double strand breaks (DSB) are the most deleterious lesions for the integrity of the genome, as their misrepair can lead to the formation of chromosome translocations. Cells have evolved two main repair pathways to suppress the formation of these genotoxic lesions: homology-dependent, error-free homologous recombination repair (HRR), and potentially error-prone, classical, DNA-PK-dependent non-homologous end-joining (c-NHEJ). The most salient feature of c-NHEJ, speed, will largely suppress chromosome translocation formation, while sequence alterations at the junction remain possible. It is now widely accepted that when c-NHEJ is inactivated, globally or locally, an alternative form of end-joining (alt-EJ) removes DSBs. Alt-EJ operates with speed and fidelity markedly lower than c-NHEJ, causing thus with higher probability chromosome translocations, and generating more extensive sequence alterations at the junction. Our working hypothesis is that alt-EJ operates as a backup to c-NHEJ. Recent results show that alt-EJ can also backup abrogated HRR in G2 phase cells, again at the cost of elevated formation of chromosome translocations. These observations raise alt-EJ to a global rescuing mechanism operating on ends that have lost their chromatin context in ways that compromise processing by HRR or c-NHEJ. While responsible for eliminating from the genome highly cytotoxic DNA ends, alt-EJ provides this function at the price of increased translocation formation. Here, we analyze recent literature on the mechanisms of chromosome translocation formation and propose a functional hierarchy among DSB processing pathways that makes alt-EJ the global backup pathway. We discuss possible ramifications of this model in cellular DSB management and pathway choice, and analyze its implications in radiation carcinogenesis and the design of novel therapeutic approaches. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. The Fanconi anemia/BRCA pathway is involved in DNA interstrand cross-link repair of adriamycin-resistant leukemia cells.

    Science.gov (United States)

    Yao, Chenjiao; Du, Wei; Chen, Haibing; Xiao, Sheng; Huang, Lihua; Chen, Fangping

    2015-03-01

    The Fanconi anemia/BRCA (FA/BRCA) pathway plays a vital role in DNA damage repair induced by DNA cross-linking agents and is closely related to drug response in cancer treatment. Here we demonstrate that the FA/BRCA pathway contributes to acquired drug resistance in adriamycin (ADR)-resistant leukemia cell lines, and disruption of this pathway partially reverses the drug resistance. We observed that ADR-resistant cells have reduced DNA interstrand cross-links (ICL) compared with ADR-sensitive cells. Western blot studies demonstrated enhanced FA protein expression in ADR-resistant cells. Using siRNA to knock down FANCF in K562/R drug-resistant cells showed increases in sensitivity to ADR and ADR-induced DNA damage, and demonstrated a direct relationship between the FA/BRCA pathway and drug sensitivity. Overexpression of FANCF in K562 drug-sensitive cells partially reproduced the drug-resistant phenotype. These results show that the FA/BRCA pathway is involved in acquired ADR resistance of leukemia cells. The FA/BRCA pathway may be a new target to reverse ADR resistance in leukemia treatment.

  13. pH-Triggered Conformational Switching along the Membrane Insertion Pathway of the Diphtheria Toxin T-Domain

    Directory of Open Access Journals (Sweden)

    Alexey S. Ladokhin

    2013-08-01

    Full Text Available The translocation (T-domain plays a key role in the action of diphtheria toxin and is responsible for transferring the catalytic domain across the endosomal membrane into the cytosol in response to acidification. Deciphering the molecular mechanism of pH-dependent refolding and membrane insertion of the T-domain, which is considered to be a paradigm for cell entry of other bacterial toxins, reveals general physicochemical principles underlying membrane protein assembly and signaling on membrane interfaces. Structure-function studies along the T-domain insertion pathway have been affected by the presence of multiple conformations at the same time, which hinders the application of high-resolution structural techniques. Here, we review recent progress in structural, functional and thermodynamic studies of the T-domain archived using a combination of site-selective fluorescence labeling with an array of spectroscopic techniques and computer simulations. We also discuss the principles of conformational switching along the insertion pathway revealed by studies of a series of T-domain mutants with substitutions of histidine residues.

  14. Human Longevity and Variation in GH/IGF-1/Insulin Signaling, DNA Damage Signaling and Repair and Pro/antioxidant Pathway Genes: Cross Sectional and Longitudinal Studies

    Science.gov (United States)

    Soerensen, Mette; Dato, Serena; Tan, Qihua; Thinggaard, Mikael; Kleindorp, Rabea; Beekman, Marian; Jacobsen, Rune; Suchiman, H. Eka D.; de Craen, Anton J.M.; Westendorp, Rudi G.J.; Schreiber, Stefan; Stevnsner, Tinna; Bohr, Vilhelm A.; Slagboom, P. Eline; Nebel, Almut; Vaupel, James W.; Christensen, Kaare; McGue, Matt; Christiansen, Lene

    2012-01-01

    Here we explore association with human longevity of common genetic variation in three major candidate pathways: GH/IGF-1/insulin signaling, DNA damage signaling and repair and pro/antioxidants by investigating 1273 tagging SNPs in 148 genes composing these pathways. In a case-control study of 1089 oldest-old (age 92–93) and 736 middle-aged Danes we found 1 pro/antioxidant SNP (rs1002149 (GSR)), 5 GH/IGF-1/INS SNPs (rs1207362 (KL), rs2267723 (GHRHR), rs3842755 (INS), rs572169 (GHSR), rs9456497 (IGF2R)) and 5 DNA repair SNPs (rs11571461 (RAD52), rs13251813 (WRN), rs1805329 (RAD23B), rs2953983 (POLB), rs3211994 (NTLH1)) to be associated with longevity after correction for multiple testing. In a longitudinal study with 11 years of follow-up on survival in the oldest-old Danes we found 2 pro/antioxidant SNPs (rs10047589 (TNXRD1), rs207444 (XDH)), 1 GH/IGF-1/INS SNP (rs26802 (GHRL)) and 3 DNA repair SNPs (rs13320360 (MLH1), rs2509049 (H2AFX) and rs705649 (XRCC5)) to be associated with mortality in late life after correction for multiple testing. When examining the 11 SNPs from the case-control study in the longitudinal data, rs3842755 (INS), rs13251813 (WRN) and rs3211994 (NTHL1) demonstrated the same directions of effect (pbased association study, the largest to date applying a pathway approach, points to potential new longevity loci, but does also underline the difficulties of replicating association findings in independent study populations and thus the difficulties in identifying universal longevity polymorphisms. PMID:22406557

  15. Pathway of membrane fusion with two tension-dependent energy barriers

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2007-01-01

    Fusion of bilayer membranes is studied via dissipative particle dynamics (DPD) simulations. A new set of DPD parameters is introduced which leads to an energy barrier for flips of lipid molecules between adhering membranes. A large number of fusion events is monitored for a vesicle in contact...

  16. Impact of two DNA repair pathways, homologous recombination and non-homologous end joining, on bacterial spore inactivation under simulated martian environmental conditions

    Science.gov (United States)

    Moeller, Ralf; Schuerger, Andrew C.; Reitz, Günther; Nicholson, Wayne L.

    2011-09-01

    Spores of Bacillus subtilis were used as a model system to study the impact of the two major DNA double-strand break (DSB) repair mechanisms [homologous recombination (HR) and non-homologous end-joining (NHEJ)] on the survivability of air-dried mono- and multilayers of bacterial spores under a simulated martian environment; i.e., an environment with low temperature (-10 °C), pure CO 2 atmosphere (99.99% CO 2), 200-1100 nm UV-VIS-NIR radiation, and 0.69 kPa pressure. Spores in multilayers exhibited low inactivation rates compared to monolayers, mainly due to shadowing effects of overlying spores. Simulated martian UV irradiation reduced dramatically spore viability, whereas when shielded from martian UV radiation, spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to simulated martian environmental conditions than were wild-type spores. In addition, NHEJ-deficient spores were consistently more sensitive than HR-deficient spores to simulated Mars environmental conditions, suggesting that DSBs were an important type of DNA damage. The results indicated that both HR and NHEJ provide an efficient set of DNA repair pathways ensuring spore survival after exposure to simulated martian environmental conditions.

  17. 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

  18. Pathways of homologous recombinantion and DNA interstrand cross-link repair : roles of mammalian RAD54 and SNMI

    NARCIS (Netherlands)

    M.L.G. Dronkert (Mies)

    2002-01-01

    textabstractThe aim of this thesis is to investigate mammalian DNA interstrand cross-link (ICL) repair. ICLs are formed by a number of agents used in tumor therapy, like mitomycin C and cisplatin. They constitute one of the most toxic damages to DNA, as they inhibit DNA strand separation. However, l

  19. Lung Basal Stem Cells Rapidly Repair DNA Damage Using the Error-Prone Nonhomologous End-Joining Pathway.

    Science.gov (United States)

    Weeden, Clare E; Chen, Yunshun; Ma, Stephen B; Hu, Yifang; Ramm, Georg; Sutherland, Kate D; Smyth, Gordon K; Asselin-Labat, Marie-Liesse

    2017-01-01

    Lung squamous cell carcinoma (SqCC), the second most common subtype of lung cancer, is strongly associated with tobacco smoking and exhibits genomic instability. The cellular origins and molecular processes that contribute to SqCC formation are largely unexplored. Here we show that human basal stem cells (BSCs) isolated from heavy smokers proliferate extensively, whereas their alveolar progenitor cell counterparts have limited colony-forming capacity. We demonstrate that this difference arises in part because of the ability of BSCs to repair their DNA more efficiently than alveolar cells following ionizing radiation or chemical-induced DNA damage. Analysis of mice harbouring a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in DNA damage repair by nonhomologous end joining (NHEJ), indicated that BSCs preferentially repair their DNA by this error-prone process. Interestingly, polyploidy, a phenomenon associated with genetically unstable cells, was only observed in the human BSC subset. Expression signature analysis indicated that BSCs are the likely cells of origin of human SqCC and that high levels of NHEJ genes in SqCC are correlated with increasing genomic instability. Hence, our results favour a model in which heavy smoking promotes proliferation of BSCs, and their predilection for error-prone NHEJ could lead to the high mutagenic burden that culminates in SqCC. Targeting DNA repair processes may therefore have a role in the prevention and therapy of SqCC.

  20. Feasibility study of collagen membranes derived from bovine pericardium and intestinal serosa for the repair of cranial defects in ovariectomised rats.

    Science.gov (United States)

    Hirata, H H; Munhoz, M A S; Plepis, A M G; Martins, V C A; Santos, G R; Galdeano, E A; Cunha, M R

    2015-07-01

    The indication of biomaterials has increased substantially in the regenerative therapy of bone defects. However, in addition to evaluating the physicochemical properties of biomaterials, the quality of the recipient tissue is also essential for the osseointegration of implants, as abnormalities in bone metabolism, such as gonadal hormone deficiency, can influence bone healing. This study evaluated the osteoregenerative capacity of collagen membranes derived from bovine pericardium and intestinal serosa in the repair of cranial defects in ovariectomised rats. Thirty female Wistar rats were submitted to surgical creation of a 5-mm cranial bone defect. The rats were divided into a control group (not ovariectomised) and an ovariectomised group. The non-ovariectomised group was divided into three subgroups: control (G1) in which the defect was not filled with the biomaterial, and two subgroups (G2 and G3) that received the bovine pericardium- and serosa-derived collagen membranes, respectively. The ovariectomised group was divided into the same subgroups (G4, G5, and G6). The animals were sacrificed 8 weeks after surgery. The calvaria were removed for macroscopic and radiographic photodocumentation and processed for histomorphometric analysis of bone healing at the surgical site. Macroscopic, radiological, and microscopic analyses demonstrated the biocompatibility of the implanted collagen membranes, as indicated by the absence of infiltration and signs of inflammation at the surgical site. Histologically, discrete immature bone neoformation projecting from the margins of the defect was observed at the surgical site in ovariectomised groups when compared to the non-ovariectomised groups. The volume of newly formed bone was significantly higher in the non-ovariectomised groups (G1: 7.83%±1.32; G2: 21.33%±1.96; and G3: 22.83%±0.98) compared to the respective ovariectomised subgroups (G4: 3.16%±0.75; G5: 16.83%±0.98; and G6: 16.16%±0.75), thus demonstrating the

  1. Regulation of the V-ATPase along the endocytic pathway occurs through reversible subunit association and membrane localization.

    Directory of Open Access Journals (Sweden)

    Céline Lafourcade

    Full Text Available The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase, a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V(0 and the cytoplasmic V(1. Here we found that the ratio of membrane associated V(1/Vo varies along the endocytic pathway, the relative abundance of V(1 being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments.

  2. Neural stem cell transplantation in a double-layer collagen membrane with unequal pore sizes for spinal cord injur y repair

    Institute of Scientific and Technical Information of China (English)

    Ning Yuan; Wei Tian; Lei Sun; Runying Yuan; Jianfeng Tao; Dafu Chen

    2014-01-01

    A novel double-layer collagen membrane with unequal pore sizes in each layer was designed and tested in this study. The inner, loose layer has about 100-μm-diameter pores, while the outer, compact layer has about 10-μm-diameter pores. In a rat model of incomplete spinal cord injury, a large number of neural stem cells were seeded into the loose layer, which was then adhered to the injured side, and the compact layer was placed against the lateral side. The results showed that the transplantation of neural stem cells in a double-layer collagen membrane with unequal pore sizes promoted the differentiation of neural stem cells, attenuated the pathological lesion, and signiifcantly improved the motor function of the rats with incomplete spinal cord injuries. These experimental ifndings suggest that the transplantation of neural stem cells in a double-lay-er collagen membrane with unequal pore sizes is an effective therapeutic strategy to repair an injured spinal cord.

  3. A Role for BLM in Double-Strand Break Repair Pathway Choice: Prevention of CtIP/Mre11-Mediated Alternative Nonhomologous End-Joining

    Directory of Open Access Journals (Sweden)

    Anastazja Grabarz

    2013-10-01

    Full Text Available The choice of the appropriate double-strand break (DSB repair pathway is essential for the maintenance of genomic stability. Here, we show that the Bloom syndrome gene product, BLM, counteracts CtIP/MRE11-dependent long-range deletions (>200 bp generated by alternative end-joining (A-EJ. BLM represses A-EJ in an epistatic manner with 53BP1 and RIF1 and is required for ionizing-radiation-induced 53BP1 focus assembly. Conversely, in the absence of 53BP1 or RIF1, BLM promotes formation of A-EJ long deletions, consistent with a role for BLM in DSB end resection. These data highlight a dual role for BLM that influences the DSB repair pathway choice: (1 protection against CtIP/MRE11 long-range deletions associated with A-EJ and (2 promotion of DNA resection. These antagonist roles can be regulated, according to cell-cycle stage, by interacting partners such as 53BP1 and TopIII, to avoid unscheduled resection that might jeopardize genome integrity.

  4. Suppression of Jab1/CSN5 induces radio- and chemo-sensitivity in nasopharyngeal carcinoma through changes to the DNA damage and repair pathways.

    Science.gov (United States)

    Pan, Y; Zhang, Q; Atsaves, V; Yang, H; Claret, F X

    2013-05-30

    Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignancy most common in East Asia and Africa. Radiotherapy and cisplatin-based chemotherapy are the main treatment options. Unfortunately, disease response to concurrent chemoradiotherapy varies among patients with NPC, and many cases are resistant to cisplatin. Increased DNA damage repair is one of the mechanisms contributing to this resistance. Jab1/CSN5 is a multifunctional protein that participates in controlling cell proliferation and the stability of multiple proteins. Jab1 overexpression has been found to correlate with poor prognosis in several tumor types. However, the biological significance of Jab1 activity in response to cancer treatment is unclear. In this study, we used three NPC cell lines (CNE1, CNE2 and HONE1) to investigate the hypothesis that Jab1 positively regulates the DNA repair protein Rad51 and, in turn, cellular response to treatment with DNA-damaging agents such as cisplatin, ionizing radiation (IR) and ultraviolet (UV) radiation. We found that Jab1 was overexpressed in two relatively cisplatin-, IR- and UV-resistant NPC cell lines, and knocking down its expression conferred sensitivity to cisplatin, IR and UV radiation. By contrast, exogenous Jab1 expression enhanced the resistance of NPC cells to cisplatin, IR and UV radiation. Moreover, we provide a mechanism by which Jab1 positively regulated Rad51 through p53-dependent pathway, and increased ectopic expression of Rad51 conferred cellular resistance to cisplatin, IR and UV radiation in Jab1-deficient cells. Taken together, our findings suggest that Jab1 has an important role in the cellular response to cisplatin and irradiation by regulating DNA damage and repair pathways. Therefore, Jab1 is a novel biomarker for predicting the outcome of patients with NPC who are treated with DNA-damaging agents.

  5. Involvement of plasma membrane peroxidases and oxylipin pathway in the recovery from phytoplasma disease in apple (Malus domestica).

    Science.gov (United States)

    Patui, Sonia; Bertolini, Alberto; Clincon, Luisa; Ermacora, Paolo; Braidot, Enrico; Vianello, Angelo; Zancani, Marco

    2013-06-01

    Apple trees (Malus domestica Borkh.) may be affected by apple proliferation (AP), caused by 'Candidatus Phytoplasma mali'. Some plants can spontaneously recover from the disease, which implies the disappearance of symptoms through a phenomenon known as recovery. In this article it is shown that NAD(P)H peroxidases of leaf plasma membrane-enriched fractions exhibited a higher activity in samples from both AP-diseased and recovered plants. In addition, an increase in endogenous SA was characteristic of the symptomatic plants, since its content increased in samples obtained from diseased apple trees. In agreement, phenylalanine ammonia lyase (PAL) activity, a key enzyme of the phenylpropanoid pathway, was increased too. Jasmonic acid (JA) increased only during recovery, in a phase subsequent to the pathological state, and in concomitance to a decline of salicylic acid (SA). Oxylipin pathway, responsible for JA synthesis, was not induced during the development of AP-disease, but it appeared to be stimulated when the recovery occurred. Accordingly, lipoxygenase (LOX) activity, detected in plasma membrane-enriched fractions, showed an increase in apple leaves obtained from recovered plants. This enhancement was paralleled by an increase of hydroperoxide lyase (HPL) activity, detected in leaf microsomes, albeit the latter enzyme was activated in either the disease or recovery conditions. Hence, a reciprocal antagonism between SA- and JA-pathways could be suggested as an effective mechanism by which apple plants react to phytoplasma invasions, thereby providing a suitable defense response leading to the establishment of the recovery phenomenon. Copyright © Physiologia Plantarum 2012.

  6. 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.

  7. Membrane properties of striatal direct and indirect pathway neurons in mouse and rat slices and their modulation by dopamine.

    Directory of Open Access Journals (Sweden)

    Henrike Planert

    Full Text Available D1 and D2 receptor expressing striatal medium spiny neurons (MSNs are ascribed to striatonigral ("direct" and striatopallidal ("indirect" pathways, respectively, that are believed to function antagonistically in motor control. Glutamatergic synaptic transmission onto the two types is differentially affected by Dopamine (DA, however, less is known about the effects on MSN intrinsic electrical properties. Using patch clamp recordings, we comprehensively characterized the two pathways in rats and mice, and investigated their DA modulation. We identified the direct pathway by retrograde labeling in rats, and in mice we used transgenic animals in which EGFP is expressed in D1 MSNs. MSNs were subjected to a series of current injections to pinpoint differences between the populations, and in mice also following bath application of DA. In both animal models, most electrical properties were similar, however, membrane excitability as measured by step and ramp current injections consistently differed, with direct pathway MSNs being less excitable than their counterparts. DA had opposite effects on excitability of D1 and D2 MSNs, counteracting the initial differences. Pronounced changes in AP shape were seen in D2 MSNs. In direct pathway MSNs, excitability increased across experimental conditions and parameters, and also when applying DA or the D1 agonist SKF-81297 in presence of blockers of cholinergic, GABAergic, and glutamatergic receptors. Thus, DA induced changes in excitability were D1 R mediated and intrinsic to direct pathway MSNs, and not a secondary network effect of altered synaptic transmission. DAergic modulation of intrinsic properties therefore acts in a synergistic manner with previously reported effects of DA on afferent synaptic transmission and dendritic processing, supporting the antagonistic model for direct vs. indirect striatal pathway function.

  8. Experimental study on cleft palate repair of rat using rat fetal membrane%鼠胎膜修复大鼠腭裂的实验研究

    Institute of Scientific and Technical Information of China (English)

    张驰; 娜荷雅; 张幸竹; 王丝墨; 唐喜乐; 马振宇; 肖晶; 李武伟

    2013-01-01

    目的 以鼠胎膜对腭裂进行整复手术,研究修复腭裂的效果.方法 建立腭裂动物模型实验.30只SD大鼠按照腭部裂隙大小分为3组,每组10只.a组:长7 mm×宽0.9 mm;b组:长7 mm×宽1.1 mm;c组:长7mm×宽1.3 mm.术后12周处死所有大鼠,观察裂隙变化.手术关闭裂隙实验.10只SD大鼠分为3组.A组大鼠5只,手术造腭裂并以新鲜双层胎膜关闭裂隙;B组大鼠3只,手术造腭裂,令裂隙自行生长;C组2只作为空白对照组.术后4周处死动物,大体观察;制作切片,行组织学观察.结果 腭裂动物模型成功建立,确立b组7mm×1.1 mm为临界缺损.A组大鼠腭裂得到成功修复,口鼻腔黏膜分别愈合,完全覆盖腭裂,镜下见腭裂处口腔黏膜层次清楚,存在黏膜下层,顶部有完整的鼻腔黏膜.结论 鼠胎膜对SD大鼠腭裂具有良好的修复效果.%Objective To explore the feasibility of repairing the defect in cleft palate using fetal membrane in a rat model. Methods To establish the animal model of cleft palate. Thirty SD rats were randomly divided into three groups with 10 rats in each group according to the different size of man - made cleft, group a: 7 mm × 0. 9 mm, group b: 7 mm × 1. 1 mm, group c: 7 mm × 1.3 mm. Animals were sacrificed at the end of 12 weeks and the changes of clefts were compared. Repairing experiment for cleft palate. Ten SD rats were randomly divided into three groups, group A was underwent reduction with 5 rats, group B was only performed model operation with 3 rats, group C was blank with 2 rats. All rats were sacrificed at the end of 4 weeks and the gross and histological observation was respectively carried out. Results The animal model of cleft palate was successfully established. Group b was determined to be the animal model group for critical - sized defect. Cleft palate was repaired successfully in group A with fetal membrane of rat, the structure of palate mucosa was similar to that of the group C in gross

  9. Epstein-Barr virus associated modulation of Wnt pathway is not dependent on latent membrane protein-1.

    Directory of Open Access Journals (Sweden)

    Natasha Webb

    Full Text Available Previous studies have indicated that Epstein-Barr virus (EBV can modulate the Wnt pathway in virus-infected cells and this effect is mediated by EBV-encoded oncogene latent membrane protein 1 (LMP1. Here we have reassessed the role of LMP1 in regulating the expression of various mediators of the canonical Wnt cascade. Contradicting the previous finding, we found that the levels of E-cadherin, beta-catenin, Glycogen Synthase Kinase 3ss (GSK3beta, axin and alpha-catenin were not affected by the expression of LMP1 sequences from normal B cells or nasopharyngeal carcinoma. Moreover, we also show that LMP1 expression had no detectable effect on the E-cadherin and beta-catenin interaction and did not induce transcriptional activation of beta-catenin. Taken together these studies demonstrate that EBV-mediated activation of Wnt pathway is not dependent on the expression of LMP1.

  10. A new probe for super-resolution imaging of membranes elucidates trafficking pathways.

    Science.gov (United States)

    Revelo, Natalia H; Kamin, Dirk; Truckenbrodt, Sven; Wong, Aaron B; Reuter-Jessen, Kirsten; Reisinger, Ellen; Moser, Tobias; Rizzoli, Silvio O

    2014-05-26

    The molecular composition of the organelles involved in membrane recycling is difficult to establish as a result of the absence of suitable labeling tools. We introduce in this paper a novel probe, named membrane-binding fluorophore-cysteine-lysine-palmitoyl group (mCLING), which labels the plasma membrane and is taken up during endocytosis. It remains attached to membranes after fixation and permeabilization and can therefore be used in combination with immunostaining and super-resolution microscopy. We applied mCLING to mammalian-cultured cells, yeast, bacteria, primary cultured neurons, Drosophila melanogaster larval neuromuscular junctions, and mammalian tissue. mCLING enabled us to study the molecular composition of different trafficking organelles. We used it to address several questions related to synaptic vesicle recycling in the auditory inner hair cells from the organ of Corti and to investigate molecular differences between synaptic vesicles that recycle actively or spontaneously in cultured neurons. We conclude that mCLING enables the investigation of trafficking membranes in a broad range of preparations.

  11. Investigations on the role of base excision repair and non-homologous end-joining pathways in sodium selenite-induced toxicity and mutagenicity in Saccharomyces cerevisiae.

    Science.gov (United States)

    Mániková, Dominika; Vlasáková, Danusa; Loduhová, Jana; Letavayová, Lucia; Vigasová, Dana; Krascsenitsová, Eva; Vlcková, Viera; Brozmanová, Jela; Chovanec, Miroslav

    2010-03-01

    Selenium (Se) belongs to nutrients that are essential for human health. Biological activity of this compound, however, mainly depends on its dose, with a potential of Se to induce detrimental effects at high doses. Although mechanisms lying behind detrimental effects of Se are poorly understood yet, they involve DNA damage induction. Consequently, DNA damage response and repair pathways may play a crucial role in cellular response to Se. Using Saccharomyces cerevisiae we showed that sodium selenite (SeL), an inorganic form of Se, can be toxic and mutagenic in this organism due to its ability to induce DNA double-strand breaks (DSBs). Moreover, we reported that a spectrum of mutations induced by this compound in the stationary phase of growth is mainly represented by 1-4 bp deletions. Consequently, we proposed that SeL acts as an oxidizing agent in yeast producing oxidative damage to DNA. As short deletions could be anticipated to arise as a result of action of non-homologous end-joining (NHEJ) and oxidative damage to DNA is primarily coped with base excision repair (BER), a contribution of these two pathways towards survival, DSB induction, mutation frequency and types of mutations following SeL exposure was examined in present study. First, we show that while NHEJ plays no role in repairing toxic DNA lesions induced by SeL, cells with impairment in BER are sensitized towards this compound. Of BER activities examined, those responsible for processing of 3'-blocking DNA termini seem to be the most crucial for manifestation of the toxic effects of SeL in yeast. Second, an impact of NHEJ and BER on DSB induction after SeL exposure turned to be inappreciable, as no increase in DNA double-strand breakage in NHEJ and BER single or NHEJ BER double mutant upon SeL exposure was observed. Finally, we demonstrate that impairment in both these pathways does not importantly change mutation frequency after SeL exposure and that NHEJ is not responsible for generation of short

  12. Esc2 and Sgs1 act in functionally distinct branches of the homologous recombination repair pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Mankouri, Hocine W; Ngo, Hien-Ping; Hickson, Ian D

    2009-01-01

    Esc2 is a member of the RENi family of SUMO-like domain proteins and is implicated in gene silencing in Saccharomyces cerevisiae. Here, we identify a dual role for Esc2 during S-phase in mediating both intra-S-phase DNA damage checkpoint signaling and preventing the accumulation of Rad51-dependen......, and sgs1esc2 cells attempt to undergo mitosis with unprocessed HRR intermediates. We propose a model whereby Esc2 acts in an Mph1-dependent process, separately from Sgs1, to influence the repair/tolerance of MMS-induced lesions during S-phase....

  13. ERCC2/XPD Lys751Gln alter DNA repair efficiency of platinum-induced DNA damage through P53 pathway.

    Science.gov (United States)

    Zhang, Guopei; Guan, Yangyang; Zhao, Yuejiao; van der Straaten, Tahar; Xiao, Sha; Xue, Ping; Zhu, Guolian; Liu, Qiufang; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Lu, Xiaobo

    2017-02-01

    Platinum-based treatment causes Pt-DNA adducts which lead to cell death. The platinum-induced DNA damage is recognized and repaired by the nucleotide excision repair (NER) system of which ERCC2/XPD is a critical enzyme. Single nucleotide polymorphisms in ERCC2/XPD have been found to be associated with platinum resistance. The aim of the present study was to investigate whether ERCC2/XPD Lys751Gln (rs13181) polymorphism is causally related to DNA repair capacity of platinum-induced DNA damage. First, cDNA clones expressing different genotypes of the polymorphism was transfected to an ERCC2/XPD defective CHO cell line (UV5). Second, all cells were treated with cisplatin. Cellular survival rate were investigated by MTT growth inhibition assay, DNA damage levels were investigated by comet assay and RAD51 staining. The distribution of cell cycle and the change of apoptosis rates were detected by a flow cytometric method (FCM). Finally, P53mRNA and phospho-P53 protein levels were further investigated in order to explore a possible explanation. As expected, there was a significantly increased in viability of UV5(ERCC2 (AA)) as compared to UV5(ERCC2 (CC)) after cisplatin treatment. The DNA damage level of UV5(ERCC2 (AA)) was significant decreased compared to UV5(ERCC2 (CC)) at 24 h of treatment. Mutation of ERCC2rs13181 AA to CC causes a prolonged S phase in cell cycle. UV5(ERCC2 (AA)) alleviated the apoptosis compared to UV5(ERCC2 (CC)), meanwhile P53mRNA levels in UV(ERCC2 (AA)) was also lower when compared UV5(ERCC2 (CC)). It co-incides with a prolonged high expression of phospho-P53, which is relevant for cell cycle regulation, apoptosis, and the DNA damage response (DDR). We concluded that ERCC2/XPD rs13181 polymorphism is possibly related to the DNA repair capacity of platinum-induced DNA damage. This functional study provides some clues to clarify the relationship between cisplatin resistance and ERCC2/XPDrs13181 polymorphism.

  14. Cross-talk between nucleotide excision and homologous recombination DNA repair pathways in the mechanism of action of antitumor trabectedin.

    Science.gov (United States)

    Herrero, Ana B; Martín-Castellanos, Cristina; Marco, Esther; Gago, Federico; Moreno, Sergio

    2006-08-15

    Trabectedin (Yondelis) is a potent antitumor drug that has the unique characteristic of killing cells by poisoning the DNA nucleotide excision repair (NER) machinery. The basis for the NER-dependent toxicity has not yet been elucidated but it has been proposed as the major determinant for the drug's cytotoxicity. To study the in vivo mode of action of trabectedin and to explore the role of NER in its cytotoxicity, we used the fission yeast Schizosaccharomyces pombe as a model system. Treatment of S. pombe wild-type cells with trabectedin led to cell cycle delay and activation of the DNA damage checkpoint, indicating that the drug causes DNA damage in vivo. DNA damage induced by the drug is mostly caused by the NER protein, Rad13 (the fission yeast orthologue to human XPG), and is mainly repaired by homologous recombination. By constructing different rad13 mutants, we show that the DNA damage induced by trabectedin depends on a 46-amino acid region of Rad13 that is homologous to a DNA-binding region of human nuclease FEN-1. More specifically, an arginine residue in Rad13 (Arg961), conserved in FEN1 (Arg314), was found to be crucial for the drug's cytotoxicity. These results lead us to propose a model for the action of trabectedin in eukaryotic cells in which the formation of a Rad13/DNA-trabectedin ternary complex, stabilized by Arg961, results in cell death.

  15. Genetic requirements for the single-strand annealing pathway of double-strand break repair in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, E.L.; Sugawara, N.; Haber, J.E. [Brandeis Univ., Waltham, MA (United States)] [and others

    1996-03-01

    HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with >80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching. 43 refs., 8 figs., 3 tabs.

  16. Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

    Directory of Open Access Journals (Sweden)

    Catherine E Smith

    2013-10-01

    Full Text Available Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

  17. Aberrant expression of proteins involved in signal transduction and DNA repair pathways in lung cancer and their association with clinical parameters.

    Directory of Open Access Journals (Sweden)

    Yong He

    Full Text Available BACKGROUND: Because cell signaling and cell metabolic pathways are executed through proteins, protein signatures in primary tumors are useful for identifying key nodes in signaling networks whose alteration is associated with malignancy and/or clinical outcomes. This study aimed to determine protein signatures in primary lung cancer tissues. METHODOLOGY/ PRINCIPAL FINDINGS: We analyzed 126 proteins and/or protein phosphorylation sites in case-matched normal and tumor samples from 101 lung cancer patients with reverse-phase protein array (RPPA assay. The results showed that 18 molecules were significantly different (p<0.05 by at least 30% between normal and tumor tissues. Most of those molecules play roles in cell proliferation, DNA repair, signal transduction and lipid metabolism, or function as cell surface/matrix proteins. We also validated RPPA results by Western blot and/or immunohistochemical analyses for some of those molecules. Statistical analyses showed that Ku80 levels were significantly higher in tumors of nonsmokers than in those of smokers. Cyclin B1 levels were significantly overexpressed in poorly differentiated tumors while Cox2 levels were significantly overexpressed in neuroendocrinal tumors. A high level of Stat5 is associated with favorable survival outcome for patients treated with surgery. CONCLUSIONS/ SIGNIFICANCE: Our results revealed that some molecules involved in DNA damage/repair, signal transductions, lipid metabolism, and cell proliferation were drastically aberrant in lung cancer tissues, and Stat5 may serve a molecular marker for prognosis of lung cancers.

  18. Biocompatibility evaluation of cigarette and carbon papers used in repair of traumatic tympanic membrane perforations: experimental study.

    Science.gov (United States)

    Altuntaş, Emine Elif; Sümer, Zeynep

    2013-01-01

    The purposes of this study were to investigate the biocompatibility of two different paper patches (carbon and cigarette papers) and compare the adhesion and proliferation features of L929 fibroblast cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT Test) test and scanning electron microscopy (SEM). In this study, time-dependent cytotoxic effects of cigarette and carbon papers used in repairing small traumatic TM perforations were investigated in vitro by using MTT test. And also adhesion and spreading of cells over disk surface were observed by SEM. Cytotoxicity test carried out by MTT analysis on leakage products collected from two types of paper patches at the end of 24 and 48 h revealed no cytotoxicity (P > 0.05). In SEM studies, it was observed that cells started to proliferate over disk surface as a result of 48-h incubation, and SEM revealed that the cell proliferation over cigarette paper was more compared to the one over carbon paper. We believe that this is the first study where biocompatibility and adhesion features of carbon and cigarette paper have been studied by using L929 fibroblast cell culture. As a result, biocompatibility of cigarette paper and also whether cigarette paper was superior to carbon paper in cell attachment and biocompatibility were studied. It was found, by MTT test and SEM test, that cigarette paper had a higher biocompatibility and cell attachment, and thus cigarette paper should be the patch to be preferred in cases where TM perforations are repaired by paper-patch method.

  19. Chapter 10 the primary cilium coordinates signaling pathways in cell cycle control and migration during development and tissue repair

    DEFF Research Database (Denmark)

    Christensen, Søren T; Pedersen, Stine F; Satir, Peter

    2008-01-01

    Cell cycle control and migration are critical processes during development and maintenance of tissue functions. Recently, primary cilia were shown to take part in coordination of the signaling pathways that control these cellular processes in human health and disease. In this review, we present...... with the extracellular matrix, coordinate Wnt signaling, and modulate cytoskeletal changes that impinge on both cell cycle control and cell migration....... an overview of the function of primary cilia and the centrosome in the signaling pathways that regulate cell cycle control and migration with focus on ciliary signaling via platelet-derived growth factor receptor alpha (PDGFRalpha). We also consider how the primary cilium and the centrosome interact...

  20. Crystallization of a member of the recFOR DNA repair pathway, RecO, with and without bound oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Aono, Shelly; Hartsch, Thomas; Schulze-Gahmen, Ursula

    2003-01-22

    RecFOR proteins are important for DNA repair by homologous recombination in bacteria. The RecO protein from Thermus thermophilus was cloned, purified and characterized for its binding to oligonucleotides. The protein was crystallized alone and in complex with a 14-mer oligonucleotide. Both crystal forms grow under different crystallization conditions in the same space group, P3121 or P3221, with almost identical unit cell parameters. Complete data sets were collected to 2.8 Angstrom and 2.5 Angstrom for RecO alone and the RecO-oligonucleotide complex, respectively. Visual comparison of the diffraction patterns between the two crystal forms and calculation of an Rmerge of 33.9 percent on F indicate that one of the crystal forms is indeed a complex of RecO with bound oligonucleotide.

  1. Do phosphoinositides regulate membrane water permeability of tobacco protoplasts by enhancing the aquaporin pathway?

    Science.gov (United States)

    Ma, Xiaohong; Shatil-Cohen, Arava; Ben-Dor, Shifra; Wigoda, Noa; Perera, Imara Y; Im, Yang Ju; Diminshtein, Sofia; Yu, Ling; Boss, Wendy F; Moshelion, Menachem; Moran, Nava

    2015-03-01

    Enhancing the membrane content of PtdInsP 2 , the already-recognized protein-regulating lipid, increased the osmotic water permeability of tobacco protoplasts, apparently by increasing the abundance of active aquaporins in their membranes. While phosphoinositides are implicated in cell volume changes and are known to regulate some ion channels, their modulation of aquaporins activity has not yet been reported for any organism. To examine this, we compared the osmotic water permeability (P f) of protoplasts isolated from tobacco (Nicotiana tabacum) cultured cells (NT1) with different (genetically lowered or elevated relative to controls) levels of inositol trisphosphate (InsP3) and phosphatidyl inositol [4,5] bisphosphate (PtdInsP2). To achieve this, the cells were transformed with, respectively, the human InsP3 5-phosphatase ('Ptase cells') or human phosphatidylinositol (4) phosphate 5-kinase ('PIPK cells'). The mean P f of the PIPK cells was several-fold higher relative to that of controls and Ptase cells. Three results favor aquaporins over the membrane matrix as underlying this excessive P f: (1) transient expression of the maize aquaporin ZmPIP2;4 in the PIPK cells increased P f by 12-30 μm s(-1), while in the controls only by 3-4 μm s(-1). (2) Cytosol acidification-known to inhibit aquaporins-lowered the P f in the PIPK cells down to control levels. (3) The transcript of at least one aquaporin was elevated in the PIPK cells. Together, the three results demonstrate the differences between the PIPK cells and their controls, and suggest a hitherto unobserved regulation of aquaporins by phosphoinositides, which could occur through direct interaction or indirect phosphoinositides-dependent cellular effects.

  2. EXPERIMENTAL STUDY ON HUMAN AMNIOTIC MEMBRANE FOR REPAIRING TENDON SHEATH DEFECT%人羊膜修复腱鞘缺损的实验研究

    Institute of Scientific and Technical Information of China (English)

    高鸣; 赵红芳; 田德虎; 于昆仑; 白江博; 董瑞一; 张国辉

    2013-01-01

    目的 探讨人羊膜修复鸡足趾腱鞘缺损后防止肌腱粘连的可行性和有效性.方法 取行剖腹产术产妇自愿捐赠的胎盘,制备大小为1.5 cm×1.0 cm的羊膜片.3~6月龄健康雄性来亨鸡40只,体重(1.86±0.04)kg,取双足第3趾制备肌腱、腱鞘损伤模型.“8”字缝合修复肌腱后,右足采用羊膜片修复缺损腱鞘(A组),左足缺损腱鞘不作处理(B组).术后1、2、4、6周各取10只实验动物行大体及组织学观察,并按照Tang等肌腱粘连大体观察分级标准进行分级,生物力学试验测定肌腱滑移度及总屈趾角度.结果 术后实验动物均存活至实验完成,切口均愈合良好.随术后时间延长,大体及组织学观察显示两组均有假鞘(新生腱鞘)形成,但A组假鞘较B组成熟、光滑.术后1、6周A组肌腱粘连分级均明显优于B组,差异有统计学意义(P< 0.05).生物力学试验测定示,术后1、2周两组肌腱滑移度比较,差异均无统计学意义(P> 0.05),4、6周时A组肌腱滑移度均较B组长(P<0.05).术后1、2、4、6周A组总屈趾角度均小于B组,差异有统计学意义(P<0.05).结论 采用人羊膜修复鸡腱鞘缺损能有效预防肌腱粘连,利于肌腱滑动功能的恢复.%Objective To investigate the feasibility and effect of human amniotic membrane in prevention of tendon adhension after tendon sheat defect repair. Methods The amniotic membrane in size of 1.5 cm × 1.0 cm was harvested from human placenta which was voluntary donated from maternal after cesarean. Forty healthy male Leghorn chicken (aged 3-6 months) were selected, weighing (1.86 ± 0.04) kg. The model of flexor digitorum profundus tendon and tendon sheath defects was established at the third toe. After repair of the flexor digitorum profundus tendon, the human amniotic membrane was used to repair the tendon sheath defect in the right foot (group A), but tendon sheath defect was not repaired in the left foot (group B) . At 1, 2, 4

  3. A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway.

    Science.gov (United States)

    Voter, Andrew F; Manthei, Kelly A; Keck, James L

    2016-07-01

    Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for resensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay, and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol.

  4. Slit-Robo Signal Pathway in Tissue Injury and Repair%Slit-Robo信号传导通路与组织损伤修复

    Institute of Scientific and Technical Information of China (English)

    刘丽华

    2012-01-01

    Slit proteins are secreted ligands that interact with Roundabout (Robo) receptors and have an important role in axon guidance , axonal migration, leukocyte chemotaxis, and cell migration. Current research of the Slit-Robo signal pathway shows expression in many types of injured tissues including traumas, inflammations and tumors. This article reviews current research of the Slit-Robo signal pathway with regards to tissue injury and repair.%Slit是一种分泌型的细胞外基质蛋白,它通过与其受体Roundabout( Roundabout,Robo)结合而发挥生物活性.早期研究发现Slit-Robo是神经轴突导向分子、神经元迁移的排斥因子,对白细胞趋化也有抑制作用;近年来发现Slit-Robo信号通路还能调节全身多个系统的多种细胞迁移.随着对Slit-Robo信号通路的深入研究,发现其在各种损伤、感染及肿瘤组织及其修复过程中都有表达,且其功能不尽相同.现就其在各种异常组织中的研究做一总结和回顾.

  5. Alternative Pathway Dysregulation and the Conundrum of Complement Activation by IgG4 Immune Complexes in Membranous Nephropathy

    Science.gov (United States)

    Borza, Dorin-Bogdan

    2016-01-01

    Membranous nephropathy (MN), a major cause of nephrotic syndrome, is a non-inflammatory immune kidney disease mediated by IgG antibodies that form glomerular subepithelial immune complexes. In primary MN, autoantibodies target proteins expressed on the podocyte surface, often phospholipase A2 receptor (PLA2R1). Pathology is driven by complement activation, leading to podocyte injury and proteinuria. This article overviews the mechanisms of complement activation and regulation in MN, addressing the paradox that anti-PLA2R1 and other antibodies causing primary MN are predominantly (but not exclusively) IgG4, an IgG subclass that does not fix complement. Besides immune complexes, alterations of the glomerular basement membrane (GBM) in MN may lead to impaired regulation of the alternative pathway (AP). The AP amplifies complement activation on surfaces insufficiently protected by complement regulatory proteins. Whereas podocytes are protected by cell-bound regulators, the GBM must recruit plasma factor H, which inhibits the AP on host surfaces carrying certain polyanions, such as heparan sulfate (HS) chains. Because HS chains present in the normal GBM are lost in MN, we posit that the local complement regulation by factor H may be impaired as a result. Thus, the loss of GBM HS in MN creates a micro-environment that promotes local amplification of complement activation, which in turn may be initiated via the classical or lectin pathways by subsets of IgG in immune complexes. A detailed understanding of the mechanisms of complement activation and dysregulation in MN is important for designing more effective therapies. PMID:27199983

  6. Membrane glycoprotein M6A promotes μ-opioid receptor endocytosis and facilitates receptor sorting into the recycling pathway

    Institute of Scientific and Technical Information of China (English)

    Ying-Jian Liang; Dai-Fei Wu; Ralf Stumm; Volker H(o)llt; Thomas Koch

    2008-01-01

    The interaction of μ-opioid receptor (MOPr) with the neuronal membrane glycoprotein M6a is known to facilitate MOPr endocytosis in human embryonic kidney 293 (HEK293) cells. To further study the role of M6a in the post-endocytotic sorting of MOPr, we investigated the agonist-induced co-internalization of MOPr and M6a and protein targeting after internalization in HEK293 cells that co-expressed HA-tagged MOPr and Myc-tagged M6a. We found that M6a, MOPr, and Rab 11, a marker for recycling endosomes, co-localized in endocytotic vesicles, indicating that MOPr and M6a are primarily targeted to recycling endosomes after endocytosis. Furthermore, co-expression of M6a augmented the post-endocytotic sorting of δ-opioid receptors into the recycling pathway, indicating that M6a might have a more general role in opioid receptor post-ndocytotic sorting. The enhanced post-endocytotic sorting of MOPr into the recycling pathway was accompanied by a decrease in agonist-induced receptor down-regulation of M6a in co-expressing cells. We tested the physiological relevance of these findings in primary cultures of cortical neurons and found that co-expression of M6a markedly increased the translocation of MOPrs from the plasma membrane to intracellular vesicles at steady state and significantly enhanced both constitutive and agonist-induced receptor endocytosis. In conclusion, our results strongly indicate that M6a modulates MOPr endocytosis and post-endocytotic sorting and has an important role in receptor regulation.

  7. Genetic evidence that the non-homologous end-joining repair pathway is involved in LINE retrotransposition.

    Directory of Open Access Journals (Sweden)

    Jun Suzuki

    2009-04-01

    Full Text Available Long interspersed elements (LINEs are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs--zebrafish ZfL2-2 and human L1--in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.

  8. Photosynthetic Membranes of Synechocystis or Plants Convert Sunlight to Photocurrent through Different Pathways due to Different Architectures.

    Directory of Open Access Journals (Sweden)

    Roy I Pinhassi

    Full Text Available Thylakoid membranes contain the redox active complexes catalyzing the light-dependent reactions of photosynthesis in cyanobacteria, algae and plants. Crude thylakoid membranes or purified photosystems from different organisms have previously been utilized for generation of electrical power and/or fuels. Here we investigate the electron transferability from thylakoid preparations from plants or the cyanobacterium Synechocystis. We show that upon illumination, crude Synechocystis thylakoids can reduce cytochrome c. In addition, this crude preparation can transfer electrons to a graphite electrode, producing an unmediated photocurrent of 15 μA/cm2. Photocurrent could be obtained in the presence of the PSII inhibitor DCMU, indicating that the source of electrons is QA, the primary Photosystem II acceptor. In contrast, thylakoids purified from plants could not reduce cyt c, nor produced a photocurrent in the photocell in the presence of DCMU. The production of significant photocurrent (100 μA/cm2 from plant thylakoids required the addition of the soluble electron mediator DCBQ. Furthermore, we demonstrate that use of crude thylakoids from the D1-K238E mutant in Synechocystis resulted in improved electron transferability, increasing the direct photocurrent to 35 μA/cm2. Applying the analogous mutation to tobacco plants did not achieve an equivalent effect. While electron abstraction from crude thylakoids of cyanobacteria or plants is feasible, we conclude that the site of the abstraction of the electrons from the thylakoids, the architecture of the thylakoid preparations influence the site of the electron abstraction, as well as the transfer pathway to the electrode. This dictates the use of different strategies for production of sustainable electrical current from photosynthetic thylakoid membranes of cyanobacteria or higher plants.

  9. Photosynthetic Membranes of Synechocystis or Plants Convert Sunlight to Photocurrent through Different Pathways due to Different Architectures.

    Science.gov (United States)

    Pinhassi, Roy I; Kallmann, Dan; Saper, Gadiel; Larom, Shirley; Linkov, Artyom; Boulouis, Alix; Schöttler, Mark-Aurel; Bock, Ralph; Rothschild, Avner; Adir, Noam; Schuster, Gadi

    2015-01-01

    Thylakoid membranes contain the redox active complexes catalyzing the light-dependent reactions of photosynthesis in cyanobacteria, algae and plants. Crude thylakoid membranes or purified photosystems from different organisms have previously been utilized for generation of electrical power and/or fuels. Here we investigate the electron transferability from thylakoid preparations from plants or the cyanobacterium Synechocystis. We show that upon illumination, crude Synechocystis thylakoids can reduce cytochrome c. In addition, this crude preparation can transfer electrons to a graphite electrode, producing an unmediated photocurrent of 15 μA/cm2. Photocurrent could be obtained in the presence of the PSII inhibitor DCMU, indicating that the source of electrons is QA, the primary Photosystem II acceptor. In contrast, thylakoids purified from plants could not reduce cyt c, nor produced a photocurrent in the photocell in the presence of DCMU. The production of significant photocurrent (100 μA/cm2) from plant thylakoids required the addition of the soluble electron mediator DCBQ. Furthermore, we demonstrate that use of crude thylakoids from the D1-K238E mutant in Synechocystis resulted in improved electron transferability, increasing the direct photocurrent to 35 μA/cm2. Applying the analogous mutation to tobacco plants did not achieve an equivalent effect. While electron abstraction from crude thylakoids of cyanobacteria or plants is feasible, we conclude that the site of the abstraction of the electrons from the thylakoids, the architecture of the thylakoid preparations influence the site of the electron abstraction, as well as the transfer pathway to the electrode. This dictates the use of different strategies for production of sustainable electrical current from photosynthetic thylakoid membranes of cyanobacteria or higher plants.

  10. Extracorporeal membrane oxygenator as a bridge to successful surgical repair of bronchopleural fistula following bilateral sequential lung transplantation: a case report and review of literature

    Directory of Open Access Journals (Sweden)

    Machaal Ali

    2007-06-01

    Full Text Available Abstract Background Lung transplantation (LTx is widely accepted as a therapeutic option for end-stage respiratory failure in cystic fibrosis. However, airway complications remain a major cause of morbidity and mortality in these patients, serious airway complications like bronchopleural fistula (BPF are rare, and their management is very difficult. Case presentation A 47-year-old man with end-stage respiratory failure due to cystic fibrosis underwent bilateral sequential lung transplantation. Severe post-operative bleeding occurred due to dense intrapleural adhesions of the native lungs. He was re-explored and packed leading to satisfactory haemostasis. He developed a bronchopleural fistula on the 14th post-operative day. The fistula was successfully repaired using pericardial and intercostal vascular flaps with veno-venous extracorporeal membrane oxygenator (VV-ECMO support. Subsequently his recovery was uneventful. Conclusion The combination of pedicled intercostal and pericardial flaps provide adequate vascular tissue for sealing a large BPF following LTx. Veno-venous ECMO allows a feasible bridge to recovery.

  11. Upregulated Ras/Raf/ERK1/2 signaling pathway: a new hope in the repair of spinal cord injury

    Directory of Open Access Journals (Sweden)

    Tao Liu

    2015-01-01

    Full Text Available An increasing number of studies report that the Ras/Raf/extracellular signal-regulated kinase 1/2 (ERK1/2 signaling pathway has a death-promoting apoptotic function in neural cells. We hypothesized that the Ras/Raf/ERK1/2 signaling pathway may be abnormally regulated in rat injured spinal cord models. The weight drop method was used to establish rat spinal cord injury at T 9 . Western blot analysis and immunohistochemical staining revealed Ras expression was dramatically elevated, and the phosphorylations of A-Raf, B-Raf and C-Raf were all upregulated in the injured spinal cord. Both mitogen-activated protein kinase kinase 1/2 and ERK1/2, which belong to the Ras/Raf signaling kinases, were upregulated. These results indicate that Ras/Raf/ERK1/2 signaling may be upregulated in injured spinal cord and are involved in recovery after spinal cord injury.

  12. KSHV Entry and Trafficking in Target Cells—Hijacking of Cell Signal Pathways, Actin and Membrane Dynamics

    Directory of Open Access Journals (Sweden)

    Binod Kumar

    2016-11-01

    Full Text Available Kaposi’s sarcoma associated herpesvirus (KSHV is etiologically associated with human endothelial cell hyperplastic Kaposi’s sarcoma and B-cell primary effusion lymphoma. KSHV infection of adherent endothelial and fibroblast cells are used as in vitro models for infection and KSHV enters these cells by host membrane bleb and actin mediated macropinocytosis or clathrin endocytosis pathways, respectively. Infection in endothelial and fibroblast cells is initiated by the interactions between multiple viral envelope glycoproteins and cell surface associated heparan sulfate (HS, integrins (α3β1, αVβ3 and αVβ5, and EphA2 receptor tyrosine kinase (EphA2R. This review summarizes the accumulated studies demonstrating that KSHV manipulates the host signal pathways to enter and traffic in the cytoplasm of the target cells, to deliver the viral genome into the nucleus, and initiate viral gene expression. KSHV interactions with the cell surface receptors is the key platform for the manipulations of host signal pathways which results in the simultaneous induction of FAK, Src, PI3-K, Rho-GTPase, ROS, Dia-2, PKC ζ, c-Cbl, CIB1, Crk, p130Cas and GEF-C3G signal and adaptor molecules that play critical roles in the modulation of membrane and actin dynamics, and in the various steps of the early stages of infection such as entry and trafficking towards the nucleus. The Endosomal Sorting Complexes Required for Transport (ESCRT proteins are also recruited to assist in viral entry and trafficking. In addition, KSHV interactions with the cell surface receptors also induces the host transcription factors NF-κB, ERK1/2, and Nrf2 early during infection to initiate and modulate viral and host gene expression. Nuclear delivery of the viral dsDNA genome is immediately followed by the host innate responses such as the DNA damage response (DDR, inflammasome and interferon responses. Overall, these studies form the initial framework for further studies of

  13. KSHV Entry and Trafficking in Target Cells—Hijacking of Cell Signal Pathways, Actin and Membrane Dynamics

    Science.gov (United States)

    Kumar, Binod; Chandran, Bala

    2016-01-01

    Kaposi’s sarcoma associated herpesvirus (KSHV) is etiologically associated with human endothelial cell hyperplastic Kaposi’s sarcoma and B-cell primary effusion lymphoma. KSHV infection of adherent endothelial and fibroblast cells are used as in vitro models for infection and KSHV enters these cells by host membrane bleb and actin mediated macropinocytosis or clathrin endocytosis pathways, respectively. Infection in endothelial and fibroblast cells is initiated by the interactions between multiple viral envelope glycoproteins and cell surface associated heparan sulfate (HS), integrins (α3β1, αVβ3 and αVβ5), and EphA2 receptor tyrosine kinase (EphA2R). This review summarizes the accumulated studies demonstrating that KSHV manipulates the host signal pathways to enter and traffic in the cytoplasm of the target cells, to deliver the viral genome into the nucleus, and initiate viral gene expression. KSHV interactions with the cell surface receptors is the key platform for the manipulations of host signal pathways which results in the simultaneous induction of FAK, Src, PI3-K, Rho-GTPase, ROS, Dia-2, PKC ζ, c-Cbl, CIB1, Crk, p130Cas and GEF-C3G signal and adaptor molecules that play critical roles in the modulation of membrane and actin dynamics, and in the various steps of the early stages of infection such as entry and trafficking towards the nucleus. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins are also recruited to assist in viral entry and trafficking. In addition, KSHV interactions with the cell surface receptors also induces the host transcription factors NF-κB, ERK1/2, and Nrf2 early during infection to initiate and modulate viral and host gene expression. Nuclear delivery of the viral dsDNA genome is immediately followed by the host innate responses such as the DNA damage response (DDR), inflammasome and interferon responses. Overall, these studies form the initial framework for further studies of simultaneous targeting of

  14. Pharmacogenomics of Methotrexate Membrane Transport Pathway: Can Clinical Response to Methotrexate in Rheumatoid Arthritis Be Predicted?

    Directory of Open Access Journals (Sweden)

    Aurea Lima

    2015-06-01

    Full Text Available Background: Methotrexate (MTX is widely used for rheumatoid arthritis (RA treatment. Single nucleotide polymorphisms (SNPs could be used as predictors of patients’ therapeutic outcome variability. Therefore, this study aims to evaluate the influence of SNPs in genes encoding for MTX membrane transport proteins in order to predict clinical response to MTX. Methods: Clinicopathological data from 233 RA patients treated with MTX were collected, clinical response defined, and patients genotyped for 23 SNPs. Genotype and haplotype analyses were performed using multivariate methods and a genetic risk index (GRI for non-response was created. Results: Increased risk for non-response was associated to SLC22A11 rs11231809 T carriers; ABCC1 rs246240 G carriers; ABCC1 rs3784864 G carriers; CGG haplotype for ABCC1 rs35592, rs2074087 and rs3784864; and CGG haplotype for ABCC1 rs35592, rs246240 and rs3784864. GRI demonstrated that patients with Index 3 were 16-fold more likely to be non-responders than those with Index 1. Conclusions: This study revealed that SLC22A11 and ABCC1 may be important to identify those patients who will not benefit from MTX treatment, highlighting the relevance in translating these results to clinical practice. However, further validation by independent studies is needed to develop the field of personalized medicine to predict clinical response to MTX treatment.

  15. Pharmacogenomics of Methotrexate Membrane Transport Pathway: Can Clinical Response to Methotrexate in Rheumatoid Arthritis Be Predicted?

    Science.gov (United States)

    Lima, Aurea; Bernardes, Miguel; Azevedo, Rita; Medeiros, Rui; Seabra, Vitor

    2015-01-01

    Background: Methotrexate (MTX) is widely used for rheumatoid arthritis (RA) treatment. Single nucleotide polymorphisms (SNPs) could be used as predictors of patients’ therapeutic outcome variability. Therefore, this study aims to evaluate the influence of SNPs in genes encoding for MTX membrane transport proteins in order to predict clinical response to MTX. Methods: Clinicopathological data from 233 RA patients treated with MTX were collected, clinical response defined, and patients genotyped for 23 SNPs. Genotype and haplotype analyses were performed using multivariate methods and a genetic risk index (GRI) for non-response was created. Results: Increased risk for non-response was associated to SLC22A11 rs11231809 T carriers; ABCC1 rs246240 G carriers; ABCC1 rs3784864 G carriers; CGG haplotype for ABCC1 rs35592, rs2074087 and rs3784864; and CGG haplotype for ABCC1 rs35592, rs246240 and rs3784864. GRI demonstrated that patients with Index 3 were 16-fold more likely to be non-responders than those with Index 1. Conclusions: This study revealed that SLC22A11 and ABCC1 may be important to identify those patients who will not benefit from MTX treatment, highlighting the relevance in translating these results to clinical practice. However, further validation by independent studies is needed to develop the field of personalized medicine to predict clinical response to MTX treatment. PMID:26086825

  16. DNA双链断裂NHEJ修复及其与肿瘤的研究%Non-homologous end joining pathway of DSB repair and cancer

    Institute of Scientific and Technical Information of China (English)

    张耀伟

    2010-01-01

    非同源末端连接是哺乳动物最主要的DNA双链断裂(DSB)连接方式.肿瘤细胞非同源末端连接能力的提高与其放化疗抵抗有关,抑制肿瘤细胞非同源末端连接能力,可能增加其对放化疗的敏感性.因此,参与非同源末端连接的修复因子可能成为肿瘤分子靶向治疗及放化疗增敏的新治疗点.%Non-homologous end joining (NHEJ) is the major pathway for repairing DNA doublestrand break (DSB) in mammalian species. The capacity of NHEJ increases in tumor cell,which plays a role in radiation/chemotherapy-resistant agent Inhibiting DSB rejoining may play a crucial role in the enhancement of cellular radiation/chemotherapy-sensitizing. Thus, the protein molecule enrolled in NHEJ may be new potential targets for radiation/chemotherapy -sensitizing.

  17. 1,4-Dihydropyridines Active on the SIRT1/AMPK Pathway Ameliorate Skin Repair and Mitochondrial Function and Exhibit Inhibition of Proliferation in Cancer Cells.

    Science.gov (United States)

    Valente, Sergio; Mellini, Paolo; Spallotta, Francesco; Carafa, Vincenzo; Nebbioso, Angela; Polletta, Lucia; Carnevale, Ilaria; Saladini, Serena; Trisciuoglio, Daniela; Gabellini, Chiara; Tardugno, Maria; Zwergel, Clemens; Cencioni, Chiara; Atlante, Sandra; Moniot, Sébastien; Steegborn, Clemens; Budriesi, Roberta; Tafani, Marco; Del Bufalo, Donatella; Altucci, Lucia; Gaetano, Carlo; Mai, Antonello

    2016-02-25

    Modulators of sirtuins are considered promising therapeutic targets for the treatment of cancer, cardiovascular, metabolic, inflammatory, and neurodegenerative diseases. Here we prepared new 1,4-dihydropyridines (DHPs) bearing changes at the C2/C6, C3/C5, C4, or N1 position. Tested with the SIRTainty procedure, some of them displayed increased SIRT1 activation with respect to the prototype 3a, high NO release in HaCat cells, and ameliorated skin repair in a mouse model of wound healing. In C2C12 myoblasts, two of them improved mitochondrial density and functions. All the effects were reverted by coadministration of compound C (9), an AMPK inhibitor, or of EX-527 (10), a SIRT1 inhibitor, highlighting the involvement of the SIRT1/AMPK pathway in the action of DHPs. Finally, tested in a panel of cancer cells, the water-soluble form of 3a, compound 8, displayed antiproliferative effects in the range of 8-35 μM and increased H4K16 deacetylation, suggesting a possible role for SIRT1 activators in cancer therapy.

  18. Effects of oxygen-containing terpenes as skin permeation enhancers on the lipoidal pathways of human epidermal membrane.

    Science.gov (United States)

    Chantasart, Doungdaw; Pongjanyakul, Thaned; Higuchi, William I; Li, S Kevin

    2009-10-01

    The present study investigated the effects of oxygen-containing terpenes as skin permeation enhancers on the lipoidal pathways of human epidermal membrane (HEM). The enhancement (E(HEM)) effects of menthol, thymol, carvacrol, menthone, and cineole on the transport of a probe permeant, corticosterone, across HEM were determined. It was found that the enhancer potencies of menthol, thymol, carvacrol, and menthone were essentially the same and higher than that of cineole based on their aqueous concentration in the diffusion cell chamber at E(HEM) = 4. Thymol and carvacrol also had the same E(HEM) = 10 concentration further supporting that they had the same enhancer potency based on the aqueous concentration. The uptake amounts of terpene into the HEM stratum corneum (SC) intercellular lipid under the same conditions indicate that the intrinsic potencies of the studied terpenes are the same based on their concentration in the SC and similar to those of n-alkanol and n-alkylphenyl alcohol. Moreover, they are all better enhancers compared to branched-chain alkanol. The approximately same uptake enhancement of beta-estradiol induced by the studied terpenes and alcohols at E(HEM) conditions into the SC intercellular lipids suggests that the mechanism of enhancement action for the terpenes and those of alcohols are essentially the same.

  19. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.

    Science.gov (United States)

    Rodriguez-Sinovas, Antonio; Boengler, Kerstin; Cabestrero, Alberto; Gres, Petra; Morente, Miriam; Ruiz-Meana, Marisol; Konietzka, Ina; Miró, Elisabet; Totzeck, Andreas; Heusch, Gerd; Schulz, Rainer; Garcia-Dorado, David

    2006-07-07

    We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.

  20. 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

  1. The classical and alternative pathways of complement activation play distinct roles in spontaneous C3 fragment deposition and membrane attack complex (MAC) formation on human B lymphocytes

    DEFF Research Database (Denmark)

    Leslie, Robert Graham Quinton; Nielsen, Claus Henrik

    2004-01-01

    The contributions of the classical (CP) and alternative (AP) pathways of complement activation to the spontaneous deposition of C3 fragments and the formation of membrane attack complexes (MAC) on human B lymphocytes, were assessed by incubating peripheral blood mononuclear cells with autologous ...... of MAC formation was also found to be highly pathway dependent, with the AP being about 15-fold more efficient at initiating this process than the CP. A model accounting for the effectiveness of the AP in both preserving C3 fragment integrity and initiating MAC is presented....

  2. Phosphorylation: The Molecular Switch of Double-Strand Break Repair

    Directory of Open Access Journals (Sweden)

    K. C. Summers

    2011-01-01

    Full Text Available Repair of double-stranded breaks (DSBs is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ, homologous recombination (HR, or the inclusive DNA damage response (DDR. These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

  3. The journey of DNA repair

    OpenAIRE

    Saini, Natalie

    2015-01-01

    21 years ago, the DNA Repair Enzyme was declared “Molecule of the Year”. Today, we are celebrating another “year of repair”, with the 2015 Nobel Prize in Chemistry being awarded to Aziz Sancar, Tomas Lindahl and Paul Modrich for their collective work on the different DNA repair pathways.

  4. 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

  5. A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

    Directory of Open Access Journals (Sweden)

    Susannah ePiek

    2012-12-01

    Full Text Available The Gram-negative bacterial cell envelope consists of an inner membrane (IM that surrounds the cytoplasm, and an asymmetrical outer-membrane (OM that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS, phospholipids, outer membrane proteins (OMPs and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the correct biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation and isomerisation pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, these conserved pathways have been modified to suit the lifestyle of each organism.

  6. Impairment of the non-homologous end joining and homologous recombination pathways of DNA double strand break repair: Impact on spontaneous and radiation-induced mammary and intestinal tumour risk in Apc min/+ mice.

    Science.gov (United States)

    Haines, Jackie W; Coster, Margaret; Bouffler, Simon D

    2015-11-01

    Female Apc(min/+) mice carrying the BALB/c variant of Prkdc or heterozygous knockout for Xrcc2, were sham- or 2 Gy X-irradiated as adults to compare the effect of mild impairments of double-strand break (DSB) repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR) respectively on spontaneous and radiation-induced mammary and intestinal tumorigenesis. Mice with impaired NHEJ showed no difference in incidence of spontaneous mammary tumours, compared with matched controls, (2.46 fold, P=0.121) and significantly less following irradiation (radiation-induced excess; 0.35 fold, P=0.008). In contrast mice with impaired HR presented with significantly less spontaneous mammary tumours than matched controls (0.33 fold, P=0.027) and significantly more following irradiation (radiation-induced excess; 3.3 fold, P=0.016). Spontaneous and radiation-induced intestinal adenoma multiplicity in the same groups were significantly greater than matched controls for mice with impaired NHEJ (sham; 1.29 fold, P<0.001, radiation-induced excess; 2.55 fold, P<0.001) and mice with impaired HR showed no significant differences (sham; 0.92 fold, P=0.166, radiation-induced excess; 1.16, P=0.274). Genetic insertion events were common in spontaneous tumours from NHEJ impaired mice compared with matched controls. γH2AX foci analysis suggests a significantly faster rate of DSB repair (MANOVA P<0.001) in intestinal than mammary tissue; apoptosis was also higher in irradiated intestine. To conclude, results suggest that pathway of choice for repair of spontaneous and radiation-induced DSBs is influenced by tissue type. NHEJ appears to play a greater role in DSB repair in intestinal tissue since impairment by functional change of Prkdc significantly increases the rate of mis-repair in intestinal but not mammary tissue. HR appears to play a greater role in DSB repair in adult mammary tissue since impaired HR results in significant changes in mammary but not in the intestinal

  7. Membrane-lipid therapy in operation: the HSP co-inducer BGP-15 activates stress signal transduction pathways by remodeling plasma membrane rafts.

    Directory of Open Access Journals (Sweden)

    Imre Gombos

    Full Text Available Aging and pathophysiological conditions are linked to membrane changes which modulate membrane-controlled molecular switches, causing dysregulated heat shock protein (HSP expression. HSP co-inducer hydroxylamines such as BGP-15 provide advanced therapeutic candidates for many diseases since they preferentially affect stressed cells and are unlikely have major side effects. In the present study in vitro molecular dynamic simulation, experiments with lipid monolayers and in vivo ultrasensitive fluorescence microscopy showed that BGP-15 alters the organization of cholesterol-rich membrane domains. Imaging of nanoscopic long-lived platforms using the raft marker glycosylphosphatidylinositol-anchored monomeric green fluorescent protein diffusing in the live Chinese hamster ovary (CHO cell plasma membrane demonstrated that BGP-15 prevents the transient structural disintegration of rafts induced by fever-type heat stress. Moreover, BGP-15 was able to remodel cholesterol-enriched lipid platforms reminiscent of those observed earlier following non-lethal heat priming or membrane stress, and were shown to be obligate for the generation and transmission of stress signals. BGP-15 activation of HSP expression in B16-F10 mouse melanoma cells involves the Rac1 signaling cascade in accordance with the previous observation that cholesterol affects the targeting of Rac1 to membranes. Finally, in a human embryonic kidney cell line we demonstrate that BGP-15 is able to inhibit the rapid heat shock factor 1 (HSF1 acetylation monitored during the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to heat shock elements. Taken together, our results indicate that BGP-15 has the potential to become a new class of pharmaceuticals for use in 'membrane-lipid therapy' to combat many various protein-misfolding diseases associated with aging.

  8. Tendon repair

    Science.gov (United States)

    Repair of tendon ... Tendon repair can be performed using: Local anesthesia (the immediate area of the surgery is pain-free) ... a cut on the skin over the injured tendon. The damaged or torn ends of the tendon ...

  9. Effect of uncoupler on assembly pathway for pigment-binding protein of bacterial photosynthetic membranes. [Rhodobacter capsulatus

    Energy Technology Data Exchange (ETDEWEB)

    Dierstein, R.; Drews, G.

    1986-10-01

    The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) was used to investigate membrane protein assembly in the phototrophic bacterium Rhodobacter capsulatus. As found for Escherichia coli and mitochondrial proteins, assembly across the bacterial photosynthetic membranes was sensitive to CCCP. At uncoupler concentrations which were sufficient to block the export of the periplasmic cytochrome c/sub 2/ and an outer membrane protein, the integration of pigment-binding protein into the photosynthetic apparatus was abolished. The unassembled protein was detected on the inner surface of the intracytoplasmic membrane. After inactivation of CCCP, accumulated protein continued insertion into the membrane. The data suggest that after binding to the cytoplasmic face of the membrane (i), translocation of protein into a transmembrane orientation takes place (ii), which is a prerequisite for the formation of a functional pigment-protein complex (iii).

  10. LncRNA pathway involved in premature preterm rupture of membrane (PPROM: an epigenomic approach to study the pathogenesis of reproductive disorders.

    Directory of Open Access Journals (Sweden)

    Xiucui Luo

    Full Text Available Preterm birth (PTB is a live birth delivered before 37 weeks of gestation (GW. About one-third of PTBs result from the preterm premature rupture of membranes (PPROM. Up to the present, the pathogenic mechanisms underlying PPROM are not clearly understood. Here, we investigated the differential expression of long chain non-coding RNAs (lncRNAs in placentas of PTBs with PPROM, and their possible involvement in the pathogenic pathways leading to PPROM. A total number of 1954, 776, and 1050 lncRNAs were identified with a microarray from placentas of PPROM (group A, which were compared to full-term birth (FTB (group B, PTB (group C, and premature rupture of membrane (PROM (group D at full-term, respectively. Instead of investigating the individual pathogenic role of each lncRNA involved in the molecular mechanism underlying PPROM, we have focused on investigating the metabolic pathways and their functions to explore what is the likely association and how they are possibly involved in the development of PPROM. Six groups, including up-regulation and down-regulation in the comparisons of A vs. B, A vs. C, and A vs. D, of pathways were analyzed. Our results showed that 22 pathways were characterized as up-regulated 7 down-regulated in A vs. C, 18 up-regulated and 15 down-regulated in A vs. D, and 33 up-regulated and 7 down-regulated in A vs. B. Functional analysis showed pathways of infection and inflammatory response, ECM-receptor interactions, apoptosis, actin cytoskeleton, and smooth muscle contraction are the major pathogenic mechanisms involved in the development of PPROM. Characterization of these pathways through identification of lncRNAs opened new avenues for further investigating the epigenomic mechanisms of lncRNAs in PPROM as well as PTB.

  11. 经耳内镜自体脂肪组织修补外伤性鼓膜穿孔%Ear endoscopic autologous fat tissue repairment of traumatic tympanic membrane perforation

    Institute of Scientific and Technical Information of China (English)

    郝中平; 肖嵩; 王杏子

    2014-01-01

    目的:探讨耳内镜下自体脂肪组织在外伤性鼓膜修补术中的临床应用。方法选取来宿州市皖北煤电集团总医院进行治疗的外伤性鼓膜穿孔患者49例,共计52患耳,所有患者均采用耳内镜下自体脂肪组织进行修补治疗。结果49例患者52患耳均顺利完成手术,所有患者均未发生术后并发症。小穿孔患者治愈率﹥中等穿孔患者治愈率﹥大穿孔患者治愈率,且组间比较差异有统计学意义( P﹤0.05)。结论耳内镜下自体脂肪组织鼓膜修补术治疗外伤性鼓膜穿孔能够取得良好的临床疗效,尤其适用于鼓膜穿孔直径较小、中央型穿孔患者的治疗。%Objective To investigate the clinical application value of ear endoscopic autologous fat tissue repairment of trau-matic tympanic membrane perforation. Methods Forty-nine cases of traumatic tympanic membrane perforation admitted in the general hospital of Wanbei coal and electricity group were chose,a total of 52 ears. All of the patients were given ear endoscopic autologous fat tissue repairment of traumatic tympanic membrane perforation. Results The 52 ears of 49 cases were successfully operated,and there were no postoperative complications. The cure rate from high to low in the order:patients with small perfora-tion﹥patients with middle perforation ﹥ patients with big perforation,and there were significant differences( P ﹤ 0. 05 ). Conclusion Ear endoscopic autologous fat tissue repairment of traumatic tympanic membrane perforation has good clinical cura-tive effect,and is especially suitable for the patients with small tympanic membrane perforation diameter and the patients with central type piercing.

  12. Molecular pathways involved in neuronal cell adhesion and membrane scaffolding contribute to schizophrenia and bipolar disorder susceptibility.

    LENUS (Irish Health Repository)

    O'Dushlaine, C

    2011-03-01

    Susceptibility to schizophrenia and bipolar disorder may involve a substantial, shared contribution from thousands of common genetic variants, each of small effect. Identifying whether risk variants map to specific molecular pathways is potentially biologically informative. We report a molecular pathway analysis using the single-nucleotide polymorphism (SNP) ratio test, which compares the ratio of nominally significant (P<0.05) to nonsignificant SNPs in a given pathway to identify the \\'enrichment\\' for association signals. We applied this approach to the discovery (the International Schizophrenia Consortium (n=6909)) and validation (Genetic Association Information Network (n=2729)) of schizophrenia genome-wide association study (GWAS) data sets. We investigated each of the 212 experimentally validated pathways described in the Kyoto Encyclopaedia of Genes and Genomes in the discovery sample. Nominally significant pathways were tested in the validation sample, and five pathways were found to be significant (P=0.03-0.001); only the cell adhesion molecule (CAM) pathway withstood conservative correction for multiple testing. Interestingly, this pathway was also significantly associated with bipolar disorder (Wellcome Trust Case Control Consortium (n=4847)) (P=0.01). At a gene level, CAM genes associated in all three samples (NRXN1 and CNTNAP2), which were previously implicated in specific language disorder, autism and schizophrenia. The CAM pathway functions in neuronal cell adhesion, which is critical for synaptic formation and normal cell signaling. Similar pathways have also emerged from a pathway analysis of autism, suggesting that mechanisms involved in neuronal cell adhesion may contribute broadly to neurodevelopmental psychiatric phenotypes.

  13. Rethinking transcription coupled DNA repair.

    Science.gov (United States)

    Kamarthapu, Venu; Nudler, Evgeny

    2015-04-01

    Nucleotide excision repair (NER) is an evolutionarily conserved, multistep process that can detect a wide variety of DNA lesions. Transcription coupled repair (TCR) is a subpathway of NER that repairs the transcribed DNA strand faster than the rest of the genome. RNA polymerase (RNAP) stalled at DNA lesions mediates the recruitment of NER enzymes to the damage site. In this review we focus on a newly identified bacterial TCR pathway in which the NER enzyme UvrD, in conjunction with NusA, plays a major role in initiating the repair process. We discuss the tradeoff between the new and conventional models of TCR, how and when each pathway operates to repair DNA damage, and the necessity of pervasive transcription in maintaining genome integrity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. DNA Repair and Genome Maintenance in Bacillus subtilis

    OpenAIRE

    2012-01-01

    Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutage...

  15. Molecular pathways involved in neuronal cell adhesion and membrane scaffolding contribute to schizophrenia and bipolar disorder susceptibility.

    Science.gov (United States)

    O'Dushlaine, C; Kenny, E; Heron, E; Donohoe, G; Gill, M; Morris, D; Corvin, A

    2011-03-01

    Susceptibility to schizophrenia and bipolar disorder may involve a substantial, shared contribution from thousands of common genetic variants, each of small effect. Identifying whether risk variants map to specific molecular pathways is potentially biologically informative. We report a molecular pathway analysis using the single-nucleotide polymorphism (SNP) ratio test, which compares the ratio of nominally significant (PSchizophrenia Consortium (n=6909)) and validation (Genetic Association Information Network (n=2729)) of schizophrenia genome-wide association study (GWAS) data sets. We investigated each of the 212 experimentally validated pathways described in the Kyoto Encyclopaedia of Genes and Genomes in the discovery sample. Nominally significant pathways were tested in the validation sample, and five pathways were found to be significant (P=0.03-0.001); only the cell adhesion molecule (CAM) pathway withstood conservative correction for multiple testing. Interestingly, this pathway was also significantly associated with bipolar disorder (Wellcome Trust Case Control Consortium (n=4847)) (P=0.01). At a gene level, CAM genes associated in all three samples (NRXN1 and CNTNAP2), which were previously implicated in specific language disorder, autism and schizophrenia. The CAM pathway functions in neuronal cell adhesion, which is critical for synaptic formation and normal cell signaling. Similar pathways have also emerged from a pathway analysis of autism, suggesting that mechanisms involved in neuronal cell adhesion may contribute broadly to neurodevelopmental psychiatric phenotypes.

  16. Processo de reparação de lesões da córnea e a membrana amniótica na oftalmologia Repair process of corneal damage and the amniotic membrane in ophthalmology

    Directory of Open Access Journals (Sweden)

    Kelly Cristine de Sousa Pontes

    2011-12-01

    Full Text Available Os eventos que fazem parte do processo de reparação de lesões da córnea ocorrem simultaneamente e envolvem proliferação, migração, diferenciação e apoptose celular, além da comunicação intercelular. Vários fatores solúveis, além de proteínas da matriz mesenquimal, proteoglicanos, enzimas proteolíticas e alguns tipos celulares são abordados nesta revisão, na qual explicam-se os processos de reparação de lesões superficiais ou penetrantes da córnea. A membrana amniótica, muito utilizada na cirurgia oftálmica, foi estudada por apresentar funções que colaboram com o processo de reparação. Entretanto, tais funções poderão ser perdidas quando tal tecido for submetido à conservação. Assim, torna-se importante conhecer o processo de reparação de lesões que envolvem, ou não, a córnea em toda a sua espessura e escolher a melhor forma de utilização da membrana amniótica quando ela for indicada na terapia para estas lesões.The events included in the process of repair of corneal damage occur simultaneously and involve proliferation, migration, differentiation, cell apoptosis and intercellular communication. Several soluble factors, mesenchymal matrix proteins, proteoglycans, proteolytic enzymes and some cell types are covered in this review, which explains the processes of repair of corneal wounds, either superficial or penetrating. The amniotic membrane, used in ophthalmic surgery, was studied because of the contribution of its functions to the repair process. However, these functions may be lost when the amniotic membrane is subjected to conservation. Therefore, it is important to understand the repair process of lesions involving or not the entire thickness of the cornea, and choose the best use of the amniotic membrane, when it is indicated for the treatment of these lesions.

  17. Interaction between small GTPase Rab7 and PI3KC3 links autophagy and endocytosis: A new Rab7 effector protein sheds light on membrane trafficking pathways.

    Science.gov (United States)

    Lin, Mary Grace; Zhong, Qing

    2011-03-01

    Endocytosis and autophagy are both membrane trafficking pathways vital for cell survival. Endocytosis, the primary means by which cells internalize material such as cell-surface receptors and their protein ligands, is essential for proper cell growth and communication. Autophagy is a catabolic process that degrades cargo ranging from organelles to protein aggregates to bacteria, and it is important for maintaining cellular homeostasis. Defects in both endosome and autophagosome maturation lead to an array of human diseases, including cancer; however, the molecular mechanisms underlying endosome and autophagosome maturation are not well characterized. In the case of endocytosis, small GTPases, key players in membrane organization, are required for endosome maturation. Specifically, activation of the small GTPase Rab7 is required for the initiation of the early-to-late endosome transition, although how this is regulated is largely unknown. Now recent findings from our laboratory show that Rubicon, a component of the PI3KC3 complex, inhibits endosome maturation by preventing activation of Rab7. Not only do our results clarify the molecular link between PI3KC3 and Rab7 function in endosome maturation, they lead us to propose new models for PI3KC3 involvement in membrane trafficking, particularly at the convergence between the endosome and autophagosome pathways.

  18. DNA repair deficiency in neurodegeneration

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A; Stevnsner, Tinna V.

    2011-01-01

    : homologous recombination and non-homologous end-joining. Ataxia telangiectasia and related disorders with defects in these pathways illustrate that such defects can lead to early childhood neurodegeneration. Aging is a risk factor for neurodegeneration and accumulation of oxidative mitochondrial DNA damage......Deficiency in repair of nuclear and mitochondrial DNA damage has been linked to several neurodegenerative disorders. Many recent experimental results indicate that the post-mitotic neurons are particularly prone to accumulation of unrepaired DNA lesions potentially leading to progressive...... neurodegeneration. Nucleotide excision repair is the cellular pathway responsible for removing helix-distorting DNA damage and deficiency in such repair is found in a number of diseases with neurodegenerative phenotypes, including Xeroderma Pigmentosum and Cockayne syndrome. The main pathway for repairing oxidative...

  19. Bladder exstrophy repair

    Science.gov (United States)

    Bladder birth defect repair; Everted bladder repair; Exposed bladder repair; Repair of bladder exstrophy ... Bladder exstrophy repair involves two surgeries. The first surgery is to repair the bladder and the second one is to attach ...

  20. 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

    , 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......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.......T. Tomicic, W.P. Roos, B. Kaina, Mechanisms of human DNA repair: an update, Toxicology 193 (2003) 3-34; N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89-108]. Protein interactions are not only important for function, but also...

  1. Analysis of the Durability of PEM FC Membrane Electrode Assemblies in Automotive Applications through the Fundamental Understanding of Membrane and MEA Degradation Pathways

    Energy Technology Data Exchange (ETDEWEB)

    Perry, Randal L. [DuPont

    2013-10-31

    The Project focused on mitigation of degradation processes on membrane electrode assemblies. The approach was to develop a model to improve understanding of the mechanisms, and to use it to focus mitigation strategies. The detailed effects of various accelerated stress tests (ASTs) were evaluated to determine the best subset to use in model development. A combination of ASTs developed by the Fuel Cell Commercialization Conference of Japan and the Fuel Cell Tech Team were selected for use. The ASTs were compared by measuring effects on performance, running in-situ diagnostics, and performing microscopic analyses of the membrane electrode assemblies after the stress tests were complete. Nissan ran FCCJ AST protocols and performed in situ and ex-situ electrochemical testing. DuPont ran FCTT and USFCC AST protocols, performed scanning and transmission electron microscopy and ran in-situ electrochemical tests. Other ex-situ testing was performed by IIT, along with much of the data analysis and model development. These tests were then modified to generate time-dependent data of the degradation mechanisms. Three different catalyst types and four membrane variants were then used to generate data for a theoretically-based degradation model. An important part of the approach was to use commercially available materials in the electrodes and membranes made in scalable semiworks processes rather than lab-based materials. This constraint ensured all materials would be practicable for full-scale testing. The initial model for the electrode layer was tested for internal consistency and agreement with the data. A Java-based computer application was developed to analyze the time-dependent AST data using polarization curves with four different cathode gas feeds and generate model parameters. Data showed very good reproducibility and good consistency as cathode catalyst loadings were varied. At the point of termination of the project, a basic electrode model was in hand with several

  2. Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin.

    Science.gov (United States)

    Collin, Séverine; Guilvout, Ingrid; Nickerson, Nicholas N; Pugsley, Anthony P

    2011-05-01

    The lipoprotein PulS is a dedicated chaperone that is required to target the secretin PulD to the outer membrane in Klebsiella or Escherichia coli, and to protect it from proteolysis. Here, we present indirect evidence that PulD protomers do not assemble into the secretin dodecamer before they reach the outer membrane, and that PulS reaches the outer membrane in a soluble heterodimer with the general lipoprotein chaperone LolA. However, we could not find any direct evidence for PulD protomer association with the PulS-LolA heterodimer. Instead, in cells producing PulD and a permanently locked PulS-LolA dimer (in which LolA carries an R43L substitution that prevents lipoprotein transfer to LolB in the outer membrane), LolAR43L was found in the inner membrane, probably still associated with PulS bound to PulD that had been incorrectly targeted because of the LolAR43L substitution. It is speculated that PulD protomers normally cross the periplasm together with PulS bound to LolA but when the latter cannot be separated (due to the mutation in lolA), the PulD protomers form dodecamers that insert into the inner membrane.

  3. Eps15 is recruited to the plasma membrane upon epidermal growth factor receptor activation and localizes to components of the endocytic pathway during receptor internalization

    DEFF Research Database (Denmark)

    Torrisi, M R; Lotti, L V; Belleudi, F;

    1999-01-01

    Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential...... role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments...... of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment...

  4. Matrix metalloproteinases in wound repair (review).

    Science.gov (United States)

    Ravanti, L; Kähäri, V M

    2000-10-01

    Wound repair is initiated with the aggregation of platelets, formation of a fibrin clot, and release of growth factors from the activated coagulation pathways, injured cells, platelets, and extracellular matrix (ECM), followed by migration of inflammatory cells to the wound site. Thereafter, keratinocytes migrate over the wound, angiogenesis is initiated, and fibroblasts deposit and remodel the granulation tissue. Cell migration, angiogenesis, degradation of provisional matrix, and remodeling of newly formed granulation tissue, all require controlled degradation of the ECM. Disturbance in the balance between ECM production and degradation leads to formation of chronic ulcers with excessive ECM degradation, or to fibrosis, for example hypertrophic scars or keloids characterized by excessive accumulation of ECM components. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which as a group can degrade essentially all ECM components. So far, 20 members of the human MMP family have been identified. Based on their structure and substrate specificity, they can be divided into subgroups of collagenases, stromelysins, stromelysin-like MMPs, gelatinases, membrane-type MMPs (MT-MMPs), and other MMPs. In this review, the role of MMPs in normal wound repair as well as in chronic ulcers is discussed. In addition, the role of signaling pathways, in particular, mitogen-activated protein kinases (MAPKs) in regulating MMP expression is discussed as possible therapeutical targets for wound healing disorders.

  5. Targeting base excision repair as a sensitization strategy in radiotherapy.

    NARCIS (Netherlands)

    Vens, C.; Begg, A.C.

    2010-01-01

    Cellular DNA repair determines survival after ionizing radiation. Human tumors commonly exhibit aberrant DNA repair since they drive mutagenesis and chromosomal instability. Recent reports have shown alterations in the base excision repair (BER) and single strand break repair (SSBR) pathways in huma

  6. T tubules and surface membranes provide equally effective pathways of carbonic anhydrase-facilitated lactic acid transport in skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Janine Hallerdei

    Full Text Available We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV-CAIX-CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.

  7. The clinical pathway of tension-free inguinal hernia repair under local anesthesia%局部麻醉下腹股沟疝无张力修补术临床路径及其应用

    Institute of Scientific and Technical Information of China (English)

    石华伟; 汤汉林; 姜海平; 祁应才; 李新军

    2014-01-01

    Objective To explore the clinical pathway of tension-free inguinal hernia repair under local anesthesia.Methods The clinical pathway of tension-free inguinal hernia repair was set up under local anesthesia.Time of hospitalization stay,hospitalization costs and complications were observed in 56 cases.Results There were totally 56 cases with 54 cases (96.4%)who completed the clinical pathway and 2 cases were withdrawal.Time of hospitalization stay was shortened and the costs were reduced in the cases with clinical pathway.Fourteen cases had postoperative short-term complications without recurrence after treatment.Conclusions The clinical pathway of tension-free inguinal hernia repair under local anesthesia is easy to operate,which can significantly shorten the time of hospitalization,reduce the medical costs,and reduce the patient and social burdens.%目的:探讨局部麻醉下腹股沟疝无张力修补术的临床路径及其应用。方法观察局部麻醉下腹股沟疝无张力修补术临床路径的56例患者住院时间、住院各项费用及并发症情况。结果进入临床路径56例患者中,54例完成临床路径,2例退出,完成率为96.4%;纳入临床路径的患者住院时间明显缩短,各项费用显著降低;有14例患者出现术后近期并发症,无复发。结论局部麻醉下腹股沟疝无张力修补术临床路径操作简单,能明显缩短住院时间、降低住院医疗成本,减轻患者和社会负担,值得推广。

  8. Gene and pathway level analyses of germline DNA-repair gene variants and prostate cancer susceptibility using the iCOGS-genotyping array

    DEFF Research Database (Denmark)

    Saunders, Edward J; Dadaev, Tokhir; Leongamornlert, Daniel A

    2016-01-01

    BACKGROUND: Germline mutations within DNA-repair genes are implicated in susceptibility to multiple forms of cancer. For prostate cancer (PrCa), rare mutations in BRCA2 and BRCA1 give rise to moderately elevated risk, whereas two of B100 common, low-penetrance PrCa susceptibility variants identif...

  9. X-ray repair cross complementing protein 1 in base excision repair

    DEFF Research Database (Denmark)

    Hanssen-Bauer, Audun; Solvang-Garten, Karin; Akbari, Mansour;

    2012-01-01

    X-ray Repair Cross Complementing protein 1 (XRCC1) acts as a scaffolding protein in the converging base excision repair (BER) and single strand break repair (SSBR) pathways. XRCC1 also interacts with itself and rapidly accumulates at sites of DNA damage. XRCC1 can thus mediate the assembly of large...

  10. An siRNA screen of membrane trafficking genes highlights pathways common to HIV-1 and M-PMV virus assembly and release.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Wen

    Full Text Available The assembly and release of retroviruses from the host cells requires a coordinated series of interactions between viral structural proteins and cellular trafficking pathways. Although a number of cellular factors involved in retrovirus assembly have been identified, it is likely that retroviruses utilize additional trafficking factors to expedite their assembly and budding that have not yet been defined. We performed a screen using an siRNA library targeting host membrane trafficking genes in order to identify new host factors that contribute to retrovirus assembly or release. We utilized two retroviruses that follow very distinct assembly pathways, HIV-1 and Mason-Pfizer monkey virus (M-PMV in order to identify host pathways that are generally applicable in retrovirus assembly versus those that are unique to HIV or M-PMV. Here we report the identification of 24 host proteins identified in the screen and subsequently validated in follow-up experiments as contributors to the assembly or release of both viruses. In addition to identifying a number of previously unsuspected individual trafficking factors, we noted multiple hits among proteins involved in modulation of the actin cytoskeleton, clathrin-mediated transport pathways, and phosphoinositide metabolism. Our study shows that distant genera of retroviruses share a number of common interaction strategies with host cell trafficking machinery, and identifies new cellular factors involved in the late stages of retroviral replication.

  11. Initial steps of the base excision repair pathway within the nuclear architecture; Les etapes initiales du mecanisme de reparation par excision de bases au sein de l'architecture nucleaire

    Energy Technology Data Exchange (ETDEWEB)

    Amouroux, R

    2009-09-15

    Oxidative stress induced lesions threaten aerobic organisms by representing a major cause of genomic instability. A common product of guanine oxidation, 8-oxo-guanine (8- oxoG) is particularly mutagenic by provoking G to T transversions. Removal of oxidised bases from DNA is initiated by the recognition and excision of the damaged base by a DNA glycosylase, initiating the base excision repair (BER) pathway. In mammals, 8-oxoG is processed by the 8-oxoG-DNA-glycosylase I (OGG1), which biochemical mechanisms has been well characterised in vitro. However how and where this enzyme finds the modified base within the complex chromatin architecture is not yet understood. We show that upon induction of 8-oxoG, OGG1, together with at least two other proteins involved in BER, is recruited from a soluble fraction to chromatin. Formation kinetics of this patches correlates with 8-oxoG excision, suggesting a direct link between presence of this chromatin-associated complexes and 8-oxoG repair. More precisely, these repair patches are specifically directed to euchromatin regions, and completely excluded from heterochromatin regions. Inducing of artificial chromatin compaction results in a complete inhibition of the in vivo repair of 8-oxoG, probably by impeding the access of OGG1 to the lesion. Using OGG1 mutants, we show that OGG1 direct recognition of 8-oxoG did not trigger its re-localisation to the chromatin. We conclude that in response to the induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions. (author)

  12. A vacuolar membrane protein affects drastically the biosynthesis of the ACV tripeptide and the beta-lactam pathway of Penicillium chrysogenum.

    Science.gov (United States)

    Fernández-Aguado, Marta; Teijeira, Fernando; Martín, Juan F; Ullán, Ricardo V

    2013-01-01

    The knowledge about enzymes' compartmentalization and transport processes involved in the penicillin biosynthesis in Penicillium chrysogenum is very limited. The genome of this fungus contains multiple genes encoding transporter proteins, but very little is known about them. A bioinformatic search was made to find major facilitator supefamily (MFS) membrane proteins related to CefP transporter protein involved in the entry of isopenicillin N to the peroxisome in Acremonium chrysogenum. No strict homologue of CefP was observed in P. chrysogenum, but the penV gene was found to encode a membrane protein that contained 10 clear transmembrane spanners and two other motifs COG5594 and DUF221, typical of membrane proteins. RNAi-mediated silencing of penV gene provoked a drastic reduction of the production of the δ-(L-α-aminoadipyl-L-cysteinyl-D-valine) (ACV) and isopenicillin N intermediates and the final product of the pathway. RT-PCR and northern blot analyses confirmed a reduction in the expression levels of the pcbC and penDE biosynthetic genes, whereas that of the pcbAB gene increased. Localization studies by fluorescent laser scanning microscopy using Dsred and GFP fluorescent fusion proteins and the FM 4-64 fluorescent dye showed clearly that the protein was located in the vacuolar membrane. These results indicate that PenV participates in the first stage of the beta-lactam biosynthesis (i.e., the formation of the ACV tripeptide), probably taking part in the supply of amino acids from the vacuolar lumen to the vacuole-anchored ACV synthetase. This is in agreement with several reports on the localization of the ACV synthetase and provides increased evidence for a compartmentalized storage of precursor amino acids for non-ribosomal peptides. PenV is the first MFS transporter of P. chrysogenum linked to the beta-lactam biosynthesis that has been located in the vacuolar membrane.

  13. On the importance of electrostatic interactions between cell penetrating peptides and membranes: a pathway toward tumor cell selectivity?

    Science.gov (United States)

    Jobin, Marie-Lise; Alves, Isabel D

    2014-12-01

    Cell-penetrating peptides (CPPs) are small molecules of major interest due to their ability to efficiently transport cargos across cell membranes in a receptor- and energy-independent way and without being cytotoxic to cells. Since their discovery 20 years ago their potential interest in drug delivery and diagnosis became undeniable. CPPs are being used to deliver inside cells a large variety of cargos such as proteins, DNA, antibodies, imaging agents and nanoparticle drug carriers. Their cellular uptake mechanisms are still debated and may vary depending on their structure, nature and size of cargo they transport and type of cell line targeted. CPPs are generally rich in positively charged residues, thus they are prone to establish electrostatic interactions with anionic membrane components (sugars and lipids). Understanding the molecular basis of CPP membrane interaction and cellular uptake is crucial to improve their in vivo efficiency target-specificity. A great number of studies demonstrated the high potential of CPPs to translocate efficiently therapeutic cargos into cells and some peptides are even in clinical phase studies. Although these molecules seem perfect for a therapeutic or diagnosis purpose, they still possess a small but non negligible drawback: a complete lack of cell type specificity. Tumor cells have recently been shown to over-express certain glycosaminoglycans at the cell membrane surface and to possess a higher amount of anionic lipids in their outer leaflet than healthy cells. Such molecules confer the cell membrane an enhanced anionic character, property that could be used by CPPs to selectively target these cells. Moreover previous studies demonstrate the importance of electrostatic interactions between basic residues in the peptide, especially Arg, and the lipid headgroups and glycosaminoglycans in the cell membrane. Electrostatic interactions put at stake in this process might be one of the keys to resolve the puzzle of CPP cell type

  14. The use of amniotic membrane in the repair of duodenal wounds in Wistar rats Uso da membrana amniótica no reparo de feridas duodenais em ratos Wistar

    Directory of Open Access Journals (Sweden)

    Luciano Rodrigues Schimidt

    2010-02-01

    Full Text Available Purpose: In the search of a new material to repair duodenal wounds, a trial was conducted to assess the behavior of human amniotic membrane in the repair of the duodenal wall in rats. METHODS: Fifty Wistar rats weighing between 250 and 350g, male, were submitted to duodenotomy and randomly distributed into two groups. Group A (n=8 had no treatment and was used as the control group. In Group B (n=42 the duodenal wound was treated with a patch of human amniotic membrane. RESULTS: All animals in Group A died. In Group B no changes were observed with regards to death or the formation of duodenal fistula. All animals presented peritoneal adherences in the region on the duodenal wall repair and intestinal obstruction was observed in two animals. Healing of the duodenal wall in the region of the patch took place progressively as the post-operatory period increased, with regeneration of the mucosa and of the smooth muscle layer. CONCLUSION: From the clinical standpoint, the amniotic membrane proved to be a biological tissue which served as a temporary seal and allowed the wound to heal by second-intention, with re-establishment of the duodenal wall structure.OBJETIVO: Na busca de um novo material para o reparo dos ferimentos duodenais, foi efetuado um estudo para avaliar o comportamento da membrana amniótica humana no reparo da parede duodenal em ratos. MÉTODOS: Foram utilizados 50 ratos Wistar, com peso entre 250 e 350g, machos, distribuídos, aleatoriamente, em dois grupos. Grupo A (n=8, submetido à duodenotomia sem tratamento, utilizados como controle. Grupo B (n=42, submetido a um remendo de membrana amniótica humana para tratamento de ferimento duodenal provocado. RESULTADOS: Todos os animais do grupo A foram a óbito. No grupo B não foram observadas alterações quanto a óbito ou formação de fístula duodenal. Observaram-se em todos os animais aderências peritoneais à região do reparo da parede do duodeno e obstrução intestinal em

  15. SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY-BASED MICROSTRUCTURAL ANALYSIS OF RETINAL ARCHITECTURE POST INTERNAL LIMITING MEMBRANE PEELING FOR SURGERY OF IDIOPATHIC MACULAR HOLE REPAIR.

    Science.gov (United States)

    Modi, Aditya; Giridhar, Anantharaman; Gopalakrishnan, Mahesh

    2017-02-01

    Spectral domain optical coherence tomography-based analysis of retinal architecture after internal limiting membrane peeling for macular hole surgery. Prospective, interventional study. Fifty eyes underwent the surgical procedure with minimum internal limiting membrane peel of 3 mm diameter. Automatic segmentation software was used to assess individual layers preoperatively and postoperatively, 1.5 millimeters medial and lateral to fovea at 3 months postoperative visit. Main outcome measures were final central macular thickness and variation in individual retinal layer thickness. Mean central macular thickness postoperatively was 201 microns. Retinal thickening was observed, 1.5 mm medial to fovea (P Internal limiting membrane peel is associated with significant alteration in inner retinal architecture, especially in ganglion cell layer, which can adversely influence functional outcome of the surgery and makes it imperative to avoid peeling internal limiting membrane over a larger surface area.

  16. A membrane microdomain-associated protein, Arabidopsis Flot1, is involved in a clathrin-independent endocytic pathway and is required for seedling development.

    Science.gov (United States)

    Li, Ruili; Liu, Peng; Wan, Yinglang; Chen, Tong; Wang, Qinli; Mettbach, Ursula; Baluska, Frantisek; Samaj, Jozef; Fang, Xiaohong; Lucas, William J; Lin, Jinxing

    2012-05-01

    Endocytosis is essential for the maintenance of protein and lipid compositions in the plasma membrane and for the acquisition of materials from the extracellular space. Clathrin-dependent and -independent endocytic processes are well established in yeast and animals; however, endocytic pathways involved in cargo internalization and intracellular trafficking remain to be fully elucidated for plants. Here, we used transgenic green fluorescent protein-flotillin1 (GFP-Flot1) Arabidopsis thaliana plants in combination with confocal microscopy analysis and transmission electron microscopy immunogold labeling to study the spatial and dynamic aspects of GFP-Flot1-positive vesicle formation. Vesicle size, as outlined by the gold particles, was ∼100 nm, which is larger than the 30-nm size of clathrin-coated vesicles. GFP-Flot1 also did not colocalize with clathrin light chain-mOrange. Variable-angle total internal reflection fluorescence microscopy also revealed that the dynamic behavior of GFP-Flot1-positive puncta was different from that of clathrin light chain-mOrange puncta. Furthermore, disruption of membrane microdomains caused a significant alteration in the dynamics of Flot1-positive puncta. Analysis of artificial microRNA Flot1 transgenic Arabidopsis lines established that a reduction in Flot1 transcript levels gave rise to a reduction in shoot and root meristem size plus retardation in seedling growth. Taken together, these findings support the hypothesis that, in plant cells, Flot1 is involved in a clathrin-independent endocytic pathway and functions in seedling development.

  17. Binding of Alphaherpesvirus Glycoprotein H to Surface α4β1-Integrins Activates Calcium-Signaling Pathways and Induces Phosphatidylserine Exposure on the Plasma Membrane

    Science.gov (United States)

    Gramatica, Andrea; Herrmann, Andreas; Osterrieder, Nikolaus

    2015-01-01

    ABSTRACT Intracellular signaling connected to integrin activation is known to induce cytoplasmic Ca2+ release, which in turn mediates a number of downstream signals. The cellular entry pathways of two closely related alphaherpesviruses, equine herpesviruses 1 and 4 (EHV-1 and EHV-4), are differentially regulated with respect to the requirement of interaction of glycoprotein H (gH) with α4β1-integrins. We show here that binding of EHV-1, but not EHV-4, to target cells resulted in a rapid and significant increase in cytosolic Ca2+ levels. EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca2+ release, while EHV-4 with gH1 triggered significant Ca2+ release. Blocking the interaction between gH1 and α4β1-integrins, inhibiting phospholipase C (PLC) activation, or blocking binding of inositol 1,4,5-triphosphate (IP3) to its receptor on the endoplasmic reticulum (ER) abrogated Ca2+ release. Interestingly, phosphatidylserine (PS) was exposed on the plasma membrane in response to cytosolic calcium increase after EHV-1 binding through a scramblase-dependent mechanism. Inhibition of both Ca2+ release from the ER and scramblase activation blocked PS scrambling and redirected virus entry to the endocytic pathway, indicating that PS may play a role in facilitating virus entry directly at the plasma membrane. PMID:26489864

  18. Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways.

    Science.gov (United States)

    Rival, Thomas; Macchi, Marc; Arnauné-Pelloquin, Laetitia; Poidevin, Mickael; Maillet, Frédéric; Richard, Fabrice; Fatmi, Ahmed; Belenguer, Pascale; Royet, Julien

    2011-03-01

    Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.

  19. The cholesterol-dependent cytolysin signature motif: a critical element in the allosteric pathway that couples membrane binding to pore assembly.

    Directory of Open Access Journals (Sweden)

    Kelley J Dowd

    Full Text Available The cholesterol-dependent cytolysins (CDCs constitute a family of pore-forming toxins that contribute to the pathogenesis of a large number of Gram-positive bacterial pathogens.The most highly conserved region in the primary structure of the CDCs is the signature undecapeptide sequence (ECTGLAWEWWR. The CDC pore forming mechanism is highly sensitive to changes in its structure, yet its contribution to the molecular mechanism of the CDCs has remained enigmatic. Using a combination of fluorescence spectroscopic methods we provide evidence that shows the undecapeptide motif of the archetype CDC, perfringolysin O (PFO, is a key structural element in the allosteric coupling of the cholesterol-mediated membrane binding in domain 4 (D4 to distal structural changes in domain 3 (D3 that are required for the formation of the oligomeric pore complex. Loss of the undecapeptide function prevents all measurable D3 structural transitions, the intermolecular interaction of membrane bound monomers and the assembly of the oligomeric pore complex. We further show that this pathway does not exist in intermedilysin (ILY, a CDC that exhibits a divergent undecapeptide and that has evolved to use human CD59 rather than cholesterol as its receptor. These studies show for the first time that the undecapeptide of the cholesterol-binding CDCs forms a critical element of the allosteric pathway that controls the assembly of the pore complex.

  20. Cellular processes and pathways that protect Saccharomyces cerevisiae cells against the plasma membrane-perturbing compound chitosan.

    NARCIS (Netherlands)

    Zakrzewska, A.M.; Boorsma, A.; Delneri, D.; Brul, S.; Oliver, S.G.; Klis, F.M.

    2007-01-01

    Global fitness analysis makes use of a genomic library of tagged deletion strains. We used this approach to study the effect of chitosan, which causes plasma membrane stress. The data were analyzed using T-profiler, which was based on determining the sensitivities of groups of deletion strains to ch

  1. Cellular processes and pathways that protect Saccharomyces cerevisiae cells against the plasma membrane-perturbing compound chitosan.

    NARCIS (Netherlands)

    Zakrzewska, A.M.; Boorsma, A.; Delneri, D.; Brul, S.; Oliver, S.G.; Klis, F.M.

    2007-01-01

    Global fitness analysis makes use of a genomic library of tagged deletion strains. We used this approach to study the effect of chitosan, which causes plasma membrane stress. The data were analyzed using T-profiler, which was based on determining the sensitivities of groups of deletion strains to

  2. Binding of alphaherpesvirus glycoprotein H to surface α4β1-integrins activates calcium-signaling pathways and induces phosphatidylserine exposure on the plasma membrane.

    Science.gov (United States)

    Azab, Walid; Gramatica, Andrea; Herrmann, Andreas; Osterrieder, Nikolaus

    2015-10-20

    Intracellular signaling connected to integrin activation is known to induce cytoplasmic Ca(2+) release, which in turn mediates a number of downstream signals. The cellular entry pathways of two closely related alphaherpesviruses, equine herpesviruses 1 and 4 (EHV-1 and EHV-4), are differentially regulated with respect to the requirement of interaction of glycoprotein H (gH) with α4β1-integrins. We show here that binding of EHV-1, but not EHV-4, to target cells resulted in a rapid and significant increase in cytosolic Ca(2+) levels. EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca(2+) release, while EHV-4 with gH1 triggered significant Ca(2+) release. Blocking the interaction between gH1 and α4β1-integrins, inhibiting phospholipase C (PLC) activation, or blocking binding of inositol 1,4,5-triphosphate (IP3) to its receptor on the endoplasmic reticulum (ER) abrogated Ca(2+) release. Interestingly, phosphatidylserine (PS) was exposed on the plasma membrane in response to cytosolic calcium increase after EHV-1 binding through a scramblase-dependent mechanism. Inhibition of both Ca(2+) release from the ER and scramblase activation blocked PS scrambling and redirected virus entry to the endocytic pathway, indicating that PS may play a role in facilitating virus entry directly at the plasma membrane. Herpesviruses are a large family of enveloped viruses that infect a wide range of hosts, causing a variety of diseases. These viruses have developed a number of strategies for successful entry into different cell types. We and others have shown that alphaherpesviruses, including EHV-1 and herpes simplex virus 1 (HSV-1), can route their entry pathway and do so by manipulation of cell signaling cascades to ensure viral genome delivery to nuclei. We show here that the interaction between EHV-1 gH and cellular α4β1-integrins is necessary to induce emptying of ER calcium stores, which induces phosphatidylserine exposure on the plasma membrane

  3. Construction of nerve guide conduits from cellulose/soy protein composite membranes combined with Schwann cells and pyrroloquinoline quinone for the repair of peripheral nerve defect

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Lihua [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Center of Molecular Medicine, School of Medicine, Hubei University of Arts and Sciences, Xiangyang 441053 (China); Gan, Li; Liu, Yongming; Tian, Weiqun; Tong, Zan [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Wang, Xiong; Huselstein, Celine [Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), UMR 7365 CNRS – Université de Lorraine, Biopôle, 54500 Vandoeuvre-lès-Nancy (France); Chen, Yun, E-mail: yunchen@whu.edu.cn [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China)

    2015-02-20

    Regeneration and functional reconstruction of peripheral nerve defects remained a significant clinical challenge. Nerve guide conduits, with seed cells or neurotrophic factors (NTFs), had been widely used to improve the repair and regeneration of injured peripheral nerve. Pyrroloquinoline quinone (PQQ) was an antioxidant that can stimulate nerve growth factors (NGFs) synthesis and accelerate the Schwann cells (SCs) proliferation and growth. In present study, three kinds of nerve guide conduits were constructed: one from cellulose/SPI hollow tube (CSC), another from CSC combined with SCs (CSSC), and the third one from CSSC combined with PQQ (CSSPC), respectively. And then they were applied to bridge and repair the sciatic nerve defect in rats, using autograft as control. Effects of different nerve guide conduits on the nerve regeneration were comparatively evaluated by general analysis, sciatic function index (SFI) and histological analysis (HE and TEM). Newly-formed regenerative nerve fibers were observed and running through the transparent nerve guide conduits 12 weeks after surgery. SFI results indicated that the reconstruction of motor function in CSSPC group was better than that in CSSC and CSC groups. HE images from the cross-sections and longitudinal-sections of the harvested regenerative nerve indicated that regenerative nerve fibers had been formed and accompanied with new blood vessels and matrix materials in the conduits. TEM images also showed that lots of fresh myelinated and non-myelinated nerve fibers had been formed. Parts of vacuolar, swollen and abnormal axons occurred in CSC and CSSC groups, while the vacuolization and swell of axons was the least serious in CSSPC group. These results indicated that CSSPC group had the most ability to repair and reconstruct the nerve structure and functions due to the comprehensive contributions from hollow CSC tube, SCs and PQQ. As a result, the CSSPC may have the potential for the applications as nerve guide

  4. On the pathway of cellular uptake: new insight into the interaction between the cell membrane and very small nanoparticles

    Directory of Open Access Journals (Sweden)

    Claudia Messerschmidt

    2016-09-01

    Full Text Available For any living cell the exchange with its environment is vital. Therefore, many different kinds of cargo are able to enter cells via energy-dependent or -independent routes. Nanoparticles are no exemption. It is known that small silica nanoparticles with a diameter below 50 nm are taken up by cells and that their uptake exerts pronounced toxic effects beyond a certain concentration threshold. However, neither the exact uptake mechanism of these particles nor the actual reason for their toxicity has yet been elucidated. In this study we examined the uptake of silica nanoparticles with a diameter of 7, 12 and 22 nm by means of transmission electron microscopy, accompanied by toxicological assays. We show that for every particle diameter tested a different membrane morphology during uptake can be observed and that the amount of particles entering in one event is different for the three sizes. Silica particles with a diameter of 22 nm show single-particle internalization with a membrane wrapped around the particles in the cytosol, whereas 12 nm particles display row-like multi-particle uptake into elongated membrane structures and those with a diameter of 7 nm or less end up in tubular endocytic structures containing many particles. These membrane morphologies proved to be highly reproducible as we found them in five different cell lines. Additionally, we performed ATP and LDH assays to determine particle toxicity. Exceeding a certain concentration threshold the nanoparticles showed a high toxic potential both in the biochemical assay measurements and from morphological findings. We could not find any hint at the induction of apoptosis, neither morphologically nor biochemically. In this regard we discuss membrane damage and consumption as one possible mechanism of toxicity, linking morphological observations to toxicological findings to bridge the gap in understanding the mechanism of toxicity of small nanoparticles.

  5. Group B streptococcal infection of the choriodecidua induces dysfunction of the cytokeratin network in amniotic epithelium: a pathway to membrane weakening.

    Directory of Open Access Journals (Sweden)

    Jeroen P Vanderhoeven

    2014-03-01

    Full Text Available Early events leading to intrauterine infection remain poorly defined, but may hold the key to preventing preterm delivery. To determine molecular pathways within fetal membranes (chorioamnion associated with early choriodecidual infection that may progress to preterm premature rupture of membranes (PPROM, we examined the effects of a Group B Streptococcus (GBS choriodecidual infection on chorioamnion in a nonhuman primate model. Ten chronically catheterized pregnant monkeys (Macaca nemestrina at 118-125 days gestation (term = 172 days received choriodecidual inoculation of either GBS (n = 5 or saline (n = 5. Cesarean section was performed in the first week after GBS or saline inoculation. RNA extracted from chorioamnion (inoculation site was profiled by microarray. Single gene, Gene Set, and Ingenuity Pathway Analysis results were validated using qRT-PCR (chorioamnion, Luminex (amniotic fluid, AF, immunohistochemistry, and transmission electron microscopy (TEM. Despite uterine quiescence in most cases, significant elevations of AF cytokines (TNF-α, IL-8, IL-1β, IL-6 were detected in GBS versus controls (p2-fold change, p<0.05. Remarkably, GBS exposure was associated with significantly downregulated expression of multiple cytokeratin (CK and other cytoskeletal genes critical for maintenance of tissue tensile strength. Immunofluorescence revealed highly significant changes in the CK network within amniocytes with dense CK aggregates and retraction from the cell periphery (all p = 0.006. In human pregnancies affected by PPROM, there was further evidence of CK network retraction with significantly shorter amniocyte foot processes (p = 0.002. These results suggest early choriodecidual infection results in decreased cellular membrane integrity and tensile strength via dysfunction of CK networks. Downregulation of CK expression and perturbations in the amniotic epithelial cell intermediate filament network occur after GBS

  6. Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

    Directory of Open Access Journals (Sweden)

    Nannenga Brent L

    2011-12-01

    Full Text Available Abstract Background Membrane proteins (MPs populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as E. coli has curtailed progress in our understanding of their structure and function. Results Using the seven transmembrane segments H. turkmenica deltarhodopsin (HtdR as a reporter, we isolated a spontaneous mutant in the arabinose-inducible PBAD promoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing htdR transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (Δtig cells, toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (N. pharonis sensory rhodopsin II was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a Δtig host. Conclusions Our system, which combines a downregulated version of the tightly repressed PBAD promoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.

  7. Plasma membrane aminoglycerolipid flippase function is required for signaling competence in the yeast mating pheromone response pathway.

    Science.gov (United States)

    Sartorel, Elodie; Barrey, Evelyne; Lau, Rebecca K; Thorner, Jeremy

    2015-01-01

    The class 4 P-type ATPases ("flippases") maintain membrane asymmetry by translocating phosphatidylethanolamine and phosphatidylserine from the outer leaflet to the cytosolic leaflet of the plasma membrane. In Saccharomyces cerevisiae, five related gene products (Dnf1, Dnf2, Dnf3, Drs2, and Neo1) are implicated in flipping of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. In MAT A: cells responding to α-factor, we found that Dnf1, Dnf2, and Dnf3, as well as the flippase-activating protein kinase Fpk1, localize at the projection ("shmoo") tip where polarized growth is occurring and where Ste5 (the central scaffold protein of the pheromone-initiated MAPK cascade) is recruited. Although viable, a MAT A: dnf1∆ dnf2∆ dnf3∆ triple mutant exhibited a marked decrease in its ability to respond to α-factor, which we could attribute to pronounced reduction in Ste5 stability resulting from an elevated rate of its Cln2⋅Cdc28-initiated degradation. Similarly, a MAT A: dnf1∆ dnf3∆ drs2∆ triple mutant also displayed marked reduction in its ability to respond to α-factor, which we could attribute to inefficient recruitment of Ste5 to the plasma membrane due to severe mislocalization of the cellular phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate pools. Thus proper remodeling of plasma membrane aminoglycerolipids and phosphoinositides is necessary for efficient recruitment, stability, and function of the pheromone signaling apparatus. © 2015 Sartorel et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  8. Construction of nerve guide conduits from cellulose/soy protein composite membranes combined with Schwann cells and pyrroloquinoline quinone for the repair of peripheral nerve defect.

    Science.gov (United States)

    Luo, Lihua; Gan, Li; Liu, Yongming; Tian, Weiqun; Tong, Zan; Wang, Xiong; Huselstein, Celine; Chen, Yun

    2015-02-20

    Regeneration and functional reconstruction of peripheral nerve defects remained a significant clinical challenge. Nerve guide conduits, with seed cells or neurotrophic factors (NTFs), had been widely used to improve the repair and regeneration of injured peripheral nerve. Pyrroloquinoline quinone (PQQ) was an antioxidant that can stimulate nerve growth factors (NGFs) synthesis and accelerate the Schwann cells (SCs) proliferation and growth. In present study, three kinds of nerve guide conduits were constructed: one from cellulose/SPI hollow tube (CSC), another from CSC combined with SCs (CSSC), and the third one from CSSC combined with PQQ (CSSPC), respectively. And then they were applied to bridge and repair the sciatic nerve defect in rats, using autograft as control. Effects of different nerve guide conduits on the nerve regeneration were comparatively evaluated by general analysis, sciatic function index (SFI) and histological analysis (HE and TEM). Newly-formed regenerative nerve fibers were observed and running through the transparent nerve guide conduits 12 weeks after surgery. SFI results indicated that the reconstruction of motor function in CSSPC group was better than that in CSSC and CSC groups. HE images from the cross-sections and longitudinal-sections of the harvested regenerative nerve indicated that regenerative nerve fibers had been formed and accompanied with new blood vessels and matrix materials in the conduits. TEM images also showed that lots of fresh myelinated and non-myelinated nerve fibers had been formed. Parts of vacuolar, swollen and abnormal axons occurred in CSC and CSSC groups, while the vacuolization and swell of axons was the least serious in CSSPC group. These results indicated that CSSPC group had the most ability to repair and reconstruct the nerve structure and functions due to the comprehensive contributions from hollow CSC tube, SCs and PQQ. As a result, the CSSPC may have the potential for the applications as nerve guide

  9. DNA repair in Chromobacterium violaceum.

    Science.gov (United States)

    Duarte, Fábio Teixeira; Carvalho, Fabíola Marques de; Bezerra e Silva, Uaska; Scortecci, Kátia Castanho; Blaha, Carlos Alfredo Galindo; Agnez-Lima, Lucymara Fassarella; Batistuzzo de Medeiros, Silvia Regina

    2004-03-31

    Chromobacterium violaceum is a Gram-negative beta-proteobacterium that inhabits a variety of ecosystems in tropical and subtropical regions, including the water and banks of the Negro River in the Brazilian Amazon. This bacterium has been the subject of extensive study over the last three decades, due to its biotechnological properties, including the characteristic violacein pigment, which has antimicrobial and anti-tumoral activities. C. violaceum promotes the solubilization of gold in a mercury-free process, and has been used in the synthesis of homopolyesters suitable for the production of biodegradable polymers. The complete genome sequence of this organism has been completed by the Brazilian National Genome Project Consortium. The aim of our group was to study the DNA repair genes in this organism, due to their importance in the maintenance of genomic integrity. We identified DNA repair genes involved in different pathways in C. violaceum through a similarity search against known sequences deposited in databases. The phylogenetic analyses were done using programs of the PHILYP package. This analysis revealed various metabolic pathways, including photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, recombinational repair, and the SOS system. The similarity between the C. violaceum sequences and those of Neisserie miningitidis and Ralstonia solanacearum was greater than that between the C. violaceum and Escherichia coli sequences. The peculiarities found in the C. violaceum genome were the absence of LexA, some horizontal transfer events and a large number of repair genes involved with alkyl and oxidative DNA damage.

  10. Base excision repair in sugarcane

    Directory of Open Access Journals (Sweden)

    Agnez-Lima Lucymara F.

    2001-01-01

    Full Text Available DNA damage can be induced by a large number of physical and chemical agents from the environment as well as compounds produced by cellular metabolism. This type of damage can interfere with cellular processes such as replication and transcription, resulting in cell death and/or mutations. The low frequency of mutagenesis in cells is due to the presence of enzymatic pathways which repair damaged DNA. Several DNA repair genes (mainly from bacteria, yeasts and mammals have been cloned and their products characterized. The high conservation, especially in eukaryotes, of the majority of genes related to DNA repair argues for their importance in the maintenance of life on earth. In plants, our understanding of DNA repair pathways is still very poor, the first plant repair genes having only been cloned in 1997 and the mechanisms of their products have not yet been characterized. The objective of our data mining work was to identify genes related to the base excision repair (BER pathway, which are present in the database of the Sugarcane Expressed Sequence Tag (SUCEST Project. This search was performed by tblastn program. We identified sugarcane clusters homologous to the majority of BER proteins used in the analysis and a high degree of conservation was observed. The best results were obtained with BER proteins from Arabidopsis thaliana. For some sugarcane BER genes, the presence of more than one form of mRNA is possible, as shown by the occurrence of more than one homologous EST cluster.

  11. Stress Signals, Mediated by Membranous Glucocorticoid Receptor, Activate PLC/PKC/GSK-3β/β-catenin Pathway to Inhibit Wound Closure.

    Science.gov (United States)

    Jozic, Ivan; Vukelic, Sasa; Stojadinovic, Olivera; Liang, Liang; Ramirez, Horacio A; Pastar, Irena; Tomic Canic, Marjana

    2017-05-01

    Glucocorticoids (GCs), key mediators of stress signals, are also potent wound healing inhibitors. To understand how stress signals inhibit wound healing, we investigated the role of membranous glucocorticoid receptor (mbGR) by using cell-impermeable BSA-conjugated dexamethasone. We found that mbGR inhibits keratinocyte migration and wound closure by activating a Wnt-like phospholipase (PLC)/ protein kinase C (PKC) signaling cascade. Rapid activation of mbGR/PLC/PKC further leads to activation of known biomarkers of nonhealing found in patients, β-catenin and c-myc. Conversely, a selective inhibitor of PKC, calphostin C, blocks mbGR/PKC pathway, and rescues GC-mediated inhibition of keratinocyte migration in vitro and accelerates wound epithelialization of human wounds ex vivo. This novel signaling mechanism may have a major impact on understanding how stress response via GC signaling regulates homeostasis and its role in development and treatments of skin diseases, including wound healing. To test tissue specificity of this nongenomic signaling mechanism, we tested retinal and bronchial human epithelial cells and fibroblasts. We found that mbGR/PLC/PKC signaling cascade exists in all cell types tested, suggesting a more general role. The discovery of this nongenomic signaling pathway, in which glucocorticoids activate Wnt pathway via mbGR, provides new insights into how stress-mediated signals may activate growth signals in various epithelial and mesenchymal tissues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Investigation of DNA repair in human oocytes and preimplantation embryos

    OpenAIRE

    Jaroudi, S.

    2010-01-01

    DNA repair genes are expressed in mammalian embryos and in human germinal vesicles, however, little is known about DNA repair in human preimplantation embryos. This project had three aims: 1) to produce a DNA repair profile of human MII oocytes and blastocysts using expression arrays and identify repair pathways that may be active before and after embryonic genome activation; 2) to design an in vitro functional assay that targeted mismatch repair and which could be applied to human oocytes...

  13. Hypospadias repair

    Science.gov (United States)

    ... the problem. If the repair is not done, problems may occur later on such as: Difficulty controlling and directing urine stream A curve in the penis during erection Decreased fertility Embarrassment about appearance of penis Surgery ...

  14. 膜缓控释给药系统促进损伤组织的修复**★%A membrane controlled release drug delivery system promotes injured tissue repair

    Institute of Scientific and Technical Information of China (English)

    李伟; 戴江华; 罗军; 戴闽; 高乾坤

    2013-01-01

      背景:目前研究多注重缓控释给药膜的缓控释效果及其生物相容性,也有开展缓控释给药膜参与损伤组织修复的机制研究,其中干细胞是损伤组织修复的关键因素,但干细胞与缓控释给药膜之间的联系尚未得到足够关注。目的:分析膜缓控释给药系统在组织损伤修复中的研究现状与进展。方法:以“缓释系统,膜,药物载体,组织损伤修复,干细胞归巢;sustained-release system,membrane, drug delivery,injuries and repairs of tissue,stem cel homing”为关键词,采用计算机检索Pubmed数据库、中国知网、Elsevier数据库1992年1月至2012年12月有关膜缓控释给药系统临床应用及实验研究的文章。结果与结论:在膜缓控释给药系统中高分子材料几乎成了药物和生长因子在传递、渗透过程中不可分割的组成部分。虽然药物缓释系统的发展与制膜技术都在不断的更新,但距离完全达到理想的应用标准还有一定的差距,如不具备主动吸引干细胞定向迁移与分布的生物学功能。近年来膜缓控释给药系统出现新的发展方向,即不仅能起到诱导干细胞定向分化的作用,也能诱导干细胞向损伤部位定向分布,从而促进损伤组织再生修复。%  BACKGROUND: At present many studies have pay attention to the sustained-release and control ed-release effects, as wel as biocompatibility, in membrane control ed release drug delivery system. There are also some studies addressing the mechanisms underlying injured tissue repair with these drug membranes, in which stem cel s are the key factors. However, the association between stem cel s and the sustained and control ed drug release membrane has not yet been paid enough attention. OBJECTIVE: To analyze the current research status and progress of membrane sustained-release system in the repair of tissue injuries. METHODS: Using the keywords of

  15. Investigation on removal pathways of Di 2-ethyl hexyl phthalate from synthetic municipal wastewater using a submerged membrane bioreactor.

    Science.gov (United States)

    Zolfaghari, Mehdi; Drogui, Patrick; Seyhi, Brahima; Brar, Satinder Kaur; Buelna, Gerardo; Dubé, Rino; Klai, Nouha

    2015-11-01

    Highly hydrophobic Di 2-ethyl hexyl phthalate (DEHP) is one of the most prevalent plasticizers in wastewaters. Since its half-life in biological treatment is around 25days, it can be used as an efficiency indicator of wastewater treatment plant for the removal of hydrophobic emerging contaminants. In this study, the performance of submerged membrane bioreactor was monitored to understand the effect of DEHP on the growth of aerobic microorganisms. The data showed that the chemical oxygen demand (COD) and ammonia concentration were detected below 10 and 1.0mg/L, respectively for operating conditions of hydraulic retention time (HRT)=4 and 6hr, sludge retention time (SRT)=140day and sludge concentration between 11.5 and 15.8g volatile solid (VS)/L. The removal efficiency of DEHP under these conditions was higher and ranged between 91% and 98%. Results also showed that the removal efficiency of DEHP in biological treatment depended on the concentration of sludge, as adsorption is the main mechanism of its removal. For the submerged membrane bioreactor, the pore size is the pivotal factor for DEHP removal, since it determines the amount of soluble microbial products coming out of the process. Highly assimilated microorganisms increase the biodegradation rate, as 74% of inlet DEHP was biodegraded; however, the concentration of DEHP inside sludge was beyond the discharge limit. Understanding the fate of DEHP in membrane bioreactor, which is one of the most promising and futuristic treatment process could provide replacement for conventional processes to satisfy the future stricter regulations on emerging contaminants.

  16. Modulation of Plant RAB GTPase-Mediated Membrane Trafficking Pathway at the Interface Between Plants and Obligate Biotrophic Pathogens.

    Science.gov (United States)

    Inada, Noriko; Betsuyaku, Shigeyuki; Shimada, Takashi L; Ebine, Kazuo; Ito, Emi; Kutsuna, Natsumaro; Hasezawa, Seiichiro; Takano, Yoshitaka; Fukuda, Hiroo; Nakano, Akihiko; Ueda, Takashi

    2016-09-01

    RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f.sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens.

  17. Modulation of RhoA GTPase Activity Sensitizes Human Cervix Carcinoma Cells to γ-Radiation by Attenuating DNA Repair Pathways

    Directory of Open Access Journals (Sweden)

    Juliana H. Osaki

    2016-01-01

    Full Text Available Radiotherapy with γ-radiation is widely used in cancer treatment to induce DNA damage reducing cell proliferation and to kill tumor cells. Although RhoA GTPase overexpression/hyperactivation is observed in many malignancies, the effect of RhoA activity modulation on cancer radiosensitivity has not been previously investigated. Here, we generated stable HeLa cell clones expressing either the dominant negative RhoA-N19 or the constitutively active RhoA-V14 and compared the responses of these cell lines with those of parental HeLa cells, after treatment with low doses of γ-radiation. HeLa-RhoA-N19 and HeLa-RhoA-V14 clones displayed reduced proliferation and survival compared to parental cells after radiation and became arrested at cell cycle stages correlated with increased cellular senescence and apoptosis. Also, Chk1/Chk2 and histone H2A phosphorylation data, as well as comet assays, suggest that the levels of DNA damage and DNA repair activation and efficiency in HeLa cell lines are correlated with active RhoA. In agreement with these results, RhoA inhibition by C3 toxin expression drastically affected homologous recombination (HR and nonhomologous end joining (NHEJ. These data suggest that modulation of RhoA GTPase activity impairs DNA damage repair, increasing HeLa cell radiosensitivity.

  18. Reciprocal bystander effect between α-irradiated macrophage and hepatocyte is mediated by cAMP through a membrane signaling pathway.

    Science.gov (United States)

    He, Mingyuan; Dong, Chen; Xie, Yuexia; Li, Jitao; Yuan, Dexiao; Bai, Yang; Shao, Chunlin

    2014-01-01

    Irradiated cells can induce biological effects on vicinal non-irradiated bystander cells, meanwhile the bystander cells may rescue the irradiated cells through a feedback signal stress. To elucidate the nature of this reciprocal effect, we examined the interaction between α-irradiated human macrophage cells U937 and its bystander HL-7702 hepatocyte cells using a cell co-culture system. Results showed that after 6h of cell co-culture, mitochondria depolarization corresponding to apoptosis was significantly induced in the HL-7702 cells, but the formation of micronuclei in the irradiated U937 cells was markedly decreased compared to that without cell co-culture treatment. This reciprocal effect was not observed when the cell membrane signaling pathway was blocked by filipin that inhibited cAMP transmission from bystander cells to irradiated cells. After treatment of cells with exogenous cAMP, forskolin (an activator of cAMP) or KH-7 (an inhibitor of cAMP), respectively, it was confirmed that cAMP communication from bystander cells to targeted cells could mitigate radiation damage in U739 cells, and this cAMP insufficiency in the bystander cells contributed to the enhancement of bystander apoptosis. Moreover, the bystander apoptosis in HL-7702 cells was aggravated by cAMP inhibition but it could not be evoked when p53 of HL-7702 cells was knocked down no matter of forskolin and KH-7 treatment. In conclusion, this study disclosed that cAMP could be released from bystander HL-7702 cells and compensated to α-irradiated U937 cells through a membrane signaling pathway and this cAMP communication played a profound role in regulating the reciprocal bystander effects.

  19. Femoral hernia repair

    Science.gov (United States)

    Femorocele repair; Herniorrhaphy; Hernioplasty - femoral ... During surgery to repair the hernia, the bulging tissue is pushed back in. The weakened area is sewn closed or strengthened. This repair ...

  20. Undescended testicle repair

    Science.gov (United States)

    Orchidopexy; Inguinal orchidopexy; Orchiopexy; Repair of undescended testicle; Cryptorchidism repair ... first year of life without treatment. Undescended testicle repair surgery is recommended for patients whose testicles do ...

  1. Genetic variants involved in oxidative stress, base excision repair, DNA methylation, and folate metabolism pathways influence myeloid neoplasias susceptibility and prognosis.

    Science.gov (United States)

    Gonçalves, Ana Cristina; Alves, Raquel; Baldeiras, Inês; Cortesão, Emília; Carda, José Pedro; Branco, Claudia C; Oliveiros, Bárbara; Loureiro, Luísa; Pereira, Amélia; Nascimento Costa, José Manuel; Sarmento-Ribeiro, Ana Bela; Mota-Vieira, Luisa

    2017-01-01

    Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) share common features: elevated oxidative stress, DNA repair deficiency, and aberrant DNA methylation. We performed a hospital-based case-control study to evaluate the association in variants of genes involved in oxidative stress, folate metabolism, DNA repair, and DNA methylation with susceptibility and prognosis of these malignancies. To that end, 16 SNPs (one per gene: CAT, CYBA, DNMT1, DNMT3A, DNMT3B, GPX1, KEAP1, MPO, MTRR, NEIL1, NFE2F2, OGG1, SLC19A1, SOD1, SOD2, and XRCC1) were genotyped in 191 patients (101 MDS and 90 AML) and 261 controls. We also measured oxidative stress (reactive oxygen species/total antioxidant status ratio), DNA damage (8-hydroxy-2'-deoxyguanosine), and DNA methylation (5-methylcytosine) in 50 subjects (40 MDS and 10 controls). Results showed that five genes (GPX1, NEIL1, NFE2L2, OGG1, and SOD2) were associated with MDS, two (DNMT3B and SLC19A1) with AML, and two (CYBA and DNMT1) with both diseases. We observed a correlation of CYBA TT, GPX1 TT, and SOD2 CC genotypes with increased oxidative stress levels, as well as NEIL1 TT and OGG1 GG genotypes with higher DNA damage. The 5-methylcytosine levels were negatively associated with DNMT1 CC, DNMT3A CC, and MTRR AA genotypes, and positively with DNMT3B CC genotype. Furthermore, DNMT3A, MTRR, NEIL1, and OGG1 variants modulated AML transformation in MDS patients. Additionally, DNMT3A, OGG1, GPX1, and KEAP1 variants influenced survival of MDS and AML patients. Altogether, data suggest that genetic variability influence predisposition and prognosis of MDS and AML patients, as well AML transformation rate in MDS patients. © 2016 Wiley Periodicals, Inc.

  2. Wortmannin induces MCF-7 breast cancer cell death via the apoptotic pathway, involving chromatin condensation, generation of reactive oxygen species, and membrane blebbing

    Directory of Open Access Journals (Sweden)

    Akter R

    2012-07-01

    Full Text Available Rozina Akter,1 Md. Zakir Hossain,2 Maurice G Kleve,3 Michael A Gealt31Applied Biosciences Emphasis, Department of Applied Science, 2Graduate Institute of Technology, 3Department of Biology, College of Science and of Mathematics, University Arkansas at Little Rock, Little Rock, AR, USABackground: Apoptosis can be used as a reliable marker for evaluating potential chemotherapeutic agents. Because wortmannin is a microbial steroidal metabolite, it specifically inhibits the phosphatidyl inositol 3-kinase pathway, and could be used as a promising apoptosis-based therapeutic agent in the treatment of cancer. The objective of this study was to investigate the biomolecular mechanisms involved in wortmannin-induced cell death of breast cancer-derived MCF-7 cells.Methods and results: Our experimental results demonstrate that wortmannin has strong apoptotic effects through a combination of different actions, including reduction of cell viability in a dose-dependent manner, inhibition of proliferation, and enhanced generation of intracellular reactive oxygen species.Conclusion: Our findings suggest that wortmannin induces MCF-7 cell death via a programmed pathway showing chromatin condensation, nuclear fragmentation, reactive oxygen species, and membrane blebbing, which are characteristics typical of apoptosis.Keywords: wortmannin, human breast adenocarcinoma, apoptosis, reactive oxygen species, flow cytometry

  3. Interference of fisetin with targets of the nuclear factor-κB signal transduction pathway activated by Epstein-Barr virus encoded latent membrane protein 1.

    Science.gov (United States)

    Li, Rong; Liang, Hong-Ying; Li, Ming-Yong; Lin, Chun-Yan; Shi, Meng-Jie; Zhang, Xiu-Juan

    2014-01-01

    Fisetin is an effective compound extracted from lacquer which has been used in the treatment of various diseases. Preliminary data indicate that it also exerts specific anti-cancer effects. However, the manner in which fisetin regulates cancer growth remains unknown. In this study, we elucidated interference of fisetin with targets of the nuclear factorκB signal transduction pathway activated by Epstein-Barr virus encoding latent membrane protein 1 (LMP1)in nasopharyngeal carcinoma (NPC) cells, Results showed that fisetin inhibited the survival rate of CNE-LMP1 cells and NF-κB activation caused by LMP1. Fisetin also suppressed nuclear translocation of NF-κB (p65) and IκBα phosphorylation, while inhibiting CyclinD1, all key targets of the NF-κB signal transduction pathway. It was suggested that interference effects of fisetin with signal transduction activated by LMP1 encoded by the Epstein-Barr virus may play an important role in its anticancer potential.

  4. Reciprocal bystander effect between α-irradiated macrophage and hepatocyte is mediated by cAMP through a membrane signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    He, Mingyuan [Institute of Radiation Medicine, Fudan University, No. 2094 Xie-Tu Road, Shanghai 200032 (China); Department of Radiation Oncology, China–Japan Union Hospital of Jilin University, Changchun 130033 (China); Dong, Chen; Xie, Yuexia; Li, Jitao; Yuan, Dexiao; Bai, Yang [Institute of Radiation Medicine, Fudan University, No. 2094 Xie-Tu Road, Shanghai 200032 (China); Shao, Chunlin, E-mail: clshao@shmu.edu.cn [Institute of Radiation Medicine, Fudan University, No. 2094 Xie-Tu Road, Shanghai 200032 (China)

    2014-05-15

    Highlights: • α-Irradiation induced reciprocal effects between macrophage and hepatocyte cells. • cAMP played a protective role in regulating the reverse bystander effect. • cAMP communication contributed to the reciprocal effects via membrane signaling. • p53 was required for cAMP-regulated bystander effect in the recipient cells. - Abstract: Irradiated cells can induce biological effects on vicinal non-irradiated bystander cells, meanwhile the bystander cells may rescue the irradiated cells through a feedback signal stress. To elucidate the nature of this reciprocal effect, we examined the interaction between α-irradiated human macrophage cells U937 and its bystander HL-7702 hepatocyte cells using a cell co-culture system. Results showed that after 6 h of cell co-culture, mitochondria depolarization corresponding to apoptosis was significantly induced in the HL-7702 cells, but the formation of micronuclei in the irradiated U937 cells was markedly decreased compared to that without cell co-culture treatment. This reciprocal effect was not observed when the cell membrane signaling pathway was blocked by filipin that inhibited cAMP transmission from bystander cells to irradiated cells. After treatment of cells with exogenous cAMP, forskolin (an activator of cAMP) or KH-7 (an inhibitor of cAMP), respectively, it was confirmed that cAMP communication from bystander cells to targeted cells could mitigate radiation damage in U739 cells, and this cAMP insufficiency in the bystander cells contributed to the enhancement of bystander apoptosis. Moreover, the bystander apoptosis in HL-7702 cells was aggravated by cAMP inhibition but it could not be evoked when p53 of HL-7702 cells was knocked down no matter of forskolin and KH-7 treatment. In conclusion, this study disclosed that cAMP could be released from bystander HL-7702 cells and compensated to α-irradiated U937 cells through a membrane signaling pathway and this cAMP communication played a profound role in

  5. [Complex control of the source of infection in sepsis : Extracorporeal membrane oxygenation (ECMO) as a bridging concept for tracheal fistula repair in sepsis-associated ARDS].

    Science.gov (United States)

    Weiterer, S; Schmidt, K; Deininger, M; Ulrich, A; Tochtermann, U; Eberhardt, R; Hofer, S; Weigand, M A; Brenner, T

    2016-09-01

    Here, we present a case of a tracheal fistula due to an anastomotic insufficiency following abdominothoracic esophageal resection. Despite immediate discontinuity resection, the tracheal fistula could not be surgically closed, resulting in incomplete control of the source of infection and an alternative treatment concept in the form of interventional fistula closure using a Y-tracheal stent. However, owing to existing severe acute respiratory distress syndrome (ARDS), which is associated with a considerable risk of peri-interventional hypoxia, a temporary bridging concept using venovenous extracorporeal membrane oxygenation (ECMO) was implemented successfully.

  6. Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection.

    Science.gov (United States)

    Heaton, Brook E; Barkan, Daniel; Bongiorno, Paola; Karakousis, Petros C; Glickman, Michael S

    2014-08-01

    Mycobacterium tuberculosis persistence within its human host requires mechanisms to resist the effector molecules of host immunity, which exert their bactericidal effects through damaging pathogen proteins, membranes, and DNA. Substantial evidence indicates that bacterial pathogens, including M. tuberculosis, require DNA repair systems to repair the DNA damage inflicted by the host during infection, but the role of double-strand DNA break (DSB) repair systems is unclear. Double-strand DNA breaks are the most cytotoxic form of DNA damage and must be repaired for chromosome replication to proceed. M. tuberculosis elaborates three genetically distinct DSB repair systems: homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). NHEJ, which repairs DSBs in quiescent cells, may be particularly relevant to M. tuberculosis latency. However, very little information is available about the phenotype of DSB repair-deficient M. tuberculosis in animal models of infection. Here we tested M. tuberculosis strains lacking NHEJ (a Δku ΔligD strain), HR (a ΔrecA strain), or both (a ΔrecA Δku strain) in C57BL/6J mice, C3HeB/FeJ mice, guinea pigs, and a mouse hollow-fiber model of infection. We found no difference in bacterial load, histopathology, or host mortality between wild-type and DSB repair mutant strains in any model of infection. These results suggest that the animal models tested do not inflict DSBs on the mycobacterial chromosome, that other repair pathways can compensate for the loss of NHEJ and HR, or that DSB repair is not required for M. tuberculosis pathogenesis.

  7. Physical association of PDK1 with AKT1 is sufficient for pathway activation independent of membrane localization and phosphatidylinositol 3 kinase.

    Directory of Open Access Journals (Sweden)

    Zhiyong Ding

    Full Text Available Frequent activation of the AKT serine-threonine kinase in cancer confers resistance to therapy. AKT is activated by a multi-step process involving phosphatidylinositide (PtdIns phosphate-mediated recruitment of AKT and its upstream kinases, including 3-Phosphoinositide-dependent kinase 1 (PDK1, to the inner surface of the cell membrane. PDK1 in the appropriate context phosphorylates AKT at threonine 308 (T308 to activate AKT. Whether PtdIns(3,4,5Ps (PtdInsP3 binding and AKT membrane translocation mediate functions other than formation of a functional PDK1::AKT complex have not been fully elucidated. We fused complementary fragments of intensely fluorescent protein (IFP to AKT1 and PDK1 to induce a stable complex to study the prerequisites of AKT1 phosphorylation and function. In the stabilized PDK1-IFPC::IFPN-AKT1 complex, AKT1 T308 phosphorylation was independent of PtdIns, as demonstrated by treatment with Phosphatidylinositol 3 Kinase (PI3K inhibitors. Further when interaction with PtdIns and the cell membrane was prevented by creating PH-domain mutants of AKT1 (R25A and PDK1 (R474A, AKT1 phosphorylation on T308 was maintained in the PDK1-IFPC::IFPN-AKT1 complex. The PDK1-IFPC::IFPN-AKT1 complex was sufficient for phosphorylation of known AKT substrates, and conferred resistance to inhibitors of PI3K (LY294002, PI103, GDC0941 and TGX286 but not inhibitors of the downstream TORC1 complex (rapamycin. Thus the locus of action of targeted therapeutics can be elucidated by the constitutively active AKT1 complex. Our data indicate that PtdIns and membrane localization are not required for AKT phosphorylation and activation, but rather serve to induce a functional physical interaction between PDK1 and AKT. The PDK1-IFPC::IFPN-AKT1 complex provides a cell-based platform to examine specificity of drugs targeting PI3K pathway components.

  8. Physical association of PDK1 with AKT1 is sufficient for pathway activation independent of membrane localization and phosphatidylinositol 3 kinase.

    Science.gov (United States)

    Ding, Zhiyong; Liang, Jiyong; Li, Jin; Lu, Yiling; Ariyaratna, Vathsala; Lu, Zhimin; Davies, Michael A; Westwick, John K; Mills, Gordon B

    2010-03-26

    Frequent activation of the AKT serine-threonine kinase in cancer confers resistance to therapy. AKT is activated by a multi-step process involving phosphatidylinositide (PtdIns) phosphate-mediated recruitment of AKT and its upstream kinases, including 3-Phosphoinositide-dependent kinase 1 (PDK1), to the inner surface of the cell membrane. PDK1 in the appropriate context phosphorylates AKT at threonine 308 (T308) to activate AKT. Whether PtdIns(3,4,5)Ps (PtdInsP3) binding and AKT membrane translocation mediate functions other than formation of a functional PDK1::AKT complex have not been fully elucidated. We fused complementary fragments of intensely fluorescent protein (IFP) to AKT1 and PDK1 to induce a stable complex to study the prerequisites of AKT1 phosphorylation and function. In the stabilized PDK1-IFPC::IFPN-AKT1 complex, AKT1 T308 phosphorylation was independent of PtdIns, as demonstrated by treatment with Phosphatidylinositol 3 Kinase (PI3K) inhibitors. Further when interaction with PtdIns and the cell membrane was prevented by creating PH-domain mutants of AKT1 (R25A) and PDK1 (R474A), AKT1 phosphorylation on T308 was maintained in the PDK1-IFPC::IFPN-AKT1 complex. The PDK1-IFPC::IFPN-AKT1 complex was sufficient for phosphorylation of known AKT substrates, and conferred resistance to inhibitors of PI3K (LY294002, PI103, GDC0941 and TGX286) but not inhibitors of the downstream TORC1 complex (rapamycin). Thus the locus of action of targeted therapeutics can be elucidated by the constitutively active AKT1 complex. Our data indicate that PtdIns and membrane localization are not required for AKT phosphorylation and activation, but rather serve to induce a functional physical interaction between PDK1 and AKT. The PDK1-IFPC::IFPN-AKT1 complex provides a cell-based platform to examine specificity of drugs targeting PI3K pathway components.

  9. Energy and Technology Review: Unlocking the mysteries of DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Quirk, W.A.

    1993-04-01

    DNA, the genetic blueprint, has the remarkable property of encoding its own repair following diverse types of structural damage induced by external agents or normal metabolism. We are studying the interplay of DNA damaging agents, repair genes, and their protein products to decipher the complex biochemical pathways that mediate such repair. Our research focuses on repair processes that correct DNA damage produced by chemical mutagens and radiation, both ionizing and ultraviolet. The most important type of DNA repair in human cells is called excision repair. This multistep process removes damaged or inappropriate pieces of DNA -- often as a string of 29 nucleotides containing the damage -- and replaces them with intact ones. We have isolated, cloned, and mapped several human repair genes associated with the nucleotide excision repair pathway and involved in the repair of DNA damage after exposure to ultraviolet light or mutagens in cooked food. We have shown that a defect in one of these repair genes, ERCC2, is responsible for the repair deficiency in one of the groups of patients with the recessive genetic disorder xeroderma pigmentosum (XP group D). We are exploring ways to purify sufficient quantities (milligrams) of the protein products of these and other repair genes so that we can understand their functions. Our long-term goals are to link defective repair proteins to human DNA repair disorders that predispose to cancer, and to produce DNA-repair-deficient mice that can serve as models for the human disorders.

  10. Intestinal obstruction repair

    Science.gov (United States)

    Repair of volvulus; Intestinal volvulus - repair; Bowel obstruction - repair ... Intestinal obstruction repair is done while you are under general anesthesia . This means you are asleep and DO NOT feel pain. ...

  11. Aortic aneurysm repair - endovascular

    Science.gov (United States)

    EVAR; Endovascular aneurysm repair - aorta; AAA repair - endovascular; Repair - aortic aneurysm - endovascular ... Endovascular aortic repair is done because your aneurysm is very large, growing quickly, or is leaking or bleeding. You may have ...

  12. Motorcycle Repair.

    Science.gov (United States)

    Hein, Jim; Bundy, Mike

    This motorcycle repair curriculum guide contains the following ten areas of study: brake systems, clutches, constant mesh transmissions, final drives, suspension, mechanical starting mechanisms, electrical systems, fuel systems, lubrication systems, and overhead camshafts. Each area consists of one or more units of instruction. Each instructional…

  13. Turbine repair process, repaired coating, and repaired turbine component

    Energy Technology Data Exchange (ETDEWEB)

    Das, Rupak; Delvaux, John McConnell; Garcia-Crespo, Andres Jose

    2015-11-03

    A turbine repair process, a repaired coating, and a repaired turbine component are disclosed. The turbine repair process includes providing a turbine component having a higher-pressure region and a lower-pressure region, introducing particles into the higher-pressure region, and at least partially repairing an opening between the higher-pressure region and the lower-pressure region with at least one of the particles to form a repaired turbine component. The repaired coating includes a silicon material, a ceramic matrix composite material, and a repaired region having the silicon material deposited on and surrounded by the ceramic matrix composite material. The repaired turbine component a ceramic matrix composite layer and a repaired region having silicon material deposited on and surrounded by the ceramic matrix composite material.

  14. Platelet-rich fibrinversus collagen membrane in the repair of gingival defects%富血小板纤维蛋白与胶原膜修复牙龈缺损创面的能力

    Institute of Scientific and Technical Information of China (English)

    董露; 肖琼; 杨琴秋; 孙勇; 陈红亮

    2016-01-01

    背景:前期实验中发现富血小板纤维蛋白具有良好的诱导牙龈软组织修复再生的能力。  目的:观察富血小板纤维蛋白膜与胶原膜对牙龈软组织愈合的影响,评价富血小板纤维蛋白膜诱导牙龈软组织缺损修复再生的能力。  方法:选因各种原因需要拔除前磨牙或磨牙同期进行位点保存的患者22例(前磨牙2颗,磨牙20颗),随机分为2组,22个软组织缺损区域,拔牙窝内植入Bio-Oss ,表面覆盖富血小板纤维蛋白膜或海奥胶原膜。通过追踪植入Bio-Oss后牙龈创面的愈合时间和植入Bio-Oss后1,2周的创面愈合率,评价富血小板纤维蛋白促进牙龈组织愈合的能力。  结果与结论:①富血小板纤维蛋白组愈合时间为(12.17±2.25) d,胶原膜组愈合时间为(17.30±2.58) d;植入Bio-Oss后1,2周富血小板纤维蛋白组愈合率明显高于胶原膜组。②结果说明,相同时间节点时富血小板纤维蛋白促进牙龈软组织的愈合能力明显优于胶原膜,达到相同愈合状态时富血小板纤维蛋白组所需时间明显短于胶原膜组。%BACKGROUND:Previous studies have found that platelet-rich fibrin has a good ability to induce gingival soft tissue repair and regeneration. OBJECTIVE:To observe the effects of platelet-rich fibrinversus colagen membrane on gingival soft tissue healing, and to evaluate the ability of platelet-rich fibrin to repair gingival defects. METHODS:Twenty-two patients (2 premolar teeth and 20 molars) scheduled for premolar or molar removal or ridge preservation due to various reasons were selected and randomized into two groups. Bio-Oss was implanted into the extraction socket folowed by covering with platelet-rich fibrin or colagen membrane. Healing time and healing rate of gingival defects were detected to evaluate the ability of platelet-rich fibrin to promote gingival tissue healing at 1-2 weeks after Bio-Oss implantation

  15. Internal Limiting Membrane Flap Techniques for the Repair of Large Macular Holes: a Short-Term Follow-up of Anatomical and Functional Outcomes.

    Science.gov (United States)

    Guber, J; Lang, C; Valmaggia, C

    2017-02-01

    Background To evaluate the technique of inverted internal limiting membrane (ILM) flaps for the management of large macular holes and autologous ILM free flaps for non-closing macular holes. Patients and methods All macular holes were treated with pars plana vitrectomy and dual blue assisted ILM flap technique. The inverted ILM flap was created as a primary procedure for large macular holes (diameter > 400 µm). On the other hand, the free ILM flap technique was used as a secondary procedure for non-closing macular holes after failed initial standard procedure. SD-OCT images were taken to assess the anatomical outcome of surgery, while best corrected visual acuity (BCVA) was used to evaluate the functional outcome during a 2-month follow-up. Results All patients underwent successful planned manipulation of the ILM flap. In seven patients/eyes, an inverted ILM flap was created, in three patients/eyes a free ILM flap translocation was performed. All patients achieved complete anatomical closure. Partial microstructural reconstruction, demonstrated on SD-OCT as restoration of the external limiting membrane and the ellipsoid zone, was observed in some cases as early as one month after surgery. Functionally, in comparison to baseline, most of the patients showed improvements in BCVA of 1 to 2 lines at the first postoperative follow-up visit. Conclusions Inverted ILM flaps for large macular holes and free flaps for non-closing macular holes appear to be a safe and effective approach, with favourable short-term anatomical and functional results.

  16. Mammalian mismatch repair

    DEFF Research Database (Denmark)

    Pena Diaz, Javier; Jiricny, Josef

    2012-01-01

    A considerable surge of interest in the mismatch repair (MMR) system has been brought about by the discovery of a link between Lynch syndrome, an inherited predisposition to cancer of the colon and other organs, and malfunction of this key DNA metabolic pathway. This review focuses on recent...... advances in our understanding of the molecular mechanisms of canonical MMR, which improves replication fidelity by removing misincorporated nucleotides from the nascent DNA strand. We also discuss the involvement of MMR proteins in two other processes: trinucleotide repeat expansion and antibody maturation...

  17. Inverted Internal Limiting Membrane Flap Technique for Repair of Large Macular Holes: A Short-term Follow-up of Anatomical and Functional Outcomes

    Institute of Scientific and Technical Information of China (English)

    Zhe Chen; Chan Zhao; Jun-Jie Ye; Xu-Qian Wang; Rui-Fang Sui

    2016-01-01

    Background:Inverted internal limiting membrane (ILM) flap technique has recently been reported in a limited number of studies as an effective surgical technique for the management of large macular holes (MHs) with fair MH closure rates as well as gains in visual acuity.In the current study,longitudinal changes in multi-focal electroretinogram (mfERG) responses,best-corrected visual acuity (BCVA) and spectral-domain optical coherence tomography (SD-OCT) were evaluated in eyes with large MHs managed by this technique.Methods:A prospective noncontrolled interventional study of eight patients (eight eyes) with large MHs (minimum diameter >400 μm) was conducted.All MHs were treated with pars plana vitrectomy and indocyanine green-assisted inverted ILM flap technique.SD-OCT images were used to assess the anatomical outcomes of surgery while BCVA and mfERG were used to evaluate the functional outcomes during a 3-month follow-up.Results:All patients underwent successful intended manipulation and translocation of the ILM flap without flap dislocation and achieved complete anatomical closure.Partial microstructural reconstruction,demonstrated on SD-OCT as restoration of the external limiting membrane and the ellipsoid zone,was observed in all cases as early as 1 month after surgery.Functionally,as compared to baseline,all patients showed improvements in BCVA and all but one in mfERG response during follow-up.However,Pearson's test revealed no significant correlations between BCVA and mfERG responses of the fovea and of the macular area at each evaluation time point.Conclusions:Inverted ILM flap technique appeares to be a safe and effective approach for the management of large idiopathic MHs with favorable short-term anatomical and functional results.Postoperative reconstruction of the microstructure generally shows good consistency with improvements in both BCVA and mfERG response,of which the latter might be a supplement for the former in postoperative functional follow-up.

  18. Databases and Bioinformatics Tools for the Study of DNA Repair

    Directory of Open Access Journals (Sweden)

    Kaja Milanowska

    2011-01-01

    Full Text Available DNA is continuously exposed to many different damaging agents such as environmental chemicals, UV light, ionizing radiation, and reactive cellular metabolites. DNA lesions can result in different phenotypical consequences ranging from a number of diseases, including cancer, to cellular malfunction, cell death, or aging. To counteract the deleterious effects of DNA damage, cells have developed various repair systems, including biochemical pathways responsible for the removal of single-strand lesions such as base excision repair (BER and nucleotide excision repair (NER or specialized polymerases temporarily taking over lesion-arrested DNA polymerases during the S phase in translesion synthesis (TLS. There are also other mechanisms of DNA repair such as homologous recombination repair (HRR, nonhomologous end-joining repair (NHEJ, or DNA damage response system (DDR. This paper reviews bioinformatics resources specialized in disseminating information about DNA repair pathways, proteins involved in repair mechanisms, damaging agents, and DNA lesions.

  19. Meningocele repair

    Science.gov (United States)

    ... or a membrane when your child is born, surgery will be done within 24 to 48 hours after birth. This is to prevent infection. If your child has hydrocephalus, a shunt (plastic tube) will be put in the child's brain ...

  20. Rapid actions of plasma membrane estrogen receptors regulate motility of mouse embryonic stem cells through a profilin-1/cofilin-1-directed kinase signaling pathway.

    Science.gov (United States)

    Yun, Seung Pil; Ryu, Jung Min; Kim, Mi Ok; Park, Jae Hong; Han, Ho Jae

    2012-08-01

    Long-term estrogen actions are vital for driving cell growth, but more recent evidence suggests that estrogen mediates more rapid cellular effects. However, the function of estradiol-17β (E(2))-BSA in mouse embryonic stem cells has not been reported. Therefore, we examined the role of E(2)-BSA in mouse embryonic stem cell motility and its related signal pathways. E(2)-BSA (10(-8) m) significantly increased motility after 24 h incubation and increased filamentous (F)-actin expression; these effects were inhibited by the estrogen receptor antagonist ICI 182,780, indicating that E(2)-BSA bound membrane estrogen receptors and initiated a signal. E(2)-BSA increased c-Src and focal adhesion kinase (FAK) phosphorylation, which was attenuated by ICI 182,780. The E(2)-BSA-induced increase in epidermal growth factor receptor (EGFR) phosphorylation was inhibited by Src inhibitor PP2. As a downstream signal molecule, E(2)-BSA activated cdc42 and increased formation of a complex with the neural Wiskott-Aldrich syndrome protein (N-WASP)/cdc42/transducer of cdc42-dependent actin assembly-1 (TOCA-1), which was inhibited by FAK small interfering RNA (siRNA) and EGFR inhibitor AG 1478. In addition, E(2)-BSA increased profilin-1 expression and cofilin-1 phosphorylation, which was blocked by cdc42 siRNA. Subsequently, E(2)-BSA induced an increase in F-actin expression, and cell motility was inhibited by each signal pathway-related siRNA molecule or inhibitors but not by cofilin-1 siRNA. A combined treatment of cofilin-1 siRNA and E(2)-BSA increased F-actin expression and cell motility more than that of E(2)-BSA alone. These data demonstrate that E(2)-BSA stimulated motility by interacting with profilin-1/cofilin-1 and F-actin through FAK- and c-Src/EGFR transactivation-dependent N-WASP/cdc42/TOCA-1 complex.

  1. Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Romain Ferru-Clément

    Full Text Available Cystic fibrosis transmembrane conductance regulator (CFTR is a chloride channel that is expressed on the apical plasma membrane (PM of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o- expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

  2. Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

    Science.gov (United States)

    Ferru-Clément, Romain; Fresquet, Fleur; Norez, Caroline; Métayé, Thierry; Becq, Frédéric; Kitzis, Alain; Thoreau, Vincent

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

  3. Involvement of the Cdc42 Pathway in CFTR Post-Translational Turnover and in Its Plasma Membrane Stability in Airway Epithelial Cells

    Science.gov (United States)

    Ferru-Clément, Romain; Fresquet, Fleur; Norez, Caroline; Métayé, Thierry; Becq, Frédéric; Kitzis, Alain; Thoreau, Vincent

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation. PMID:25768293

  4. NPRL2 sensitizes human non-small cell lung cancer (NSCLC cells to cisplatin treatment by regulating key components in the DNA repair pathway.

    Directory of Open Access Journals (Sweden)

    Gitanjali Jayachandran

    Full Text Available NPRL2, one of the tumor suppressor genes residing in a 120-kb homozygous deletion region of human chromosome band 3p21.3, has a high degree of amino acid sequence homology with the nitrogen permease regulator 2 (NPR2 yeast gene, and mutations of NPRL2 in yeast cells are associated with resistance to cisplatin-mediated cell killing. Previously, we showed that restoration of NPRL2 in NPRL2-negative and cisplatin-resistant cells resensitize lung cancer cells to cisplatin treatment in vitro and in vivo. In this study, we show that sensitization of non-small cell lung cancer (NSCLC cells to cisplatin by NPRL2 is accomplished through the regulation of key components in the DNA-damage checkpoint pathway. NPRL2 can phosphorylate ataxia telangiectasia mutated (ATM kinase activated by cisplatin and promote downstream gamma-H2AX formation in vitro and in vivo, which occurs during apoptosis concurrently with the initial appearance of high-molecular-weight DNA fragments. Moreover, this combination treatment results in higher Chk1 and Chk2 kinase activity than does treatment with cisplatin alone and can activate Chk2 in pleural metastases tumor xenograft in mice. Activated Chk1 and Chk2 increase the expression of cell cycle checkpoint proteins, including Cdc25A and Cdc25C, leading to higher levels of G2/M arrest in tumor cells treated with NPRL2 and cisplatin than in tumor cells treated with cisplatin only. Our results therefore suggest that ectopic expression of NPRL2 activates the DNA damage checkpoint pathway in cisplatin-resistant and NPRL2-negative cells; hence, the combination of NPRL2 and cisplatin can resensitize cisplatin nonresponders to cisplatin treatment through the activation of the DNA damage checkpoint pathway, leading to cell arrest in the G2/M phase and induction of apoptosis. The direct implication of this study is that combination treatment with NPRL2 and cisplatin may overcome cisplatin resistance and enhance therapeutic efficacy.

  5. Targeting the CD80/CD86 costimulatory pathway with CTLA4-Ig directs microglia toward a repair phenotype and promotes axonal outgrowth.

    Science.gov (United States)

    Louveau, Antoine; Nerrière-Daguin, Véronique; Vanhove, Bernard; Naveilhan, Philippe; Neunlist, Michel; Nicot, Arnaud; Boudin, Hélène

    2015-12-01

    Among the costimulatory factors widely studied in the immune system is the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA4)-CD80/CD86 pathway, which critically controls the nature and duration of the T-cell response. In the brain, up-regulated expression of CD80/CD86 during inflammation has consistently been reported in microglia. However, the role of CD80/CD86 molecules has mainly been studied in a context of microglia-T cell interactions in pathological conditions, while the function of CD80/CD86 in the regulation of intrinsic brain cells remains largely unknown. In this study, we used a transgenic pig line in which neurons express releasable CTLA4-Ig, a synthetic molecule mimicking CTLA4 and binding to CD80/CD86. The effects of CTLA4-Ig on brain cells were analyzed after intracerebral transplantation of CTLA4-Ig-expressing neurons or wild-type neurons as control. This model provided in vivo evidence that CTLA4-Ig stimulated axonal outgrowth, in correlation with a shift of the nearby microglia from a compact to a ramified morphology. In a culture system, we found that the CTLA4-Ig-induced morphological change of microglia was mediated through CD86, but not CD80. This was accompanied by microglial up-regulated expression of the anti-inflammatory molecule Arginase 1 and the neurotrophic factor BDNF, in an astrocyte-dependent manner through the purinergic P2Y1 receptor pathway. Our study identifies for the first time CD86 as a key player in the modulation of microglia phenotype and suggests that CTLA4-Ig-derived compounds might represent new tools to manipulate CNS microglia.

  6. DNA-Protein Crosslink Proteolysis Repair.

    Science.gov (United States)

    Vaz, Bruno; Popovic, Marta; Ramadan, Kristijan

    2017-06-01

    Proteins that are covalently bound to DNA constitute a specific type of DNA lesion known as DNA-protein crosslinks (DPCs). DPCs represent physical obstacles to the progression of DNA replication. If not repaired, DPCs cause stalling of DNA replication forks that consequently leads to DNA double-strand breaks, the most cytotoxic DNA lesion. Although DPCs are common DNA lesions, the mechanism of DPC repair was unclear until now. Recent work unveiled that DPC repair is orchestrated by proteolysis performed by two distinct metalloproteases, SPARTAN in metazoans and Wss1 in yeast. This review summarizes recent discoveries on two proteases in DNA replication-coupled DPC repair and establishes DPC proteolysis repair as a separate DNA repair pathway for genome stability and protection from accelerated aging and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Binding of SEC11 indicates its role in SNARE recycling after vesicle fusion and identifies two pathways for vesicular traffic to the plasma membrane.

    Science.gov (United States)

    Karnik, Rucha; Zhang, Ben; Waghmare, Sakharam; Aderhold, Christin; Grefen, Christopher; Blatt, Michael R

    2015-03-01

    SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.

  8. Sub-lethal oxidative stress induces lysosome biogenesis via a lysosomal membrane permeabilization-cathepsin-caspase 3-transcription factor EB-dependent pathway.

    Science.gov (United States)

    Leow, San Min; Chua, Shu Xian Serene; Venkatachalam, Gireedhar; Shen, Liang; Luo, Le; Clement, Marie-Veronique

    2016-12-18

    Here we provide evidence to link sub-lethal oxidative stress to lysosomal biogenesis. Exposure of cells to sub-lethal concentrations of exogenously added hydrogen peroxide resulted in cytosol to nuclear translocation of the Transcription Factor EB (TFEB), the master controller of lysosome biogenesis and function. Nuclear translocation of TFEB was dependent upon the activation of a cathepsin-caspase 3 signaling pathway, downstream of a lysosomal membrane permeabilization and accompanied by a significant increase in lysosome numbers as well as induction of TFEB dependent lysosome-associated genes expression such as Ctsl, Lamp2 and its spliced variant Lamp2a, Neu1and Ctsb and Sqstm1 and Atg9b. The effects of sub-lethal oxidative stress on lysosomal gene expression and biogenesis were rescued upon gene silencing of caspase 3 and TFEB. Notably, caspase 3 activation was not associated with phenotypic hallmarks of apoptosis, evidenced by the absence of caspase 3 substrate cleavage, such as PARP, Lamin A/C or gelsolin. Taken together, these data demonstrate for the first time an unexpected and non-canonical role of a cathepsin-caspase 3 axis in the nuclear translocation of TFEB leading to lysosomes biogenesis under conditions of sub-lethal oxidative stress.

  9. Hydroxylation of multi-walled carbon nanotubes: Enhanced biocompatibility through reduction of oxidative stress initiated cell membrane damage, cell cycle arrestment and extrinsic apoptotic pathway.

    Science.gov (United States)

    Liu, Zhenbao; Liu, Yanfei; Peng, Dongming

    2016-10-01

    Modification of CNTs with hydroxyl group promotes their applications in biomedical area. However, the impact of hydroxylation on their biocompatibility is far from being completely understood. In this study, we carried out a comprehensive evaluation of hydroxylated multi-walled carbon nanotubes (MWCNTs-OH) on the human normal liver L02 cell line, and compared it with that of pristine multi-walled carbon nanotubes (p-MWCNTs). Results demonstrated that compared with p-MWCNTs, MWCNTs-OH induced significantly lower oxidative stress as indicated by the level of intracellular antioxidant glutathione (GSH), subsequently lead to less cell membrane damage as demonstrated by lactate dehydrogenase (LDH) leakage assay, and showed slightly decreased arrestment of cell cycle distribution at G0/G1. More interestingly, MWCNTs-OH exhibited significantly lower tendency to activate caspase-8, a key molecule involved in the extrinsic apoptotic pathway. All these in vitro results demonstrated that hydroxylation of MWCNTs enhanced their biocompatibility compare with p-MWCNTs.

  10. Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1 (MTCBP-1) acts as an eukaryotic aci-reductone dioxygenase (ARD) in the methionine salvage pathway.

    Science.gov (United States)

    Hirano, Wakako; Gotoh, Isamu; Uekita, Takamasa; Seiki, Motoharu

    2005-06-01

    MTCBP-1 was identified as a protein that binds the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14). Since MTCBP-1 has a putative beta-barrel structure, it is presumably a member of the recently proposed cupin superfamily that contains tremendously diverged functions of proteins in spite of their well-conserved beta-barrel structure. MTCBP-1 shows significant homology to the bacterial aci-reductone dioxygenase (ARD) in the cupin family, which is an enzyme in the methionine salvage pathway (MTA cycle). Since it is difficult to speculate the functions of cupin proteins simply based on their sequence homology, we examined whether the eukaryotic ARD homologs surely function in the methionine metabolism. Under sulfur-depleted conditions, yeast could grow when substrate of MTA cycle was provided. Disruption of the yeast ARD homolog, YMR009w gene, abolished ability of the cells to grow in this culture condition. Re-expression of either the YMR009w or MTCBP-1 gene restored the cell growth. Mutation analysis revealed that the glutamic acid residue in the beta-barrel fold and the N-terminal extension from the beta-barrel fold were found to be important for the activity to restore the growth. Thus, MTCBP-1 isolated as a binding protein for MT1-MMP was demonstrated to function as an ARD-like enzyme in the MTA cycle in yeast.

  11. DNA repair in species with extreme lifespan differences

    Science.gov (United States)

    MacRae, Sheila L.; Croken, Matthew McKnight; Calder, R.B.; Aliper, Alexander; Milholland, Brandon; White, Ryan R.; Zhavoronkov, Alexander; Gladyshev, Vadim N.; Seluanov, Andrei; Gorbunova, Vera; Zhang, Zhengdong D.; Vijg, Jan

    2015-01-01

    Differences in DNA repair capacity have been hypothesized to underlie the great range of maximum lifespans among mammals. However, measurements of individual DNA repair activities in cells and animals have not substantiated such a relationship because utilization of repair pathways among animals—depending on habitats, anatomical characteristics, and life styles—varies greatly between mammalian species. Recent advances in high-throughput genomics, in combination with increased knowledge of the genetic pathways involved in genome maintenance, now enable a comprehensive comparison of DNA repair transcriptomes in animal species with extreme lifespan differences. Here we compare transcriptomes of liver, an organ with high oxidative metabolism and abundant spontaneous DNA damage, from humans, naked mole rats, and mice, with maximum lifespans of ∼120, 30, and 3 years, respectively, with a focus on genes involved in DNA repair. The results show that the longer-lived species, human and naked mole rat, share higher expression of DNA repair genes, including core genes in several DNA repair pathways. A more systematic approach of signaling pathway analysis indicates statistically significant upregulation of several DNA repair signaling pathways in human and naked mole rat compared with mouse. The results of this present work indicate, for the first time, that DNA repair is upregulated in a major metabolic organ in long-lived humans and naked mole rats compared with short-lived mice. These results strongly suggest that DNA repair can be considered a genuine longevity assurance system. PMID:26729707

  12. Benzo[a]pyrene induced p53-mediated cell cycle arrest, DNA repair, and apoptosis pathways in Chinese rare minnow (Gobiocypris rarus).

    Science.gov (United States)

    Yuan, Lilai; Lv, Biping; Zha, Jinmiao; Wang, Zijian

    2017-03-01

    The p53 pathways play an important role in carcinogenesis. In mammals, p53 and p53 target genes have been extensively studied, but little is known about their functions and regulation in fish. In this study, the cDNA fragments of p53 network genes, including p53, p21, mdm2, gadd45α, gadd45β, igfbp-3, and bax, were cloned from Chinese rare minnow (Gobiocypris rarus). These genes displayed high amino acid sequence identities with their zebrafish orthologs. The mRNA levels of p53 network genes and pathological changes in the liver were determined after adult rare minnow were exposed to 0.4, 2, and 10 µg/L of benzo[a]pyrene (BaP) for 28 days. The results showed that p53, p21, mdm2, gadd45α, and bax mRNA expressions in the livers from males and females were significantly upregulated compared with those of the controls (p p53 network genes in the livers suggest that rare minnow is suitable as an experimental fish to screen environmental carcinogens. In addition, the p53 network genes in rare minnow could feasibly be used to identify the mechanism of environmental carcinogenesis. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 979-988, 2017.

  13. Understanding the molecular pathways associated with seed vigor.

    Science.gov (United States)

    Ventura, Lorenzo; Donà, Mattia; Macovei, Anca; Carbonera, Daniela; Buttafava, Armando; Mondoni, Andrea; Rossi, Graziano; Balestrazzi, Alma

    2012-11-01

    Farmers and growers are constantly looking for high quality seeds able to ensure uniform field establishment and increased production. Seed priming is used to induce pre-germinative metabolism and then enhance germination efficiency and crop yields. It has been hypothesized that priming treatments might also improve stress tolerance in germinating seeds, leaving a sort of 'stress memory'. However, the molecular bases of priming still need to be clarified and the identification of molecular indicators of seed vigor is nowadays a relevant goal for the basic and applied research in seed biology. It is generally acknowledged that enhanced seed vigor and successful priming depend on DNA repair mechanisms, activated during imbibition. The complexity of the networks of DNA damage control/repair functions has been only partially elucidated in plants and the specific literature that address seeds remains scanty. The DNA repair pathways hereby described (Nucleotide and Base Excision Repair, Non-Homologous End Joining, Homologous Recombination) play specific roles, all of them being critical to ensure genome stability. This review also focuses on some novel regulatory mechanisms of DNA repair (chromatin remodeling and small RNAs) while the possible use of telomere sequences as markers of aging in seed banks is discussed. The significant contribution provided by Electron Paramagnetic Resonance in elucidating the kinetics of seed aging, in terms of free radical profiles and membrane integrity is reported.

  14. Kaposi Sarcoma Herpesvirus (KSHV Latency-Associated Nuclear Antigen (LANA recruits components of the MRN (Mre11-Rad50-NBS1 repair complex to modulate an innate immune signaling pathway and viral latency.

    Directory of Open Access Journals (Sweden)

    Giuseppe Mariggiò

    2017-04-01

    Full Text Available Kaposi Sarcoma Herpesvirus (KSHV, a γ2-herpesvirus and class 1 carcinogen, is responsible for at least three human malignancies: Kaposi Sarcoma (KS, Primary Effusion Lymphoma (PEL and Multicentric Castleman's Disease (MCD. Its major nuclear latency protein, LANA, is indispensable for the maintenance and replication of latent viral DNA in infected cells. Although LANA is mainly a nuclear protein, cytoplasmic isoforms of LANA exist and can act as antagonists of the cytoplasmic DNA sensor, cGAS. Here, we show that cytosolic LANA also recruits members of the MRN (Mre11-Rad50-NBS1 repair complex in the cytosol and thereby inhibits their recently reported role in the sensing of cytoplasmic DNA and activation of the NF-κB pathway. Inhibition of NF-κB activation by cytoplasmic LANA is accompanied by increased lytic replication in KSHV-infected cells, suggesting that MRN-dependent NF-κB activation contributes to KSHV latency. Cytoplasmic LANA may therefore support the activation of KSHV lytic replication in part by counteracting the activation of NF-κB in response to cytoplasmic DNA. This would complement the recently described role of cytoplasmic LANA in blocking an interferon response triggered by cGAS and thereby promoting lytic reactivation. Our findings highlight a second point at which cytoplasmic LANA interferes with the innate immune response, as well as the importance of the recently discovered role of cytoplasmic MRN complex members as innate sensors of cytoplasmic DNA for the control of KSHV replication.

  15. Large Extremity Peripheral Nerve Repair

    Science.gov (United States)

    2015-10-01

    IL, Kochevar IE, Redmond RW. Large extremity peripheral nerve repair. Military Health System Research Symposium (MHSRS) Fort Lauderdale, FL. August...some notable discoveries that may impact military health care in the near future. There is a clear need in military medicine to improve outcomes in...membranes or “caul” intact was considered extremely lucky. Children were gifted with life-long happiness , the ability to see spirits, and protection

  16. Large Extremity Peripheral Nerve Repair

    Science.gov (United States)

    2016-12-01

    71. Burman S, Tejwani S, Vemuganti GK. Ophthalmic applications of preserved human amniotic membrane: a review of current indications. Cell Tissue Bank...segmental nerve deficit repair using isograft show the best performing wrap/ fixation method to be sutureless photochemical tissue bonding with the...crosslinked amnion wrap. Autograft is often unavailable in wounded warriors, due to extensive tissue damage and amputation and, importantly, we also

  17. DMPD: CR3 (CD11b, CD18): a phagocyte and NK cell membrane receptor with multipleligand specificities and functions. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 8485905 CR3 (CD11b, CD18): a phagocyte and NK cell membrane receptor with multipleligand specificities...) (.html) (.csml) Show CR3 (CD11b, CD18): a phagocyte and NK cell membrane receptor with multipleligand specificities...d NK cell membrane receptor with multipleligand specificities and functions. Authors Ross GD, Vetvicka V. Pu

  18. Herpes Simplex Virus 1 Enters Human Keratinocytes by a Nectin-1-Dependent, Rapid Plasma Membrane Fusion Pathway That Functions at Low Temperature.

    Science.gov (United States)

    Sayers, Charlotte L; Elliott, Gillian

    2016-11-15

    Herpes simplex virus 1 (HSV-1) infects humans through stratified epithelia that are composed primarily of keratinocytes. The route of HSV-1 entry into keratinocytes has been the subject of limited investigation, but it is proposed to involve pH-dependent endocytosis, requiring the gD-binding receptor nectin-1. Here, we have utilized the nTERT human keratinocyte cell line as a new model for dissecting the mechanism of HSV-1 entry into the host. Although immortalized, these cells nonetheless retain normal growth and differentiation properties of primary cells. Using short interfering RNA (siRNA) depletion studies, we confirm that, despite nTERT cells expressing high levels of the alternative gD receptor HVEM, HSV-1 requires nectin-1, not HVEM, to enter these cells. Strikingly, virus entry into nTERT cells occurred with unusual rapidity, such that maximum penetration was achieved within 5 min. Moreover, HSV-1 was able to enter keratinocytes but not other cell types at temperatures as low as 7°C, conditions where endocytosis was shown to be completely inhibited. Transmission electron microscopy of early entry events at both 37°C and 7°C identified numerous examples of naked virus capsids located immediately beneath the plasma membrane, with no evidence of virions in cytoplasmic vesicles. Taken together, these results imply that HSV-1 uses the nectin-1 receptor to enter human keratinocyte cells via a previously uncharacterized rapid plasma membrane fusion pathway that functions at low temperature. These studies have important implications for current understanding of the relationship between HSV-1 and its relevant in vivo target cell. The gold standard of antiviral treatment for any human virus infection is the prevention of virus entry into the host cell. In the case of HSV-1, primary infection in the human begins in the epidermis of the skin or the oral mucosa, where the virus infects keratinocytes, and it is therefore important to understand the molecular events

  19. Experimental Research on the Combination of PHBV Membrane and Coralline Hydroxyapatite on Repair of Mandibular Defects%PHBV膜与珊瑚羟基磷灰石联合修复颌骨缺损的研究

    Institute of Scientific and Technical Information of China (English)

    鲁琴艳; 陈黄琴; 黄彬; 冯蕊

    2015-01-01

    Objective To investigate the effect of polyhydroxybutyrate -hydroxyvalerate ( PHBV) mem-brane combined with coralline hydroxyapatite ( CHA) on maxillary defects and its mechanism .Methods Forty-eight white healthy rabbits were divided into experimental group ( left side ) and control group ( right side ) . 12mm ×5mm ×4mm mandibular defect of bilateral mandibular corpus in the manufacture was made ,the PHBV membrane combined with CHA repaired defects in the experimental group , and only CHA repaired defects in the control group.Left and right sides of 12mm ×5mm ×4mm mandibular defects in six rabbits were used as blank group.Sixteen rabbits were sacrificed in4,8,12 weeks after operation,respectively.two in each period of control group .Results New bone could be observed in the center of defect area of experimental group after 4 weeks,but there hada large number of fibrous connective tissue in control group and new bone formation could only be seen in the bone-bed .There had many small bone cells in new bone tissue of experimental group after 12 weeks ,and some new bone cells arrange regularly as mature bone tissue .In control group ,part of bone tissue formation and the fibrous connective tissue was still visible in defect area of control group after 12 weeks.Con-clusion The combination of PHBV membrane and CHA has faster speed in bone formation of mandibular defects than that of only CHA treatment .PHBV membrane can inhibit fibrous tissue permeability and facilitate new bone formation through the mechanical property of membrane .%目的:通过聚羟基丁酸酯-羟基戊酸酯(PHBV)膜与珊瑚羟基磷灰石(CHA)联合修复颌骨缺损,来观察和探讨骨缺损的修复方式及成骨机制,为PHBV膜将来应用于临床提供理论依据。方法实验选择54只实验动物日本大耳白兔,随机抽取48只左侧为PHBV膜与CHA复合组(实验组),右侧为单纯CHA(对照组)。在下颌骨体部双侧造成12mm ×5mm ×4

  20. Eye muscle repair - discharge

    Science.gov (United States)

    ... Lazy eye repair - discharge; Strabismus repair - discharge; Extraocular muscle surgery - discharge ... You or your child had eye muscle repair surgery to correct eye muscle ... term for crossed eyes is strabismus. Children most often ...

  1. Ventral hernia repair

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/007661.htm Ventral hernia repair To use the sharing features on this page, please enable JavaScript. Ventral hernia repair is surgery to repair a ventral hernia. ...

  2. Brain aneurysm repair

    Science.gov (United States)

    ... aneurysm repair; Dissecting aneurysm repair; Endovascular aneurysm repair - brain; Subarachnoid hemorrhage - aneurysm ... Your scalp, skull, and the coverings of the brain are opened. A metal clip is placed at ...

  3. The Fanconi anaemia pathway: new players and new functions.

    Science.gov (United States)

    Ceccaldi, Raphael; Sarangi, Prabha; D'Andrea, Alan D

    2016-06-01

    The Fanconi anaemia pathway repairs DNA interstrand crosslinks (ICLs) in the genome. Our understanding of this complex pathway is still evolving, as new components continue to be identified and new biochemical systems are used to elucidate the molecular steps of repair. The Fanconi anaemia pathway uses components of other known DNA repair processes to achieve proper repair of ICLs. Moreover, Fanconi anaemia proteins have functions in genome maintenance beyond their canonical roles of repairing ICLs. Such functions include the stabilization of replication forks and the regulation of cytokinesis. Thus, Fanconi anaemia proteins are emerging as master regulators of genomic integrity that coordinate several repair processes. Here, we summarize our current understanding of the functions of the Fanconi anaemia pathway in ICL repair, together with an overview of its connections with other repair pathways and its emerging roles in genome maintenance.

  4. Changes in DNA repair during aging

    Science.gov (United States)

    Gorbunova, Vera; Seluanov, Andrei; Mao, Zhiyong; Hine, Christpher

    2007-01-01

    DNA is a precious molecule. It encodes vital information about cellular content and function. There are only two copies of each chromosome in the cell, and once the sequence is lost no replacement is possible. The irreplaceable nature of the DNA sets it apart from other cellular molecules, and makes it a critical target for age-related deterioration. To prevent DNA damage cells have evolved elaborate DNA repair machinery. Paradoxically, DNA repair can itself be subject to age-related changes and deterioration. In this review we will discuss the changes in efficiency of mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER) and double-strand break (DSB) repair systems during aging, and potential changes in DSB repair pathway usage that occur with age. Mutations in DNA repair genes and premature aging phenotypes they cause have been reviewed extensively elsewhere, therefore the focus of this review is on the comparison of DNA repair mechanisms in young versus old. PMID:17913742

  5. Biological membrane for repair of different sizes of bone defects surrounding BLB implants%生物膜对不同范围BLB种植体周骨缺损修复的影像学观察

    Institute of Scientific and Technical Information of China (English)

    孟维艳; 周延民; 储顺礼; 杨立明; 蔡青

    2008-01-01

    was 3 mm or less.no significant difference in bone density and bone trabecular morphology and orientating was found between newly formed bone and host bone no matter whether biological membrane was used or not;in the 4-mm-length bone defect areas.implants contacted with newly formed bone directly,but the calcified degree ofnewly formed bone was poor,bone trabecula was thin,and bone trabecular course was irregular,nevertheless,the calcified degree of newly formed bone was better under the condition of being with biological membrane than without biological membrane.CONCLUSION:Biological membrane exhibits strong capacity to promote the regeneration and repair of bone defect tissue with a horizontal length of 3 mm or less,and plays an important role in repatr of large sizes of bone detect%背景:即刻种植体周与骨之间常存在着间隙,究竟是否需要应用生物膜进行引导骨再生目前仍无定论.目的:通过在犬的股骨植入种植体创造不同的骨缺损间隙,观察生物膜对不同范围骨缺损骨组织再生修复的影响.设计、时间及地点:同体对照动物实验,于2005-03/12在吉林大学白求恩医学院实验动物中心及吉林大学口腔医院完成.材料:BLB羟基磷灰石涂层种植体由北京莱顿公司提供,BME-10X医用胶原膜由福建省博特生物科技有限公司提供.方法:在犬两侧股骨近心端植入种植体,并形成水平宽度3mm,垂直深度约5mm、水平长度分别为0,1,2,3,4 mm的标准梯度骨缺损.左侧直接分层严密缝合,右侧于骨缺损区覆盖生物膜后分层缝合.术后3个月处死动物,取含种植体的骨段,制成组织块进行影像学观察.主要观察指标:表面及剖面观察种植体周围骨缺损区新骨形成的量、色泽、质地等情况.应用软X射线观察种植体与骨组织界面的骨结合情况以及骨缺损区新骨形成情况.结果:大体观察:3 mm以下骨缺损区与原皮质骨无界限,有膜组与无膜组基本相同:4

  6. Molecular mechanism of cellular reception of ionizing radiation and of activation of signal transduction pathway

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Keiji [Nagasaki Univ. (Japan). Faculty of Pharmaceutical Sciences

    1997-03-01

    The author reviewed what in cells receives ionizing radiation as a stress and which signal transduction pathway is activated to induce the stress reaction in the following order: Activation of protein kinase C (PKC) pathway by radiation, activation of MAP kinase superfamily by radiation, induction of p53 function by radiation, and radiation exposure and stress reaction pathway. Conclusion was as follows: Cellular receptors to radiation can be cell membrane and DNA. Membrane reception of radiation induces activation of tyrosine kinase and sphingomyelinase, which resulting in activation of PKC- and MAP kinase-mediated signal transduction. The signal generated in the nucleus participates in regulation of cell cycle and in DNA repair. Therefore, it seems that irradiation of ionizing radiation gives energy to various cellular receptor sites as well as DNA, which generate various independent signals to be transduced and accumulated in the nucleus, and leading to cellular response. (K.H.). 63 refs.

  7. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells [v1; ref status: indexed, http://f1000r.es/yx

    Directory of Open Access Journals (Sweden)

    Michael Hartmann

    2013-08-01

    Full Text Available We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL. By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1, shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.

  8. The role of polymorphisms of genes repair pathway to the radiotoxicity in patients with cancer of the cervix; O papel dos polimorfismos de genes da via de reparo com a radiotoxidade em pacientes com cancer de colo uterino

    Energy Technology Data Exchange (ETDEWEB)

    Carvalho, Ana Terra Silva

    2012-07-01

    Background: In Brazil, cervical cancer is the second most common among women. Radiation therapy is part of its interdisciplinary management, playing an important role in their loco regional control. The major challenge of modern medicine in radiotherapy is to develop predictive methods that can determine the level of radiosensitivity of the patient and the healthy surrounding tissue in order to individualize the prescribed radiation dose, to prevent severe side effects and promoting better local tumor control. This study evaluated the acute and chronic adverse effects on the skin, lower gastrointestinal tract and urinary tract of radiotherapy in 47 cervical cancer patients. Methods and Materials: Biological material was collected and DNA from peripheral blood was extracted of ali patients studied. The fragments of TP53 and ATM were amplified to be sequenced, to verify if there are any polymorphisms witch could be responsible to the radiosensitivity of the patients. Results and Discussion: In a univariate analysis, the variable age was strongly associated with a risk of acute toxicity skin (p=O,023). Patients that received a high dose of external beam radiation and patients who have undergone brachytherapy, showed a significantly higher incidence of chronic urinary tract toxicity (p=O,031) and (p=O,019), respectively. The exchange G>A in the position 5557 of the A TM gene was significantly associated with the risk of acute lower gastrointestinal tract (p=O,008). There wasn't association between the other TP53 polymorphisms analyzed and the frequency of side effects (p>O,05). Our data revealed that patients who evolved significant association presented death (p=O,019) with the increase of chronic skin radiossensitivity. Conclusions: These observations corroborate the importance of investigating the genetic profile to predict adverse side effects in cervical cancer patients undergoing radiotherapy. These genes have an important role in DNA repair pathways and

  9. Prostate specific membrane antigen knockdown impairs the tumorigenicity of LNCaP prostate cancer cells by inhibiting the phosphatidylinositol 3-kinase/Akt signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Guo Zhenghui; Lai Yiming; Du Tao; Zhang Yiming; Chen Jieqing; Bi Liangkuan; Lin Tianxin

    2014-01-01

    Background Prostate specific membrane antigen (PSMA) can facilitate the growth,migration,and invasion of the LNCaP prostate cancer cell lines,but the underlying molecular mechanisms have not yet been clearly defined.Here,we investigated whether PSMA serves as a novel regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling by employing PSMA knockdown model and PI3K pharmacological inhibitor (LY294002) in LNCaP prostate cancer cells.Methods PSMA knockdown had been stably established by transfecting with lentivirus-mediated siRNA in our previous study.Then,LNCaP cells were divided into interference,non-interference,and blank groups.We first testified the efficacy of PSMA knockdown in our LNCaP cell line.Then,we compared the expression of PSMA and total/activated Akt by Westem blotting in the above three groups with or without LY294002 treatment.Furthermore,immunocytochemistry was performed to confirm the changes of activated Akt (p-Akt,Ser473) in groups.Besides,cell proliferation,migration,and cell cycle were measured by CCK-8 assay,Transwell analysis,and Flow cytometry respectively.Results After PSMA knockdown,the level of p-Akt (Ser473) but not of total-Akt (Akt1/2) was significantly decreased when compared with the non-interference and blank groups.However,LY294002 administration significantly reduced the expression of p-Akt (Ser473) in all the three groups.The results of immunocytochemistry further confirmed that PSMA knockdown or LY294002 treatment was associated with p-Akt (Ser473) down-regulation.Decrease of cell proliferation,migration,and survival were also observed upon PSMA knockdown and LY294002 treatment.Conclusions Taken together,our results reveal that PI3K/Akt signaling pathway inhibition may serve as a novel molecular mechanism in LNCaP prostate cancer cells of PSMA knockdown and suggest that Akt (Ser473) may play a critical role as a downstream signaling target effector of PSMA in this cellular model.

  10. DNA repair. [UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, R.

    1978-01-01

    Some topics discussed are as follows: difficulty in extrapolating data from E. coli to mammalian systems; mutations caused by UV-induced changes in DNA; mutants deficient in excision repair; other postreplication mechanisms; kinds of excision repair systems; detection of repair by biochemical or biophysical means; human mutants deficient in repair; mutagenic effects of UV on XP cells; and detection of UV-repair defects among XP individuals. (HLW)

  11. Chromatin challenges during DNA replication and repair

    DEFF Research Database (Denmark)

    Groth, Anja; Rocha, Walter; Verreault, Alain

    2007-01-01

    Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet...... the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic...

  12. Pore dynamics in lipid membranes

    Science.gov (United States)

    Gozen, I.; Dommersnes, P.

    2014-09-01

    Transient circular pores can open in plasma membrane of cells due to mechanical stress, and failure to repair such pores lead to cell death. Similar pores in the form of defects also exist among smectic membranes, such as in myelin sheaths or mitochondrial membranes. The formation and growth of membrane defects are associated with diseases, for example multiple sclerosis. A deeper understanding of membrane pore dynamics can provide a more refined picture of membrane integrity-related disease development, and possibly also treatment options and strategies. Pore dynamics is also of great importance regarding healthcare applications such as drug delivery, gene or as recently been implied, cancer therapy. The dynamics of pores significantly differ in stacks which are confined in 2D compared to those in cells or vesicles. In this short review, we will summarize the dynamics of different types of pores that can be observed in biological membranes, which include circular transient, fusion and hemi-fusion pores. We will dedicate a section to floral and fractal pores which were discovered a few years ago and have highly peculiar characteristics. Finally, we will discuss the repair mechanisms of large area pores in conjunction with the current cell membrane repair hypotheses.

  13. Envisioning the molecular choreography of DNA base excision repair.

    Science.gov (United States)

    Parikh, S S; Mol, C D; Hosfield, D J; Tainer, J A

    1999-02-01

    Recent breakthroughs integrate individual DNA repair enzyme structures, biochemistry and biology to outline the structural cell biology of the DNA base excision repair pathways that are essential to genome integrity. Thus, we are starting to envision how the actions, movements, steps, partners and timing of DNA repair enzymes, which together define their molecular choreography, are elegantly controlled by both the nature of the DNA damage and the structural chemistry of the participating enzymes and the DNA double helix.

  14. DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

    Energy Technology Data Exchange (ETDEWEB)

    Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A., E-mail: sarah.martin@qmul.ac.uk [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ (United Kingdom)

    2014-08-05

    Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting.

  15. Suppressed expression of non-DSB repair genes inhibits gamma-radiation-induced cytogenetic repair and cell cycle arrest.

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish K; Jeevarajan, Antony S; Pierson, Duane L; Wu, Honglu

    2008-11-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in regulating DSB repair and cell cycle progression. In this study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequency of micronuclei (MN) formation and chromosome aberrations were measured to determine efficiency of cytogenetic repair, especially DSB repair. In response to IR, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that

  16. Mfd as a central partner of transcription coupled repair.

    Science.gov (United States)

    Monnet, Jordan; Grange, Wilfried; Strick, Terence R; Joly, Nicolas

    2013-01-01

    Transcription-coupled repair (TCR) is one of the key of the nucleotide excision repair (NER) pathways required to preserve genome integrity. Although understanding TCR is still a major challenge, recent single-molecule experiments have brought new insights into the initial steps of TCR leading to new perspectives.

  17. Transcription-coupled repair: an update.

    Science.gov (United States)

    Spivak, Graciela

    2016-11-01

    Nucleotide excision repair (NER) is a versatile pathway that removes helix-distorting DNA lesions from the genomes of organisms across the evolutionary scale, from bacteria to humans. The serial steps in NER involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. Transcription-coupled repair (TCR) is a subpathway of NER dedicated to the repair of lesions that, by virtue of their location on the transcribed strands of active genes, encumber elongation by RNA polymerases. In this review, I report on recent findings that contribute to the elucidation of TCR mechanisms in the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae and human cells. I review general models for the biochemical pathways and how and when cells might choose to utilize TCR or other pathways for repair or bypass of transcription-blocking DNA alterations.

  18. Epigenetic reduction of DNA repair in progression togastrointestinal cancer

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Deficiencies in DNA repair due to inherited germ-linemutations in DNA repair genes cause increased risk ofgastrointestinal (GI) cancer. In sporadic GI cancers,mutations in DNA repair genes are relatively rare.However, epigenetic alterations that reduce expressionof DNA repair genes are frequent in sporadic GI cancers.These epigenetic reductions are also found in fielddefects that give rise to cancers. Reduced DNA repairlikely allows excessive DNA damages to accumulatein somatic cells. Then either inaccurate translesionsynthesis past the un-repaired DNA damages or errorproneDNA repair can cause mutations. ErroneousDNA repair can also cause epigenetic alterations (i.e. ,epimutations, transmitted through multiple replicationcycles). Some of these mutations and epimutations maycause progression to cancer. Thus, deficient or absentDNA repair is likely an important underlying cause ofcancer. Whole genome sequencing of GI cancers showthat between thousands to hundreds of thousands ofmutations occur in these cancers. Epimutations thatreduce DNA repair gene expression and occur early inprogression to GI cancers are a likely source of this highgenomic instability. Cancer cells deficient in DNA repairare more vulnerable than normal cells to inactivation byDNA damaging agents. Thus, some of the most clinicallyeffective chemotherapeutic agents in cancer treatmentare DNA damaging agents, and their effectivenessoften depends on deficient DNA repair in cancer cells.Recently, at least 18 DNA repair proteins, each activein one of six DNA repair pathways, were found to besubject to epigenetic reduction of expression in GIcancers. Different DNA repair pathways repair differenttypes of DNA damage. Evaluation of which DNA repairpathway(s) are deficient in particular types of GI cancerand/or particular patients may prove useful in guidingchoice of therapeutic agents in cancer therapy.

  19. 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.

  20. Application of Hydroxyapatite Bioceramics and Oral Repair Membrane in the Surgery of Apical Syst%羟基磷灰石生物陶瓷及口腔修复膜在根尖囊肿手术中的应用

    Institute of Scientific and Technical Information of China (English)

    钱楚凡; 李小琳; 王栋

    2015-01-01

    Objective:To study the application of hydroxyapatite bioceramics and oral repair membrane in the surgery of apical syst and provide theory basis for jaw bone defect repair surgery of apical syst.Method:64 patients with apical syst in our hospital from June 2013 to May 2014 were selected and randomly divided into the control group and the observation group,with 32 cases in each group.The control group was treated with gelatin sponge filling repair,the observation group was treated with hydroxyapatite bioceramics filling and oral repair membrane for film repair.The clinical efficacy of the two groups was compared. Result:6 months after surgery,the height and thickness of bone graft and the bone growth effect in the observation group were better than those in the control group,the new bone density increased rate of the observation group was higher than that of the control group,the differences above were all statistically significant(P<0.05).Conclusion:Hydroxyapatite bioceramics and oral repair membrane applied in the jaw bone defect repair surgery of apical syst has a good effect,it is worth spreading.%目的:探讨羟基磷灰石生物陶瓷及口腔修复膜在根尖囊肿手术中的应用,为根尖囊肿颌骨缺损修复手术提供理论依据。方法:本研究选取2013年6月-2014年5月本院收治的64例根尖囊肿患者作为研究对象,采用随机数字表法将其分为对照组和观察组,每组32例,对照组采用明胶海棉填充修复,观察组采用羟基磷灰石生物陶瓷填充及口腔修复膜覆盖修复,比较两组患者的治疗效果。结果:手术后6个月,观察组植骨高度、厚度及骨生长效果均优于对照组,观察组新生骨密度明显增加比率高于对照组,比较差异均有统计学意义(P<0.05)。结论:羟基磷灰石生物陶瓷及口腔修复膜应用于根尖囊肿颌骨缺损修复手术具有良好效果,值得推广。

  1. DNA repair genes in the Megavirales pangenome.

    Science.gov (United States)

    Blanc-Mathieu, Romain; Ogata, Hiroyuki

    2016-06-01

    The order 'Megavirales' represents a group of eukaryotic viruses with a large genome encoding a few hundred up to two thousand five hundred genes. Several members of Megavirales possess genes involved in major DNA repair pathways. Some of these genes were likely inherited from an ancient virus world and some others were derived from the genomes of their hosts. Here we examine molecular phylogenies of key DNA repair enzymes in light of recent hypotheses on the origin of Megavirales, and propose that the last common ancestors of the individual families of the order Megavirales already possessed DNA repair functions to achieve and maintain a moderately large genome and that this repair capacity gradually increased, in a family-dependent manner, during their recent evolution.

  2. Pectus excavatum repair

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/002949.htm Pectus excavatum repair To use the sharing features on this page, please enable JavaScript. Pectus excavatum repair is surgery to correct pectus excavatum . This ...

  3. 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.

  4. When "Other" Initiate Repair.

    Science.gov (United States)

    Schegloff, Emanuel A.

    2000-01-01

    Elaborates on the locus of other-initiated repair, and reports on a number of environments in which others initiate repair turns later than the one directly following the trouble-source turn. Describes several ways that other initiation of repair, which occurs in next-turn position, may be delayed within that position. (Author/VWL)

  5. Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.

    Science.gov (United States)

    Swaggart, Kayleigh A; Demonbreun, Alexis R; Vo, Andy H; Swanson, Kaitlin E; Kim, Ellis Y; Fahrenbach, John P; Holley-Cuthrell, Jenan; Eskin, Ascia; Chen, Zugen; Squire, Kevin; Heydemann, Ahlke; Palmer, Abraham A; Nelson, Stanley F; McNally, Elizabeth M

    2014-04-22

    Many monogenic disorders, including the muscular dystrophies, display phenotypic variability despite the same disease-causing mutation. To identify genetic modifiers of muscular dystrophy and its associated cardiomyopathy, we used quantitative trait locus mapping and whole genome sequencing in a mouse model. This approach uncovered a modifier locus on chromosome 11 associated with sarcolemmal membrane damage and heart mass. Whole genome and RNA sequencing identified Anxa6, encoding annexin A6, as a modifier gene. A synonymous variant in exon 11 creates a cryptic splice donor, resulting in a truncated annexin A6 protein called ANXA6N32. Live cell imaging showed that annexin A6 orchestrates a repair zone and cap at the site of membrane disruption. In contrast, ANXA6N32 dramatically disrupted the annexin A6-rich cap and the associated repair zone, permitting membrane leak. Anxa6 is a modifier of muscular dystrophy and membrane repair after injury.

  6. Double-strand break repair and G4 DNA stability in Caenorhabditis elegans

    NARCIS (Netherlands)

    Pontier, D.B.

    2010-01-01

    DNA double-strand breaks (DSBs) can be repaired by three canonical repair pathways. Homologous recombination (HR) uses the sister chromatid or homologous chromosome as a template to repair the DSB in an error-free manner. In non-homologous end-joining (NHEJ), the broken ends are ligated with little

  7. Double-strand break repair and G4 DNA stability in Caenorhabditis elegans

    NARCIS (Netherlands)

    Pontier, D.B.

    2010-01-01

    DNA double-strand breaks (DSBs) can be repaired by three canonical repair pathways. Homologous recombination (HR) uses the sister chromatid or homologous chromosome as a template to repair the DSB in an error-free manner. In non-homologous end-joining (NHEJ), the broken ends are ligated with little

  8. DNA repair and gene targeting in plant end-joining mutants

    NARCIS (Netherlands)

    Jia, Qi

    2011-01-01

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or by non-homologous end joining (NHEJ). The latter mechanism is the major route for DSB repair in the somatic cells of higher eukaryotes, including plants. If we could manipulate the balance of the DSB repair pathways

  9. DNA repair and gene targeting in plant end-joining mutants

    NARCIS (Netherlands)

    Jia, Qi

    2011-01-01

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or by non-homologous end joining (NHEJ). The latter mechanism is the major route for DSB repair in the somatic cells of higher eukaryotes, including plants. If we could manipulate the balance of the DSB repair pathways

  10. Understanding the molecular mechanism of formaldehyde-induced DNA-protein crosslink repair

    Science.gov (United States)

    Formaldehyde induces DNA-protein crosslinks (DPCs) in several experimental in vitro and in vivo test systems, as well as in exposed human workers. DPCs are repaired by several DNA repair pathways in different species, but the molecular understanding of DPC repair in human tissues...

  11. Targeting the Fanconi Anemia Pathway to Identify Tailored Anticancer Therapeutics

    Directory of Open Access Journals (Sweden)

    Chelsea Jenkins

    2012-01-01

    Full Text Available The Fanconi Anemia (FA pathway consists of proteins involved in repairing DNA damage, including interstrand cross-links (ICLs. The pathway contains an upstream multiprotein core complex that mediates the monoubiquitylation of the FANCD2 and FANCI heterodimer, and a downstream pathway that converges with a larger network of proteins with roles in homologous recombination and other DNA repair pathways. Selective killing of cancer cells with an intact FA pathway but deficient in certain other DNA repair pathways is an emerging approach to tailored cancer therapy. Inhibiting the FA pathway becomes selectively lethal when certain repair genes are defective, such as the checkpoint kinase ATM. Inhibiting the FA pathway in ATM deficient cells can be achieved with small molecule inhibitors, suggesting that new cancer therapeutics could be developed by identifying FA pathway inhibitors to treat cancers that contain defects that are synthetic lethal with FA.

  12. The Vasopressin Type-2 Receptor and Prostaglandin Receptors EP2 and EP4 can Increase Aquaporin-2 Plasma Membrane Targeting Through a cAMP Independent Pathway

    DEFF Research Database (Denmark)

    Olesen, Emma Tina Bisgaard; Moeller, Hanne Bjerregaard; Assentoft, Mette

    2016-01-01

    Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be cA...

  13. Identification of Pathways Required for the Coordination of Late Mitotic Events in Animal Cells

    Science.gov (United States)

    2006-08-01

    prevent the recognition of chromosome ends by DNA repair pathways. Repair of telomeres as DSBs can lead to dicentric chromosomes , which are very...a more appropriate telomere-specific pathway. If telomeres are repaired by NHEJ, dicentric chromosomes are created, which lead to breakage-fusion...chromatin structures that protect chromosomes ends from the DNA repair pathways. Telomeres are re-formed after each round of DNA replication. The

  14. Human longevity and variation in DNA damage response and repair

    DEFF Research Database (Denmark)

    Debrabant, Birgit; Soerensen, Mette; Flachsbart, Friederike

    2014-01-01

    others. Data were applied on 592 SNPs from 77 genes involved in nine sub-processes: DNA-damage response, base excision repair (BER), nucleotide excision repair, mismatch repair, non-homologous end-joining, homologous recombinational repair (HRR), RecQ helicase activities (RECQ), telomere functioning...... and mitochondrial DNA processes. The study population was 1089 long-lived and 736 middle-aged Danes. A self-contained set-based test of all SNPs displayed association with longevity (P-value=9.9 × 10-5), supporting that the overall pathway could affect longevity. Investigation of the nine sub-processes using...

  15. Repair of large tympanic membrane perforation with inner graft using Portmann' s posterior-superior incision retro-auricular approach: a report of 45 cases%Portmann氏耳后上进路全翻内植法修补鼓膜大穿孔45例报告

    Institute of Scientific and Technical Information of China (English)

    董和霞; 王晓辉

    2011-01-01

    目的 探讨Portmann氏耳后上进路全翻内植法修补鼓膜大穿孔的方法及疗效.方法 采用耳后上进路,自体颞肌筋膜作为移植材料,对45例鼓膜大穿孔病人进行全翻外耳道后壁皮瓣鼓膜成形术,并对其临床资料进行回顾性分析.结果 术后6个月随访,43例鼓膜穿孔愈合,2例出现鼓膜中央小穿孔,听力均有不同程度的提高.结论 本术式术野暴露清晰,取颞肌筋膜在同一切口进行,修补鼓膜大穿孔成功率高,具有较高的临床应用价值.%Objective To report techniques and effects of myringoplasty using Portmann' s posterior-superior incision retro-auricular approach with complete tympanic membrane elevation and inner graft for repair large size perforations. Methods Myringoplasty was performed in 45 patients. Following tympanic membrane elevation, temporal fascia was used as the autograft and placed on the inner side. Results Forty-three cases acheieved first stage healing of perforation At six month follow up, the tympanic membrane remained healed in 43 cases and showed small central perforation in 2 cases. Hearing in all patients was improved after operations. Conclusion This operative approach allows clear visualization and harvesting of temporal fascia in the same field. The success rate of repairing large perforation with this technique is higher, deserving extensive use.

  16. Percutaneous mitral valve repair.

    Science.gov (United States)

    Gillinov, A Marc; Liddicoat, John R

    2006-01-01

    Surgical mitral valve repair is the procedure of choice to treat mitral regurgitation of all etiologies. Whereas annuloplasty is the cornerstone of mitral valve repair, a variety of other surgical techniques are utilized to correct dysfunction of the leaflets and subvalvular apparatus; in most cases, surgical repair entails application of multiple repair techniques in each patient. Preclinical studies and early human experience have demonstrated that some of these surgical repair techniques can be performed using percutaneous approaches. Specifically, there has been great progress in the development of novel technology to facilitate percutaneous annuloplasty and percutaneous edge-to-edge repair. The objectives of this report were to (1) discuss the surgical foundations for these percutaneous approaches; (2) review device design and experimental and clinical results of percutaneous valve repair; and (3) address future directions, including the key challenges of patient selection and clinical trial design.

  17. Replication protein A: single-stranded DNA's first responder: dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair.

    Science.gov (United States)

    Chen, Ran; Wold, Marc S

    2014-12-01

    Replication protein A (RPA), the major single-stranded DNA-binding protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high-affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error-prone repair of double-stranded breaks and promote error-free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis. © 2014 WILEY Periodicals, Inc.

  18. DNA repair in neurons: So if they don't divide what's to repair?

    Energy Technology Data Exchange (ETDEWEB)

    Fishel, Melissa L. [Department of Pediatrics (Section of Hematology/Oncology), Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States); Vasko, Michael R. [Department of Pharmacology and Toxicology, Indiana University School of Medicine, 1044 W. Walnut St., Indianapolis, IN 46202 (United States); Kelley, Mark R. [Department of Pediatrics (Section of Hematology/Oncology), Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States) and Department of Pharmacology and Toxicology, Indiana University School of Medicine, 1044 W. Walnut St., Indianapolis, IN 46202 (United States) and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 1044 W. Walnut, Room 302C, Indianapolis, IN 46202 (United States)]. E-mail: mkelley@iupui.edu

    2007-01-03

    Neuronal DNA repair remains one of the most exciting areas for investigation, particularly as a means to compare the DNA repair response in mitotic (cancer) vs. post-mitotic (neuronal) cells. In addition, the role of DNA repair in neuronal cell survival and response to aging and environmental insults is of particular interest. DNA damage caused by reactive oxygen species (ROS) such as generated by mitochondrial respiration includes altered bases, abasic sites, and single- and double-strand breaks which can be prevented by the DNA base excision repair (BER) pathway. Oxidative stress accumulates in the DNA of the human brain over time especially in the mitochondrial DNA (mtDNA) and is proposed to play a critical role in aging and in the pathogenesis of several neurological disorders including Parkinson's disease, ALS, and Alzheimer's diseases. Because DNA damage accumulates in the mtDNA more than nuclear DNA, there is increased interest in DNA repair pathways and the consequence of DNA damage in the mitochondria of neurons. The type of damage that is most likely to occur in neuronal cells is oxidative DNA damage which is primarily removed by the BER pathway. Following the notion that the bulk of neuronal DNA damage is acquired by oxidative DNA damage and ROS, the BER pathway is a likely area of focus for neuronal studies of DNA repair. BER variations in brain aging and pathology in various brain regions and tissues are presented. Therefore, the BER pathway is discussed in greater detail in this review than other repair pathways. Other repair pathways including direct reversal, nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination and non-homologous end joining are also discussed. Finally, there is a growing interest in the role that DNA repair pathways play in the clinical arena as they relate to the neurotoxicity and neuropathy associated with cancer treatments. Among the numerous side effects of cancer treatments, major

  19. Membrane dynamics

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...... for the lateral organization of membranes as wells as for physical properties like bending, permeability and elasticity...

  20. Type IV Collagens and Basement Membrane Diseases: Cell Biology and Pathogenic Mechanisms.

    Science.gov (United States)

    Mao, Mao; Alavi, Marcel V; Labelle-Dumais, Cassandre; Gould, Douglas B

    2015-01-01

    Basement membranes are highly specialized extracellular matrices. Once considered inert scaffolds, basement membranes are now viewed as dynamic and versatile environments that modulate cellular behaviors to regulate tissue development, function, and repair. Increasing evidence suggests that, in addition to providing structural support to neighboring cells, basement membranes serve as reservoirs of growth factors that direct and fine-tune cellular functions. Type IV collagens are a major component of all basement membranes. They evolved along with the earliest multicellular organisms and have been integrated into diverse fundamental biological processes as time and evolution shaped the animal kingdom. The roles of basement membranes in humans are as complex and diverse as their distributions and molecular composition. As a result, basement membrane defects result in multisystem disorders with ambiguous and overlapping boundaries that likely reflect the simultaneous interplay and integration of multiple cellular pathways and processes. Consequently, there will be no single treatment for basement membrane disorders, and therapies are likely to be as varied as the phenotypes. Understanding tissue-specific pathology and the underlying molecular mechanism is the present challenge; personalized medicine will rely upon understanding how a given mutation impacts diverse cellular functions.

  1. DNA repair variants and breast cancer risk.

    Science.gov (United States)

    Grundy, Anne; Richardson, Harriet; Schuetz, Johanna M; Burstyn, Igor; Spinelli, John J; Brooks-Wilson, Angela; Aronson, Kristan J

    2016-05-01

    A functional DNA repair system has been identified as important in the prevention of tumour development. Previous studies have hypothesized that common polymorphisms in DNA repair genes could play a role in breast cancer risk and also identified the potential for interactions between these polymorphisms and established breast cancer risk factors such as physical activity. Associations with breast cancer risk for 99 single nucleotide polymorphisms (SNPs) from genes in ten DNA repair pathways were examined in a case-control study including both Europeans (644 cases, 809 controls) and East Asians (299 cases, 160 controls). Odds ratios in both additive and dominant genetic models were calculated separately for participants of European and East Asian ancestry using multivariate logistic regression. The impact of multiple comparisons was assessed by correcting for the false discovery rate within each DNA repair pathway. Interactions between several breast cancer risk factors and DNA repair SNPs were also evaluated. One SNP (rs3213282) in the gene XRCC1 was associated with an increased risk of breast cancer in the dominant model of inheritance following adjustment for the false discovery rate (P breast cancer risk or their modification by breast cancer risk factors were observed.

  2. Sulforaphane, a cancer chemopreventive agent, induces pathways associated with membrane biosynthesis in response to tissue damage by aflatoxin B{sub 1}

    Energy Technology Data Exchange (ETDEWEB)

    Techapiesancharoenkij, Nirachara [Laboratory of Environmental Toxicology, Chulabhorn Research Institute, Bangkok 10210 (Thailand); Fiala, Jeannette L.A. [Department of Biological Engineering and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Navasumrit, Panida [Laboratory of Environmental Toxicology, Chulabhorn Research Institute, Bangkok 10210 (Thailand); Croy, Robert G.; Wogan, Gerald N. [Department of Biological Engineering and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Groopman, John D. [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205 (United States); Ruchirawat, Mathuros [Laboratory of Environmental Toxicology, Chulabhorn Research Institute, Bangkok 10210 (Thailand); Essigmann, John M., E-mail: jessig@mit.edu [Department of Biological Engineering and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States)

    2015-01-01

    Aflatoxin B{sub 1} (AFB{sub 1}) is one of the major risk factors for liver cancer globally. A recent study showed that sulforaphane (SF), a potent inducer of phase II enzymes that occurs naturally in widely consumed vegetables, effectively induces hepatic glutathione S-transferases (GSTs) and reduces levels of hepatic AFB{sub 1}-DNA adducts in AFB{sub 1}-exposed Sprague Dawley rats. The present study characterized the effects of SF pre-treatment on global gene expression in the livers of similarly treated male rats. Combined treatment with AFB{sub 1} and SF caused reprogramming of a network of genes involved in signal transduction and transcription. Changes in gene regulation were observable 4 h after AFB{sub 1} administration in SF-pretreated animals and may reflect regeneration of cells in the wake of AFB{sub 1}-induced hepatotoxicity. At 24 h after AFB{sub 1} administration, significant induction of genes that play roles in cellular lipid metabolism and acetyl-CoA biosynthesis was detected in SF-pretreated AFB{sub 1}-dosed rats. Induction of this group of genes may indicate a metabolic shift toward glycolysis and fatty acid synthesis to generate and maintain pools of intermediate molecules required for tissue repair, cell growth and compensatory hepatic cell proliferation. Collectively, gene expression data from this study provide insights into molecular mechanisms underlying the protective effects of SF against AFB{sub 1} hepatotoxicity and hepatocarcinogenicity, in addition to the chemopreventive activity of this compound as a GST inducer. - Highlights: • This study revealed sulforaphane (SF)-deregulated gene sets in aflatoxin B{sub 1} (AFB{sub 1})-treated rat livers. • SF redirects biochemical networks toward lipid biosynthesis in AFB{sub 1}-dosed rats. • SF enhanced gene sets that would be expected to favor cell repair and regeneration.

  3. The complex choreography of transcription-coupled repair.

    Science.gov (United States)

    Spivak, Graciela; Ganesan, Ann K

    2014-07-01

    A quarter of a century has elapsed since the discovery of transcription-coupled repair (TCR), and yet our fascination with this process has not diminished. Nucleotide excision repair (NER) is a versatile pathway that removes helix-distorting DNA lesions from the genomes of organisms across the evolutionary scale, from bacteria to humans. TCR, defined as a subpathway of NER, is dedicated to the repair of lesions that, by virtue of their location on the transcribed strands of active genes, encumber elongation by RNA polymerases. In this review, we will report on newly identified proteins, protein modifications, and protein complexes that participate in TCR in Escherichia coli and in human cells. We will discuss general models for the biochemical pathways and how and when cells might choose to utilize TCR or other pathways for repair or bypass of transcription-blocking DNA alterations.

  4. Immunobiology of Facial Nerve Repair and Regeneration

    Institute of Scientific and Technical Information of China (English)

    QUAN Shi-ming; GAO Zhi-qiang

    2006-01-01

    Immunobiological study is a key to revealing the important basis of facial nerve repair and regeneration for both research and development of clinic treatments. The microenvironmental changes around an injuried facial motoneuron, i.e., the aggregation and expression of various types of immune cells and molecules in a dynamic equilibrium, impenetrate from the start to the end of the repair of an injured facial nerve. The concept of "immune microenvironment for facial nerve repair and regeneration", mainly concerns with the dynamic exchange between expression and regulation networks and a variaty of immune cells and immune molecules in the process of facial nerve repair and regeneration for the maintenance of a immune microenvironment favorable for nerve repair.Investigation on microglial activation and recruitment, T cell behavior, cytokine networks, and immunological cellular and molecular signaling pathways in facial nerve repair and regeneration are the current hot spots in the research on immunobiology of facial nerve injury. The current paper provides a comprehensive review of the above mentioned issues. Research of these issues will eventually make immunological interventions practicable treatments for facial nerve injury in the clinic.

  5. Exosomes and cardiac repair after myocardial infarction.

    Science.gov (United States)

    Sahoo, Susmita; Losordo, Douglas W

    2014-01-17

    Myocardial infarction is a leading cause of death among all cardiovascular diseases. The analysis of molecular mechanisms by which the ischemic myocardium initiates repair and remodeling indicates that secreted soluble factors are key players in communication to local and distant tissues, such as bone marrow. Recently, actively secreted membrane vesicles, including exosomes, are being recognized as new candidates with important roles in intercellular and tissue-level communication. In this review, we critically examine the emerging role of exosomes in local and distant microcommunication mechanisms after myocardial infarction. A comprehensive understanding of the role of exosomes in cardiac repair after myocardial infarction could bridge a major gap in knowledge of the repair mechanism after myocardial injury.

  6. Choreography of oxidative damage repair in mammalian genomes.

    Science.gov (United States)

    Mitra, Sankar; Izumi, Tadahide; Boldogh, Istvan; Bhakat, Kishor K; Hill, Jeff W; Hazra, Tapas K

    2002-07-01

    The lesions induced by reactive oxygen species in both nuclear and mitochondrial genomes include altered bases, abasic (AP) sites, and single-strand breaks, all repaired primarily via the base excision repair (BER) pathway. Although the basic BER process (consisting of five sequential steps) could be reconstituted in vitro with only four enzymes, it is now evident that repair of oxidative damage, at least in mammalian cell nuclei, is more complex, and involves a number of additional proteins, including transcription- and replication-associated factors. These proteins may be required in sequential repair steps in concert with other cellular changes, starting with nuclear targeting of the early repair enzymes in response to oxidative stress, facilitation of lesion recognition, and access by chromatin unfolding via histone acetylation, and formation of metastable complexes of repair enzymes and other accessory proteins. Distinct, specific subclasses of protein complexes may be formed for repair of oxidative lesions in the nucleus in transcribed vs. nontranscribed sequences in chromatin, in quiescent vs. cycling cells, and in nascent vs. parental DNA strands in replicating cells. Characterizing the proteins for each repair subpathway, their signaling-dependent modifications and interactions in the nuclear as well as mitochondrial repair complexes, will be a major focus of future research in oxidative damage repair.

  7. Optimality in DNA repair.

    Science.gov (United States)

    Richard, Morgiane; Fryett, Matthew; Miller, Samantha; Booth, Ian; Grebogi, Celso; Moura, Alessandro

    2012-01-07

    DNA within cells is subject to damage from various sources. Organisms have evolved a number of mechanisms to repair DNA damage. The activity of repair enzymes carries its own risk, however, because the repair of two nearby lesions may lead to the breakup of DNA and result in cell death. We propose a mathematical theory of the damage and repair process in the important scenario where lesions are caused in bursts. We use this model to show that there is an optimum level of repair enzymes within cells which optimises the cell's response to damage. This optimal level is explained as the best trade-off between fast repair and a low probability of causing double-stranded breaks. We derive our results analytically and test them using stochastic simulations, and compare our predictions with current biological knowledge.

  8. Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

    Science.gov (United States)

    Rossi, Omar; Pesce, Isabella; Giannelli, Carlo; Aprea, Susanna; Caboni, Mariaelena; Citiulo, Francesco; Valentini, Sara; Ferlenghi, Ilaria; MacLennan, Calman Alexander; D'Oro, Ugo; Saul, Allan; Gerke, Christiane

    2014-09-05

    Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.

  9. Cordyceps militaris fruit body extract ameliorates membranous glomerulonephritis by attenuating oxidative stress and renal inflammation via the NF-κB pathway.

    Science.gov (United States)

    Song, Jingjing; Wang, Yingwu; Liu, Chungang; Huang, Yan; He, Liying; Cai, Xueying; Lu, Jiahui; Liu, Yan; Wang, Di

    2016-04-01

    Membranous glomerulonephritis (MGN) is a common pathogenesis of nephritic syndrome in adult patients. Nuclear factor kappa B (NF-κB) serves as the main transcription factor for the inflammatory response mediated nephropathy. Cordyceps militaris, containing various pharmacological components, has been used as a kind of crude drug and folk tonic food for improving immunity and reducing inflammation. The current study aims to investigate the renoprotective activity of Cordyceps militaris aqueous extract (CM) in the cationic bovine serum albumin (C-BSA)-induced rat model of membranous glomerulonephritis. Significant renal dysfunction was observed in MGN rats; comparatively, 4-week CM administration strongly decreased the levels of 24 h urine protein, total cholesterol, triglyceride, blood urea nitrogen and serum creatinine, and increased the levels of serum albumin and total serum protein. Strikingly, recovery of the kidney histological architecture was noted in CM-treated MGN rats. A significant improvement in the glutathione peroxidase and superoxide dismutase levels, and a reduced malondialdehyde concentration were observed in the serum and kidney of CM-treated rats. Altered levels of inflammatory cytokines including interleukins, monocyte chemoattractant protein-1, intercellular adhesion molecule 1, vascular adhesion molecule 1, tumor necrosis factor-α, 6-keto-prostaglandin F1α, and nuclear transcriptional factor subunit NF-κB p65 reverted to normal levels upon treatment with CM. The present data suggest that CM protects rats against membranous glomerulonephritis via the normalization of NF-κB activity, thereby inhibiting oxidative damage and reducing inflammatory cytokine levels, which further provide experimental evidence in support of the clinical use of CM as an effective renoprotective agent.

  10. The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H

    OpenAIRE

    Rauna eRiva; Korhonen, Timo K.; Seppo eMeri

    2015-01-01

    The virulence factor PgtE is an outer membrane protease (omptin) of the zoonotic pathogen Salmonella enterica that causes diseases ranging from gastroenteritis to severe enteric fever. It is surface exposed in bacteria that have a short-chain, i.e., rough LPS, as observed e.g., in bacteria residing inside macrophages or just emerging from them. We investigated whether PgtE cleaves the complement factors B (B) and H (H), key proteins controlling formation and inactivation of the complement pro...

  11. Membrane-impermeable estrogen is involved in regulation of calbindin-D9k expression via non-genomic pathways in a rat pituitary cell line, GH3 cells.

    Science.gov (United States)

    Dang, Vu Hoang; Choi, Kyung-Chul; Jeung, Eui-Bae

    2010-06-01

    Estrogen (E2) has been shown to regulate various functions for many pituitary hormones. Recently, the potential roles of non-genomic pathways in E2-induced actions have been proposed in the previous studies, however, the effects of E2 remain to be elucidated in regard to non-genomic induction of cytosolic protein calbindin-D9k (CaBP-9k). To gain a better understanding of the molecular events underlying E2-induced expression of CaBP-9k, rat pituitary tumor cells (GH3 cells) were treated with E-BSA (membrane impermeable E2-conjugated with BSA). Non-genomic induction of CaBP-9k by E-BSA was determined using RT-PCR and western blot analysis. The significant increase in CaBP-9k mRNA level was observed as early as 15 min following treatment with a high concentration of E-BSA (10(-6)M), whereas rapid and significant induction of CaBP-9k protein was noted at 5, 15 and 30 min after E-BSA exposure (p<0.05). In order to determine the potential involvement of different signaling pathways, several inhibitors were employed, i.e., ICI 182,780 for the estrogen receptor (ER) pathway, pertussis toxin (PTX) for the G-protein-coupled signaling pathway, U0126 (U) for the ERK (extracellular regulated kinase) and wortmannin (W) for the Akt (protein kinase B). Co-treatment with ICI 182, 780 and PTX reversed an E-BSA-induced increase in CaBP-9k mRNA and protein. Although neither U nor W alone attenuated E-BSA-induced effects, these inhibitors together abolished E-BSA-induced CaBP-9k expression, suggesting their involvement in its regulation. Taken together, these results demonstrate the involvement of various signaling pathways in E2-induced regulation of CaBP-9k. In addition, ER and G-protein-coupled signaling pathways may play central roles in the non-genomic activities of E2 and that downstream signaling via ERK and Akt are required to evoke ER-mediated induction of CaBP-9k in vitro. Copyright 2010 Elsevier Ltd. All rights reserved.

  12. Nano-hydroxyapatite/collagen composited with recombinant human bone morphogenetic protein-2 and titanium membrane in repairing peripheral bone defects of instant dental implants%胶原基纳米骨复合重组人骨形成蛋白2及钛膜修复即刻钛种植体周围骨缺损

    Institute of Scientific and Technical Information of China (English)

    刘冰; 陈鹏; 王忠义; 柯杰; 李晓华; 汪正文

    2009-01-01

    BACKGROUND:Recently,with the rapid development of material science and bioscience,the technology of dental implant has made great progress,especially the immediate implant technology.But the size and shape of implant are usually not fit for tooth extraction wound,so it is an important factor that leads to failure when implant and tooth extraction wound can not form close tangency.Guided bone regeneration or bone grafting materials are usually used to solve this problem.OBJECTIVE:To study the effects of nano-hydroxyapatite/collogen (nHAC) with recombinant human bone morphogenetic protein-2(rhBMP-2) and titanium (Ti) membrane on repairing peripheral bone defects of instant implant.DESIGN,TIME AND SETTING:A randomized,controlled animal study was performed at the Central Laboratory,the Fourth Military Medical University of Chinese PLA between January 2005 and January 2006.MATERIALS:Ti screw implants (diameter 2 mm,length 10 mm,and pitch 0.4 mm) without the part that went through gum were offered by Nonferrous Metal Academy in Baoji,China.The nonabsorbable Ti membranes (2 cm×2 cm) were offered by Zhongbang Biomaterial Limited Company in Xi'an,China.The nHAC materials were gifted by professor Cui Fu-zhai from Material Science and Engineering Department of Tsinghua University and fabricated into 0.5 mm×0.5 mm×0.5 mm small blocks.rhBMP-2 was offered by the Academy of Military Medical Sciences in Beijing,China.rhBMP-2 was dissolved with hydrochloric carbamidine and then nHAC was immersed in it.Vacuumization,freeze-drying,and Ekibon degermation were followed.Each gram of nHAC compounds required approximately 1 mg rhBMP-2.METHODS:Four healthy purebred male dogs were included in this study.According to the methods to repair bone defects rhBMP-2+Ti membrane,nHAC composited with rhBMP-2 was implanted,covering Ti membrane.Six defects were made on the mandible on each side.MAIN OUTCOME MEASURES:At 6 and 12 weeks after implantation,new bone formation and the correlation of new

  13. Vascular smooth muscle progenitor cells: building and repairing blood vessels.

    Science.gov (United States)

    Majesky, Mark W; Dong, Xiu Rong; Regan, Jenna N; Hoglund, Virginia J

    2011-02-04

    Molecular pathways that control the specification, migration, and number of available smooth muscle progenitor cells play key roles in determining blood vessel size and structure, capacity for tissue repair, and progression of age-related disorders. Defects in these pathways produce malformations of developing blood vessels, depletion of smooth muscle progenitor cell pools for vessel wall maintenance and repair, and aberrant activation of alternative differentiation pathways in vascular disease. A better understanding of the molecular mechanisms that uniquely specify and maintain vascular smooth muscle cell precursors is essential if we are to use advances in stem and progenitor cell biology and somatic cell reprogramming for applications directed to the vessel wall.

  14. Platinum drugs and DNA repair mechanisms in lung cancer.

    Science.gov (United States)

    Bonanno, Laura; Favaretto, Adolfo; Rosell, Rafael

    2014-01-01

    The standard first-line treatment for around 80% of newly-diagnosed advanced non-small cell lung cancer (NSCLC) is chemotherapy. Currently, patients are allocated to chemotherapy on the basis of clinical conditions, comorbidities and histology. If feasible, platinum-based chemotherapy is considered as the most efficacious option. Due to the heterogeneity in terms of platinum-sensitivity among patients with NSCLC, great efforts have been made in order to identify molecular predictive markers of platinum resistance. Based on the mechanism of action of platinum, several components of DNA repair pathways have been investigated as potential predictive markers. The main DNA repair pathways involved in the repair of platinum-induced DNA damage are nucleotide excision repair and homologous recombination. The most studied potential predictive markers of platinum-sensitivity are Excision Repair Cross Complementing-1 (ERCC1) and Brest Cancer Type-I Susceptibility protein (BRCA1); however, increasing biological knowledge about DNA repair pathways suggests the potential clinical usefulness of integrated analysis of multiple DNA repair components.

  15. Repairs of composite structures

    Science.gov (United States)

    Roh, Hee Seok

    Repair on damaged composite panels was conducted. To better understand adhesively bonded repair, the study investigates the effect of design parameters on the joint strength. The design parameters include bondline length, thickness of adherend and type of adhesive. Adhesives considered in this study were tested to measure their tensile material properties. Three types of adhesively bonded joints, single strap, double strap, and single lap joint were considered under changing bondline lengths, thickness of adherend and type of adhesive. Based on lessons learned from bonded joints, a one-sided patch repair method for composite structures was conducted. The composite patch was bonded to the damaged panel by either film adhesive FM-73M or paste adhesive EA-9394 and the residual strengths of the repaired specimens were compared under varying patch sizes. A new repair method using attachments has been suggested to enhance the residual strength. Results obtained through experiments were analyzed using finite element analysis to provide a better repair design and explain the experimental results. It was observed that the residual strength of the repaired specimen was affected by patch length. Method for rapid repairs of damaged composite structures was investigated. The damage was represented by a circular hole in a composite laminated plate. Pre-cured composite patches were bonded with a quick-curing commercial adhesive near (rather than over) the hole. Tensile tests were conducted on specimens repaired with various patch geometries. The test results showed that, among the methods investigated, the best repair method restored over 90% of the original strength of an undamaged panel. The interfacial stresses in the adhesive zone for different patches were calculated in order to understand the efficiencies of the designs of these patch repairs. It was found that the composite patch that yielded the best strength had the lowest interfacial peel stress between the patch and

  16. Membrane Phosphoproteomics of Yeast Early Response to Acetic Acid: Role of Hrk1 Kinase and Lipid Biosynthetic Pathways, in Particular Sphingolipids.

    Science.gov (United States)

    Guerreiro, Joana F; Mira, Nuno P; Santos, Aline X S; Riezman, Howard; Sá-Correia, Isabel

    2017-01-01

    Saccharomyces cerevisiae response and tolerance to acetic acid is critical in industrial biotechnology and in acidic food and beverages preservation. The HRK1 gene, encoding a protein kinase of unknown function belonging to the "Npr1-family" of kinases known to be involved in the regulation of plasma membrane transporters, is an important determinant of acetic acid tolerance. This study was performed to identify the alterations occurring in yeast membrane phosphoproteome profile during the adaptive early response to acetic acid stress (following 1 h of exposure to a sub-lethal inhibitory concentration; 50 mM at pH 4.0) and the effect of HRK1 expression on the phosphoproteome. Results from mass spectrometry analysis following the prefractionation and specific enrichment of phosphorylated peptides using TiO2 beads highlight the contribution of processes related with translation, protein folding and processing, transport, and cellular homeostasis in yeast response to acetic acid stress, with particular relevance for changes in phosphorylation of transport-related proteins, found to be highly dependent on the Hrk1 kinase. Twenty different phosphoproteins known to be involved in lipid and sterol metabolism were found to be differently phosphorylated in response to acetic acid stress, including several phosphopeptides that had not previously been described as being phosphorylated. The suggested occurrence of cellular lipid composition remodeling during the short term yeast response to acetic acid was confirmed: Hrk1 kinase-independent reduction in phytoceramide levels and a reduction in phosphatidylcholine and phosphatidylinositol levels under acetic acid stress in the more susceptible hrk1Δ strain were revealed by a lipidomic analysis.

  17. Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes.

    Directory of Open Access Journals (Sweden)

    Maria E Morales

    Full Text Available Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl2 favored resolution of DSBs through homologous recombination (HR and single strand annealing (SSA, which were inhibited by higher NiCl2 doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the "error prone" non-homologous end joining (alt-NHEJ while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair.

  18. Workshop on DNA repair.

    NARCIS (Netherlands)

    A.R. Lehmann (Alan); J.H.J. Hoeijmakers (Jan); A.A. van Zeeland (Albert); C.M.P. Backendorf (Claude); B.A. Bridges; A. Collins; R.P.D. Fuchs; G.P. Margison; R. Montesano; E. Moustacchi; A.T. Natarajan; M. Radman; A. Sarasin; E. Seeberg; C.A. Smith; M. Stefanini (Miria); L.H. Thompson; G.P. van der Schans; C.A. Weber (Christine); M.Z. Zdzienika

    1992-01-01

    textabstractA workshop on DNA repair with emphasis on eukaryotic systems was held, under the auspices of the EC Concerted Action on DNA Repair and Cancer, at Noordwijkerhout (The Netherlands) 14-19 April 1991. The local organization of the meeting was done under the auspices of the Medical Genetic C

  19. Laparoscopic lumbar hernia repair.

    Science.gov (United States)

    Madan, Atul K; Ternovits, Craig A; Speck, Karen E; Pritchard, F Elizabeth; Tichansky, David S

    2006-04-01

    Lumbar hernias are rare clinical entities that often pose a challenge for repair. Because of the surrounding anatomy, adequate surgical herniorraphy is often difficult. Minimally invasive surgery has become an option for these hernias. Herein, we describe two patients with lumbar hernias (one with a recurrent traumatic hernia and one with an incisional hernia). Both of these hernias were successfully repaired laparoscopically.

  20. VARIATION IN MEMBRANE PROPERTIES FROM THE ACTION OF LAMININ ON MEMBRANE RECEPTORS

    Institute of Scientific and Technical Information of China (English)

    苏雅娴; 薛燕玲; 肖军军

    1995-01-01

    Biophysical studies were conducted on the action of laminin through membrane receptors of cancer cells. The results showed that variations occurred in the thermodynamic properties of membrane proteins, the mobility of hydrocarbon chains of membrane lipids, and the permeshility and transportation pathways of the membrane.

  1. Epstein-Barr virus encoded latent membrane protein 1 induces TRAF1 expression to promote anti-apoptosis activity via NF-κB signaling pathway in nasopharyngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    王承兴; 艾米丹; 任维; 肖绘; 李小燕; 唐发清; 顾焕华; 易薇; 翁新宪; 邓锡云; 曹亚

    2003-01-01

    Objectives To identify whether Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) can induce tumor necrosis factor receptor-associated factor 1 (TRAF1) expression and promote its anti-apoptosis activity via the NF-κB signaling pathway, and assess that LMP1 suppresses apoptosis in nasopharyngeal carcinoma (NPC). Methods A stable transfected cell line HNE2-LMP1 was established by introducing LMP1 cDNA into HNE2 cells. Transactivation of TRAF1 was determined by luciferase reporter assay, while expression of TRAF1 mRNA was detected by RT-PCR and expression of TRAF1 protein and caspase 3 by Western blot analysis. Apoptosis activity was observed through fluorescence staining. Results LMP1 induced TRAF1 expression in NPC cells and caused a decrease in apoptosis. This induction could be blocked by antisense LMP1. Moreover, LMP1-mediated induction of a TRAF1 promoter-driven reporter gene was significantly impaired when the κB site κB1 or κB5 was disrupted, whereas mutation of κB3 had only a minor effect on LMP1 dependent up-regulation of the reporter gene. Conclusion LMP1 induces TRAF1 expression and promotes its anti-apoptosis activity via the NF-κB signaling pathway, which may be one of the mechanisms that LMP1 uses to suppress apoptosis in NPC cells.

  2. DNA repair protocols

    DEFF Research Database (Denmark)

    Bjergbæk, Lotte

    In its 3rd edition, this Methods in Molecular Biology(TM) book covers the eukaryotic response to genomic insult including advanced protocols and standard techniques in the field of DNA repair. Offers expert guidance for DNA repair, recombination, and replication. Current knowledge of the mechanisms...... that regulate DNA repair has grown significantly over the past years with technology advances such as RNA interference, advanced proteomics and microscopy as well as high throughput screens. The third edition of DNA Repair Protocols covers various aspects of the eukaryotic response to genomic insult including...... recent advanced protocols as well as standard techniques used in the field of DNA repair. Both mammalian and non-mammalian model organisms are covered in the book, and many of the techniques can be applied with only minor modifications to other systems than the one described. Written in the highly...

  3. INTERNAL REPAIR OF PIPELINES

    Energy Technology Data Exchange (ETDEWEB)

    Bill Bruce; Nancy Porter; George Ritter; Matt Boring; Mark Lozev; Ian Harris; Bill Mohr; Dennis Harwig; Robin Gordon; Chris Neary; Mike Sullivan

    2005-07-20

    The two broad categories of fiber-reinforced composite liner repair and deposited weld metal repair technologies were reviewed and evaluated for potential application for internal repair of gas transmission pipelines. Both are used to some extent for other applications and could be further developed for internal, local, structural repair of gas transmission pipelines. Principal conclusions from a survey of natural gas transmission industry pipeline operators can be summarized in terms of the following performance requirements for internal repair: (1) Use of internal repair is most attractive for river crossings, under other bodies of water, in difficult soil conditions, under highways, under congested intersections, and under railway crossings. (2) Internal pipe repair offers a strong potential advantage to the high cost of horizontal direct drilling when a new bore must be created to solve a leak or other problem. (3) Typical travel distances can be divided into three distinct groups: up to 305 m (1,000 ft.); between 305 m and 610 m (1,000 ft. and 2,000 ft.); and beyond 914 m (3,000 ft.). All three groups require pig-based systems. A despooled umbilical system would suffice for the first two groups which represents 81% of survey respondents. The third group would require an onboard self-contained power unit for propulsion and welding/liner repair energy needs. (4) The most common size range for 80% to 90% of operators surveyed is 508 mm (20 in.) to 762 mm (30 in.), with 95% using 558.8 mm (22 in.) pipe. Evaluation trials were conducted on pipe sections with simulated corrosion damage repaired with glass fiber-reinforced composite liners, carbon fiber-reinforced composite liners, and weld deposition. Additional un-repaired pipe sections were evaluated in the virgin condition and with simulated damage. Hydrostatic failure pressures for pipe sections repaired with glass fiber-reinforced composite liner were only marginally greater than that of pipe sections without

  4. Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1 enhance Gag-specific immune response.

    Directory of Open Access Journals (Sweden)

    Rodrigo Maciel da Costa Godinho

    Full Text Available We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1 elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

  5. Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

    Science.gov (United States)

    Godinho, Rodrigo Maciel da Costa; Matassoli, Flavio Lemos; Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J Thomas; Marques, Ernesto Torres de Azevedo; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

  6. Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

    Science.gov (United States)

    Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

  7. The Functions of BMP3 in Rabbit Articular Cartilage Repair

    Directory of Open Access Journals (Sweden)

    Zhe Zhang

    2015-10-01

    Full Text Available Bone morphogenetic proteins (BMPs play important roles in skeletal development and repair. Previously, we found fibroblast growth factor 2 (FGF2 induced up-regulation of BMP2, 3, 4 in the process of rabbit articular cartilage repair, which resulted in satisfactory repair effects. As BMP2/4 show a clearly positive effect for cartilage repair, we investigated the functions of BMP3 in rabbit articular cartilage repair. In this paper, we find that BMP3 inhibits the repair of partial-thickness defect of articular cartilage in rabbit by inducing the degradation of extracellular matrix, interfering with the survival of chondrocytes surrounding the defect, and directly inhibiting the expression of BMP2 and BMP4. Meanwhile BMP3 suppress the repair of full-thickness cartilage defect by destroying the subchondral bone through modulating the proliferation and differentiation of bone marrow stem cells (BMSCs, and directly increasing the expression of BMP4. Although BMP3 has different functions in the repair of partial and full-thickness defects of articular cartilage in rabbit, the regulation of BMP expression is involved in both of them. Together with our previous findings, we suggest the regulation of the BMP signaling pathway by BMP3 is essential in articular cartilage repair.

  8. Cancer TARGETases: DSB repair as a pharmacological target.

    Science.gov (United States)

    Samadder, Pounami; Aithal, Rakesh; Belan, Ondrej; Krejci, Lumir

    2016-05-01

    Cancer is a disease attributed to the accumulation of DNA damages due to incapacitation of DNA repair pathways resulting in genomic instability and a mutator phenotype. Among the DNA lesions, double stranded breaks (DSBs) are the most toxic forms of DNA damage which may arise as a result of extrinsic DNA damaging agents or intrinsic replication stress in fast proliferating cancer cells. Accurate repair of DSBs is therefore paramount to the cell survival, and several classes of proteins such as kinases, nucleases, helicases or core recombinational proteins have pre-defined jobs in precise execution of DSB repair pathways. On one hand, the proper functioning of these proteins ensures maintenance of genomic stability in normal cells, and on the other hand results in resistance to various drugs employed in cancer therapy and therefore presents a suitable opportunity for therapeutic targeting. Higher relapse and resistance in cancer patients due to non-specific, cytotoxic therapies is an alarming situation and it is becoming more evident to employ personalized treatment based on the genetic landscape of the cancer cells. For the success of personalized treatment, it is of immense importance to identify more suitable targetable proteins in DSB repair pathways and also to explore new synthetic lethal interactions with these pathways. Here we review the various alternative approaches to target the various protein classes termed as cancer TARGETases in DSB repair pathway to obtain more beneficial and selective therapy.

  9. INTERNAL REPAIR OF PIPELINES

    Energy Technology Data Exchange (ETDEWEB)

    Robin Gordon; Bill Bruce; Ian Harris; Dennis Harwig; George Ritter; Bill Mohr; Matt Boring; Nancy Porter; Mike Sullivan; Chris Neary

    2004-12-31

    The two broad categories of fiber-reinforced composite liner repair and deposited weld metal repair technologies were reviewed and evaluated for potential application for internal repair of gas transmission pipelines. Both are used to some extent for other applications and could be further developed for internal, local, structural repair of gas transmission pipelines. Principal conclusions from a survey of natural gas transmission industry pipeline operators can be summarized in terms of the following performance requirements for internal repair: (1) Use of internal repair is most attractive for river crossings, under other bodies of water, in difficult soil conditions, under highways, under congested intersections, and under railway crossings. (2) Internal pipe repair offers a strong potential advantage to the high cost of horizontal direct drilling when a new bore must be created to solve a leak or other problem. (3) Typical travel distances can be divided into three distinct groups: up to 305 m (1,000 ft.); between 305 m and 610 m (1,000 ft. and 2,000 ft.); and beyond 914 m (3,000 ft.). All three groups require pig-based systems. A despooled umbilical system would suffice for the first two groups which represents 81% of survey respondents. The third group would require an onboard self-contained power unit for propulsion and welding/liner repair energy needs. (4) The most common size range for 80% to 90% of operators surveyed is 508 mm (20 in.) to 762 mm (30 in.), with 95% using 558.8 mm (22 in.) pipe. Evaluation trials were conducted on pipe sections with simulated corrosion damage repaired with glass fiber-reinforced composite liners, carbon fiber-reinforced composite liners, and weld deposition. Additional un-repaired pipe sections were evaluated in the virgin condition and with simulated damage. Hydrostatic failure pressures for pipe sections repaired with glass fiber-reinforced composite liner were only marginally greater than that of pipe sections without

  10. 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.

  11. Membrane reactor. Membrane reactor

    Energy Technology Data Exchange (ETDEWEB)

    Shindo, Y.; Wakabayashi, K. (National Chemical Laboratory for Industry, Tsukuba (Japan))

    1990-08-05

    Many reaction examples were introduced of membrane reactor, to be on the point of forming a new region in the field of chemical technology. It is a reactor to exhibit excellent function, by its being installed with membrane therein, and is generally classified into catalyst function type and reaction promotion type. What firstly belongs to the former is stabilized zirconia, where oxygen, supplied to the cathodic side of membrane with voltage, impressed thereon, becomes O {sup 2 {minus}} to be diffused through the membrane and supplied, as variously activated oxygenous species, on the anodic side. Examples with many advantages can be given such as methane coupling, propylene oxidation, methanating reaction of carbon dioxide, etc. Apart, palladium film and naphion film also belong to the former. While examples of the latter comprise, among others, decomposition of hydrogen sulfide by porous glass film and dehydrogenation of cyclohexane or palladium alloy film, which are expected to be developed and materialized in the industry. 33 refs., 8 figs.

  12. Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining

    OpenAIRE

    Bowater, Richard; Doherty, Aidan J.

    2006-01-01

    DNA double-strand breaks (DSBs) are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ). Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHE...

  13. Enhancement of glucose uptake in muscular cell by soybean charged peptides isolated by electrodialysis with ultrafiltration membranes (EDUF): activation of the AMPK pathway.

    Science.gov (United States)

    Roblet, Cyril; Doyen, Alain; Amiot, Jean; Pilon, Geneviève; Marette, André; Bazinet, Laurent

    2014-03-15

    Soy peptides consumption has been associated with beneficial effects in type 2 diabetes patients. However, the peptide fractions responsible for these effects, and their mechanisms of action, have not been identified yet. In this study, we have isolated soybean peptides by electrodialysis with an ultrafiltration membrane (EDUF) at 50 V/100 kDa, and tested them for their capacity to improve glucose uptake in L6 muscle cells. We observed that these fractions were able to significantly enhance glucose uptake in the presence of insulin. The reported bioactivity would be due to the low molecular weight peptides (300-500 Da) recovered. Moreover, we observed that an enhancement of glucose uptake was correlated to the activation of the AMPK enzyme, well known for its capacity to increase glucose uptake in muscle cells. To our knowledge, this is the first time that bioactive peptides with glucose uptake activity have been isolated from a complex soy matrix, and that the implication of AMPK in it is demonstrated. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. The impact of heterochromatin on DSB repair.

    Science.gov (United States)

    Goodarzi, Aaron A; Noon, Angela T; Jeggo, Penny A

    2009-06-01

    DNA NHEJ (non-homologous end-joining) is the major DNA DSB (double-strand break) repair pathway in mammalian cells. Although NHEJ-defective cell lines show marked DSB-repair defects, cells defective in ATM (ataxia telangiectasia mutated) repair most DSBs normally. Thus NHEJ functions independently of ATM signalling. However, approximately 15% of radiation-induced DSBs are repaired with slow kinetics and require ATM and the nuclease Artemis. DSBs persisting in the presence of an ATM inhibitor, ATMi, localize to heterochromatin, suggesting that ATM is required for repairing DSBs arising within or close to heterochromatin. Consistent with this, we show that siRNA (small interfering RNA) of key heterochromatic proteins, including KAP-1 [KRAB (Krüppel-associated box) domain-associated protein 1], HP1 (heterochromatin protein 1) and HDAC (histone deacetylase) 1/2, relieves the requirement for ATM for DSB repair. Furthermore, ATMi addition to cell lines with genetic alterations that have an impact on heterochromatin, including Suv39H1/2 (suppressor of variegation 3-9 homologue 1/2)-knockout, ICFa (immunodeficiency, centromeric region instability, facial anomalies syndrome type a) and Hutchinson-Guilford progeria cell lines, fails to have an impact on DSB repair. KAP-1 is a highly dose-dependent, transient and ATM-specific substrate, and mutation of the ATM phosphorylation site on KAP-1 influences DSB repair. Collectively, the findings show that ATM functions to overcome the barrier to DSB repair posed by heterochromatin. However, even in the presence of ATM, gamma-H2AX (phosphorylated histone H2AX) foci form on the periphery rather than within heterochromatic centres. Finally, we show that KAP-1's association with heterochromatin is diminished as cells progress through mitosis. We propose that KAP-1 is a critical heterochromatic factor that undergoes specific modifications to promote DSB repair and mitotic progression in a manner that allows localized and transient

  15. Repair of traumatic plasmalemmal damage to neurons and other eukar yotic cells

    Institute of Scientific and Technical Information of China (English)

    George D. Bittner; Christopher S. Spaeth§; Andrew D. Poon; Zachary S. Burgess; Christopher H. McGill

    2016-01-01

    The repair (sealing) of plasmalemmal damage, consisting of small holes to complete transections, is criti-cal for cell survival, especially for neurons that rarely regenerate cell bodies. We ifrst describe and evaluate different measures of cell sealing. Some measures, including morphological/ultra-structural observations, membrane potential, and input resistance, provide very ambiguous assessments of plasmalemmal sealing. In contrast, measures of ionic current lfow and dye barriers can, if appropriately used, provide more ac-curate assessments. We describe the effects of various substances (calcium, calpains, cytoskeletal proteins, ESCRT proteins, mUNC-13, NSF, PEG) and biochemical pathways (PKA, PKC, PLC, Epac, cytosolic ox-idation) on plasmalemmal sealing probability, and suggest that substances, pathways, and cellular events associated with plasmalemmal sealing have undergone a very conservative evolution. During sealing, calcium ion inlfux mobilizes vesicles and other membranous structures (lysosomes, mitochondria, etc.) in a continuous fashion to form a vesicular plug that gradually restricts diffusion of increasingly smaller molecules and ions over a period of seconds to minutes. Furthermore, we find no direct evidence that sealing occurs through the collapse and fusion of severed plasmalemmal lealfets, or in a single step involv-ing the fusion of one large wound vesicle with the nearby, undamaged plasmalemma. We describe how increases in perikaryal calcium levels following axonal transection account for observations that cell body survival decreases the closer an axon is transected to the perikaryon. Finally, we speculate on relationships between plasmalemmal sealing, Wallerian degeneration, and the ability of polyethylene glycol (PEG) to seal cell membranes and rejoin severed axonal ends–an important consideration for the future treatment of trauma to peripheral nerves. A better knowledge of biochemical pathways and cytoplasmic structures in-volved in

  16. Repair of traumatic plasmalemmal damage to neurons and other eukaryotic cells

    Directory of Open Access Journals (Sweden)

    George D Bittner

    2016-01-01

    Full Text Available The repair (sealing of plasmalemmal damage, consisting of small holes to complete transections, is critical for cell survival, especially for neurons that rarely regenerate cell bodies. We first describe and evaluate different measures of cell sealing. Some measures, including morphological/ultra-structural observations, membrane potential, and input resistance, provide very ambiguous assessments of plasmalemmal sealing. In contrast, measures of ionic current flow and dye barriers can, if appropriately used, provide more accurate assessments. We describe the effects of various substances (calcium, calpains, cytoskeletal proteins, ESCRT proteins, mUNC-13, NSF, PEG and biochemical pathways (PKA, PKC, PLC, Epac, cytosolic oxidation on plasmalemmal sealing probability, and suggest that substances, pathways, and cellular events associated with plasmalemmal sealing have undergone a very conservative evolution. During sealing, calcium ion influx mobilizes vesicles and other membranous structures (lysosomes, mitochondria, etc. in a continuous fashion to form a vesicular plug that gradually restricts diffusion of increasingly smaller molecules and ions over a period of seconds to minutes. Furthermore, we find no direct evidence that sealing occurs through the collapse and fusion of severed plasmalemmal leaflets, or in a single step involving the fusion of one large wound vesicle with the nearby, undamaged plasmalemma. We describe how increases in perikaryal calcium levels following axonal transection account for observations that cell body survival decreases the closer an axon is transected to the perikaryon. Finally, we speculate on relationships between plasmalemmal sealing, Wallerian degeneration, and the ability of polyethylene glycol (PEG to seal cell membranes and rejoin severed axonal ends – an important consideration for the future treatment of trauma to peripheral nerves. A better knowledge of biochemical pathways and cytoplasmic structures

  17. Repair of ultraviolet-light-induced damage

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, B.M.

    1981-01-01

    Studies are reviewed which present three major new findings in the photobiology of skin. First, detectable numbers of dimers are formed even at sub-erythymal doses. Second, excision of dimers is much more rapid than would be predicted from results obtained in cell culture. Third, comparison of the rates of excision and photoreactivation in skin indicates that in normal sunlight exposure, photoreactivation may well be the predominant repair pathway in skin. (ACR)

  18. INTERNAL REPAIR OF PIPELINES

    Energy Technology Data Exchange (ETDEWEB)

    Robin Gordon; Bill Bruce; Ian Harris; Dennis Harwig; George Ritter; Bill Mohr; Matt Boring; Nancy Porter; Mike Sullivan; Chris Neary

    2004-08-17

    The two broad categories of fiber-reinforced composite liner repair and deposited weld metal repair technologies were reviewed and evaluated for potential application for internal repair of gas transmission pipelines. Both are used to some extent for other applications and could be further developed for internal, local, structural repair of gas transmission pipelines. Principal conclusions from a survey of natural gas transmission industry pipeline operators can be summarized in terms of the following performance requirements for internal repair: (1) Use of internal repair is most attractive for river crossings, under other bodies of water, in difficult soil conditions, under highways, under congested intersections, and under railway. (2) Internal pipe repair offers a strong potential advantage to the high cost of horizontal direct drilling when a new bore must be created to solve a leak or other problem. (3) Typical travel distances can be divided into three distinct groups: up to 305 m (1,000 ft.); between 305 m and 610 m (1,000 ft. and 2,000 ft.); and beyond 914 m (3,000 ft.). All three groups require pig-based systems. A despooled umbilical system would suffice for the first two groups which represents 81% of survey respondents. The third group would require an onboard self-contained power unit for propulsion and welding/liner repair energy needs. (4) The most common size range for 80% to 90% of operators surveyed is 508 mm (20 in.) to 762 mm (30 in.), with 95% using 558.8 mm (22 in.) pipe. Evaluation trials were conducted on pipe sections with simulated corrosion damage repaired with glass fiber-reinforced composite liners, carbon fiber-reinforced composite liners, and weld deposition. Additional un-repaired pipe sections were evaluated in the virgin condition and with simulated damage. Hydrostatic failure pressures for pipe sections repaired with glass fiber-reinforced composite liner were only marginally greater than that of pipe sections without liners

  19. Control of gene editing by manipulation of DNA repair mechanisms.

    Science.gov (United States)

    Danner, Eric; Bashir, Sanum; Yumlu, Saniye; Wurst, Wolfgang; Wefers, Benedikt; Kühn, Ralf

    2017-04-03

    DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.

  20. DNA repair mechanisms in eukaryotes: Special focus in Entamoeba histolytica and related protozoan parasites.

    Science.gov (United States)

    López-Camarillo, César; Lopez-Casamichana, Mavil; Weber, Christian; Guillen, Nancy; Orozco, Esther; Marchat, Laurence A

    2009-12-01

    Eukaryotic cell viability highly relies on genome stability and DNA integrity maintenance. The cellular response to DNA damage mainly consists of six biological conserved pathways known as homologous recombination repair (HRR), non-homologous end-joining (NHEJ), base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), and methyltransferase repair that operate in a concerted way to minimize genetic information loss due to a DNA lesion. Particularly, protozoan parasites survival depends on DNA repair mechanisms that constantly supervise chromosomes to correct damaged nucleotides generated by cytotoxic agents, host immune pressure or cellular processes. Here we reviewed the current knowledge about DNA repair mechanisms in the most relevant human protozoan pathogens. Additionally, we described the recent advances to understand DNA repair mechanisms in Entamoeba histolytica with special emphasis in the use of genomic approaches based on bioinformatic analysis of parasite genome sequence and microarrays technology.

  1. Salvage hypospadias repairs

    Directory of Open Access Journals (Sweden)

    Sripathi V

    2008-01-01

    Full Text Available Aim: Review of our experience and to develop an algorithm for salvage procedures in the management of hypospadias cripples and treatment of urethral strictures following hypospadias repair. Methods: This is a retrospective review of hypospadias surgeries over a 41-month period. Out of a total 168 surgeries, 20 were salvage/re-operative repairs. In three children a Duplay repair was feasible, while in four others a variety of single-stage repairs could be done. The repair was staged in seven children - buccal mucosal grafts (BMGs in five, buccal mucosal tube in one, and skin graft in one. Five children with dense strictures were managed by dorsal BMG inlay grafting in one, vascularized tunical onlay grafting on the ventrum in one, and a free tunical patch in one. Three children were treated by internal urethrotomy and stenting for four weeks with a poor outcome. Results: The age of children ranged from 1.5-15 years (mean 4.5. Follow-up ranged from 3 months to 3.5 years. Excellent results were obtained in 10 children (50% with a well-surfaced erect penis and a slit-like meatus. Glans closure could not be achieved and meatus was coronal in three. Two children developed fistulae following a Duplay repair and following a staged BMG. Three repairs failed completely - a composite repair broke down, a BMG tube stenosed with a proximal leak, and a stricture recurred with loss of a ventral free tunical graft. Conclusions: In salvage procedures performed on hypospadias cripples, a staged repair with buccal mucosa as an inlay in the first stage followed by tubularization 4-6 months later provides good results. A simple algorithm to plan corrective surgery in failed hypospadias cases and obtain satisfactory results is devised.

  2. The GacS/A-RsmA Signal Transduction Pathway Controls the Synthesis of Alkylresorcinol Lipids that Replace Membrane Phospholipids during Encystment of Azotobacter vinelandii SW136

    Science.gov (United States)

    Romero, Yanet; Guzmán, Josefina; Moreno, Soledad; Cocotl-Yañez, Miguel; Vences-Guzmán, Miguel Ángel; Castañeda, Miguel; Espín, Guadalupe; Segura, Daniel

    2016-01-01

    Azotobacter vinelandii is a soil bacterium that undergoes a differentiation process that forms cysts resistant to desiccation. During encystment, a family of alkylresorcinols lipids (ARs) are synthesized and become part of the membrane and are also components of the outer layer covering the cyst, where they play a structural role. The synthesis of ARs in A. vinelandii has been shown to occur by the activity of enzymes encoded in the arsABCD operon. The expression of this operon is activated by ArpR, a LysR-type transcriptional regulator whose transcription occurs during encystment and is dependent on the alternative sigma factor RpoS. In this study, we show that the two component response regulator GacA, the small RNA RsmZ1 and the translational repressor protein RsmA, implicated in the control of the synthesis of other cysts components (i.e., alginate and poly-ß-hydroxybutyrate), are also controlling alkylresorcinol synthesis. This control affects the expression of arsABCD and is exerted through the regulation of arpR expression. We show that RsmA negatively regulates arpR expression by binding its mRNA, repressing its translation. GacA in turn, positively regulates arpR expression through the activation of transcription of RsmZ1, that binds RsmA, counteracting its repressor activity. This regulatory cascade is independent of RpoS. We also show evidence suggesting that GacA exerts an additional regulation on arsABCD expression through an ArpR independent route. PMID:27055016

  3. The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H

    Directory of Open Access Journals (Sweden)

    Rauna eRiva

    2015-02-01

    Full Text Available The virulence factor PgtE is an outer membrane protease (omptin of the zoonotic pathogen Salmonella enterica that causes diseases ranging from gastroenteritis to severe enteric fever. It is surface exposed in bacteria that have a short-chain, i.e. rough LPS, as observed e.g. in bacteria residing inside macrophages or just emerging from them. We investigated whether PgtE cleaves the complement factors B (B and H (H, key proteins controlling formation and inactivation of the complement protein C3b and thereby the activity of the complement system. S. enterica serovar Typhimurium or omptin-expressing recombinant E. coli bacteria were incubated with purified human complement proteins or recombinant H fragments. PgtE cleaved both B and H, whereas its close homolog Pla of Yersinia pestis cleaved only H. H was cleaved at both N- and C-termini, while the central region resisted proteolysis. Because of multiple effects of PgtE on complement components (cleavage of C3, C3b, B and H we assessed its effect on the opsonophagocytosis of Salmonella. In human serum, C3 cleavage was dependent on proteolytically active PgtE. Human neutrophils interacted less with serum-opsonized FITC-stained S. enterica 14028R than with the isogenic ΔpgtE strain, as analyzed by flow cytometry. In conclusion, cleavage of B and H by PgtE, together with C3 cleavage, affects the C3-mediated recognition of S. enterica by human neutrophils, thus thwarting the immune protection against Salmonella.

  4. Biobased Membrane

    NARCIS (Netherlands)

    Koenders, E.A.B.; Zlopasa, J.; Picken, S.J.

    2015-01-01

    The present invention is in the field of a composition for forming a bio-compatible membrane applicable to building material, such as concrete, cement, etc., to a meth od of applying said composition for forming a bio-compatible membrane, a biocompatible membrane, use of said membrane for various pu

  5. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  6. Genetic and physiological factors affecting repair and mutagenesis in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Lemontt, J F

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described, particularly in relation to their imvolvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus are shown to also play a role in repair and mutagenesis.

  7. Targeting DNA Repair in Cancer: Beyond PARP Inhibitors.

    Science.gov (United States)

    Brown, Jessica S; O'Carrigan, Brent; Jackson, Stephen P; Yap, Timothy A

    2017-01-01

    Germline aberrations in critical DNA-repair and DNA damage-response (DDR) genes cause cancer predisposition, whereas various tumors harbor somatic mutations causing defective DDR/DNA repair. The concept of synthetic lethality can be exploited in such malignancies, as exemplified by approval of poly(ADP-ribose) polymerase inhibitors for treating BRCA1/2-mutated ovarian cancers. Herein, we detail how cellular DDR processes engage various proteins that sense DNA damage, initiate signaling pathways to promote cell-cycle checkpoint activation, trigger apoptosis, and coordinate DNA repair. We focus on novel therapeutic strategies targeting promising DDR targets and discuss challenges of patient selection and the development of rational drug combinations.

  8. Genetic and physiological factors affecting repair and mutagenesis in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Lemontt, J F

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data, and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described particularly in relation to their involvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus, are shown to also play a role in repair and mutagenesis.

  9. Meningocele repair - series (image)

    Science.gov (United States)

    ... containing a portion of the spinal cord membrane (meninges), spinal fluid, and a portion of spinal cord ... The spinal cord is covered with the membranes (meninges) and the skin is closed over the protruding ...

  10. Progress of peripheral nerve repair

    Institute of Scientific and Technical Information of China (English)

    陈峥嵘

    2002-01-01

    Study on repair of peripheral nerve injury has been proceeding over a long period of time. With the use of microsurgery technique since 1960s,the quality of nerve repair has been greatly improved. In the past 40 years, with the continuous increase of surgical repair methods, more progress has been made on the basic research of peripheral nerve repair.

  11. Achilles tendon repair

    Science.gov (United States)

    Achilles tendon rupture-surgery; Percutaneous Achilles tendon rupture repair ... To fix your torn Achilles tendon, the surgeon will: Make a cut down the back of your heel Make several small cuts rather than one large cut ...

  12. Diaphragmatic hernia repair - slideshow

    Science.gov (United States)

    ... presentations/100014.htm Diaphragmatic hernia repair - series—Normal anatomy To use the sharing ... Overview The chest cavity includes the heart and lungs. The abdominal cavity includes the liver, the stomach, ...

  13. Eye muscle repair - slideshow

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/presentations/100062.htm Eye muscle repair - series—Normal anatomy To use the sharing ... the eyeball to the eye socket. The external muscles of the eye are found behind the conjunctiva. ...

  14. Tracheoesophageal fistula repair - slideshow

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/presentations/100103.htm Tracheoesophageal fistula repair - series—Normal anatomy To use the sharing ... Editorial team. Related MedlinePlus Health Topics Esophagus Disorders Fistulas Tracheal Disorders A.D.A.M., Inc. is ...

  15. Inguinal hernia repair - slideshow

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/presentations/100027.htm Inguinal hernia repair - series—Normal anatomy To use the sharing ... to slide 4 out of 4 Overview A hernia occurs when part of an organ protrudes through ...

  16. INTERNAL REPAIR OF PIPELINES

    Energy Technology Data Exchange (ETDEWEB)

    Robin Gordon; Bill Bruce; Ian Harris; Dennis Harwig; Nancy Porter; Mike Sullivan; Chris Neary

    2004-04-12

    The two broad categories of deposited weld metal repair and fiber-reinforced composite liner repair technologies were reviewed for potential application for internal repair of gas transmission pipelines. Both are used to some extent for other applications and could be further developed for internal, local, structural repair of gas transmission pipelines. Preliminary test programs were developed for both deposited weld metal repair and for fiber-reinforced composite liner repair. Evaluation trials have been conducted using a modified fiber-reinforced composite liner provided by RolaTube and pipe sections without liners. All pipe section specimens failed in areas of simulated damage. Pipe sections containing fiber-reinforced composite liners failed at pressures marginally greater than the pipe sections without liners. The next step is to evaluate a liner material with a modulus of elasticity approximately 95% of the modulus of elasticity for steel. Preliminary welding parameters were developed for deposited weld metal repair in preparation of the receipt of Pacific Gas & Electric's internal pipeline welding repair system (that was designed specifically for 559 mm (22 in.) diameter pipe) and the receipt of 559 mm (22 in.) pipe sections from Panhandle Eastern. The next steps are to transfer welding parameters to the PG&E system and to pressure test repaired pipe sections to failure. A survey of pipeline operators was conducted to better understand the needs and performance requirements of the natural gas transmission industry regarding internal repair. Completed surveys contained the following principal conclusions: (1) Use of internal weld repair is most attractive for river crossings, under other bodies of water, in difficult soil conditions, under highways, under congested intersections, and under railway crossings. (2) Internal pipe repair offers a strong potential advantage to the high cost of horizontal direct drilling (HDD) when a new bore must be created

  17. Pectus excavatum repair - slideshow

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/presentations/100035.htm Pectus excavatum repair - series—Normal anatomy To use the sharing ... Go to slide 4 out of 4 Overview Pectus excavatum is a deformity of the front of the ...

  18. Hiatal hernia repair - slideshow

    Science.gov (United States)

    ... presentations/100028.htm Hiatal hernia repair - series—Normal anatomy To use the sharing features on ... Overview The esophagus runs through the diaphragm to the stomach. It functions to carry food from the mouth ...

  19. Repairing ceramic insulating tiles

    Science.gov (United States)

    Dunn, B. R.; Laymance, E. L.

    1980-01-01

    Fused-silica tiles containing large voids or gauges are repaired without adhesives by plug insertion method. Tiles are useful in conduits for high-temperature gases, in furnaces, and in other applications involving heat insulation.

  20. Rotator cuff repair - slideshow

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/presentations/100229.htm Rotator cuff repair - series—Normal anatomy To use the sharing ... to slide 4 out of 4 Overview The rotator cuff is a group of muscles and tendons that ...

  1. Cleft lip repair - slideshow

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/presentations/100010.htm Cleft lip repair - series—Normal anatomy To use the sharing ... abnormal opening in the middle of the upper lip. A cleft palate is an opening in the roof of ...

  2. Grey Repairable System Analysis

    Institute of Scientific and Technical Information of China (English)

    Renkuan Guo; Charles Ernie Love

    2006-01-01

    In this paper, we systematically discuss the basic concepts of grey theory, particularly the grey differential equation and its mathematical foundation, which is essentially unknown in the reliability engineering community. Accordingly,we propose a small-sample based approach to estimate repair improvement effects by partitioning system stopping times into intrinsic functioning times and repair improvement times. An industrial data set is used for illustrative purposes in a stepwise manner.

  3. Recycling from endosomes to the plasma membrane

    NARCIS (Netherlands)

    Dam, E.M. van

    2001-01-01

    Summary V Chapter?Summary Many membrane proteins are, after endocytic uptake, efficiently recycled back to the plasma membrane. The aim of the studies presented in this thesis was to determine pathways and molecular mechanisms that are involved in recycling. Plasma membrane-derived clathrin-coated v

  4. Recycling from endosomes to the plasma membrane

    NARCIS (Netherlands)

    Dam, E.M. van

    2001-01-01

    Summary V Chapter?Summary Many membrane proteins are, after endocytic uptake, efficiently recycled back to the plasma membrane. The aim of the studies presented in this thesis was to determine pathways and molecular mechanisms that are involved in recycling. Plasma membrane-derived clathrin-coated

  5. Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology

    Institute of Scientific and Technical Information of China (English)

    Sarah C Shuck; Emily A Short; John J Turchi

    2008-01-01

    Repair of bulky DNA adducts by the nucleotide excision repair (NER) pathway is one of the more versatile DNA repair pathways for the removal of DNA lesions. There are two subsets of the NER pathway, global genomic-NER (GG-NER) and transcription-coupled NER (TC-NER), which differ only in the step involving recognition of the DNA lesion. Following recognition of the damage, the sub-pathways then converge for the incision/excision steps and subsequent gap filling and ligation steps. This review will focus on the GGR sub-pathway of NER while the TCR sub-pathway will be covered in another article in this issue. The ability of the NER pathway to repair a wide array of adducts stems, in part, from the mechanisms involved in the initial recognition step of the damaged DNA and results in NER impacting an equally wide array of human physiological responses and events. In this review, the impact of NER on carcinogenesis, neurological function, sensitivity to environmental factors and sensitivity to cancer therapeutics will be discussed. The knowledge generated in our understanding of the NER pathway over the past 40 years has resulted from advances in the fields of animal model systems, mammalian genetics and in vitro biochemistry, as well as from reconstitution studies and structural analyses of the proteins and enzymes that participate in this pathway. Each of these avenues of research has contributed significantly to our understanding of how the NER pathway works and how alterations in NER activity, both positive and negative, influence human biology.

  6. Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Andrew H Lloyd

    Full Text Available DNA double strand breaks (DSBs occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ. We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp deletions occurred at a minority (25-45% of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.

  7. Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

    Science.gov (United States)

    Lloyd, Andrew H; Wang, Dong; Timmis, Jeremy N

    2012-01-01

    DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.

  8. Repair of a Bacterial Small β-Barrel Toxin Pore Depends on Channel Width.

    Science.gov (United States)

    von Hoven, Gisela; Rivas, Amable J; Neukirch, Claudia; Meyenburg, Martina; Qin, Qianqian; Parekh, Sapun; Hellmann, Nadja; Husmann, Matthias

    2017-02-14

    Membrane repair emerges as an innate defense protecting target cells against bacterial pore-forming toxins. Here, we report the first paradigm of Ca(2+)-dependent repair following attack by a small β-pore-forming toxin, namely, plasmid-encoded phobalysin of Photobacterium damselae subsp. damselae In striking contrast, Vibrio cholerae cytolysin, the closest ortholog of phobalysin, subverted repair. Mutational analysis uncovered a role of channel width in toxicity and repair. Thus, the replacement of serine at phobalysin´s presumed channel narrow point with the bulkier tryptophan, the corresponding residue in Vibrio cholerae cytolysin (W318), modulated Ca(2+) influx, lysosomal exocytosis, and membrane repair. And yet, replacing tryptophan (W318) with serine in Vibrio cholerae cytolysin enhanced toxicity. The data reveal divergent strategies evolved by two related small β-pore-forming toxins to manipulate target cells: phobalysin leads to fulminant perturbation of ion concentrations, closely followed by Ca(2+) influx-dependent membrane repair. In contrast, V. cholerae cytolysin causes insidious perturbations and escapes control by the cellular wounded membrane repair-like response.IMPORTANCE Previous studies demonstrated that large transmembrane pores, such as those formed by perforin or bacterial toxins of the cholesterol-dependent cytolysin family, trigger rapid, Ca(2+) influx-dependent repair mechanisms. In contrast, recovery from attack by the small β-pore-forming Staphylococcus aureus alpha-toxin or aerolysin is slow in comparison and does not depend on extracellular Ca(2+) To further elucidate the scope of Ca(2+) influx-dependent repair and understand its limitations, we compared the cellular responses to phobalysin and V. cholerae cytolysin, two related small β-pore-forming toxins which create membrane pores of slightly different sizes. The data indicate that the channel width of a small β-pore-forming toxin is a critical determinant of both primary

  9. Effects of PDT on the endocytic pathway

    Science.gov (United States)

    Kessel, David

    2010-02-01

    Two lines of evidence point to an early effect of photodamage on membrane trafficking. [1] Internalization of a fluorescent probe for hydrophobic membrane loci was impaired by prior photodamage. [2] Interference with the endocytic pathway by the PI-3 kinase antagonist wortmannin led to accumulation of cytoplasmic vacuoles suggesting a block in the recycling of plasma membrane components. Prior photodamage blocked this pathway so that no vacuoles were formed upon exposure of cells to wortmannin. In a murine hepatoma line, the endocytic pathway was preferentially sensitive to lysosomal photodamage. The role of photodamage to the endocytic pathway as a factor in PDT efficacy remains to be assessed.

  10. Resveratrol mediated cell death in cigarette smoke transformed breast epithelial cells is through induction of p21Waf1/Cip1 and inhibition of long patch base excision repair pathway

    Energy Technology Data Exchange (ETDEWEB)

    Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Das, Dipon; Siddharth, Sumit [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India); Choudhuri, Tathagata [Institute of Life Sciences, Nalco Square, Bhubaneswar, Orissa 751023 (India); Department of Biotechnology, Visva Bharati University, Santiniketan, West Bengal (India); Kundu, Chanakya Nath, E-mail: cnkundu@gmail.com [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India)

    2014-03-15

    Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Res mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-β, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21. - Highlights: • Resveratrol (Res) caused reduction of MCF-10A-Tr cell growth by inducing apoptosis. • Res caused cell cycle arrest and DNA damage in p21 dependent manner. • Res mediated LP-BER reduction in MCF-10A-Tr cells was a p21 dependent phenomenon. • Res inhibits BER and PI

  11. Carbon nanotubes in neuroregeneration and repair.

    Science.gov (United States)

    Fabbro, Alessandra; Prato, Maurizio; Ballerini, Laura

    2013-12-01

    In the last decade, we have experienced an increasing interest and an improved understanding of the application of nanotechnology to the nervous system. The aim of such studies is that of developing future strategies for tissue repair to promote functional recovery after brain damage. In this framework, carbon nanotube based technologies are emerging as particularly innovative tools due to the outstanding physical properties of these nanomaterials together with their recently documented ability to interface neuronal circuits, synapses and membranes. This review will discuss the state of the art in carbon nanotube technology applied to the development of devices able to drive nerve tissue repair; we will highlight the most exciting findings addressing the impact of carbon nanotubes in nerve tissue engineering, focusing in particular on neuronal differentiation, growth and network reconstruction.

  12. The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

    Energy Technology Data Exchange (ETDEWEB)

    Bajinskis, Ainars, E-mail: ainars.bajinskis@gmt.su.se [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden); Olsson, Gunilla; Harms-Ringdahl, Mats [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden)

    2012-03-01

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO{sub 3}). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

  13. Polymorphic membrane protein (PMP) 20 and PMP 21 of Chlamydia pneumoniae induce proinflammatory mediators in human endothelial cells in vitro by activation of the nuclear factor-kappaB pathway.

    Science.gov (United States)

    Niessner, Alexander; Kaun, Christoph; Zorn, Gerlinde; Speidl, Walter; Türel, Zeynep; Christiansen, Gunna; Pedersen, Anna-Sofie; Birkelund, Svend; Simon, Susan; Georgopoulos, Apostolos; Graninger, Wolfgang; de Martin, Rainer; Lipp, Joachim; Binder, Bernd R; Maurer, Gerald; Huber, Kurt; Wojta, Johann

    2003-07-01

    We tested whether polymorphic membrane proteins (PMPs) of Chlamydia pneumoniae might play a role in triggering an inflammatory response in human endothelial cells. Of 15 purified, recombinant chlamydial PMPs tested, 2 (PMP 20 and PMP 21) dose-dependently increased the production of the inflammatory mediators interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1), in cultured human endothelial cells; production of IL-8 was also increased. When endothelial cells were infected by live C. pneumoniae, an increase in the production of IL-6, IL-8, and MCP-1 was seen. We used adenovirus-induced overexpression of IkappaBalpha-an inhibitor of nuclear factor (NF)-kappaB-to demonstrate that PMP 20 and PMP 21 increase the production of IL-6 and MCP-1 in human endothelial cells by activation of the NF-kappaB pathway, because, in cells overexpressing IkappaBalpha, treatment with the respective PMP did not result in increased production of IL-6 and MCP-1. Thus, C. pneumoniae could, by interactions of its PMPs with the endothelium, contribute to the process of vascular injury during the development and progression of atherosclerotic lesions.

  14. Membranous nephropathy

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/000472.htm Membranous nephropathy To use the sharing features on this page, please enable JavaScript. Membranous nephropathy is a kidney disorder that leads to changes ...

  15. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1)