Pir51, a Rad51-interacting protein with high expression in aggressive lymphoma, controls mitomycin C sensitivity and prevents chromosomal breaks

International Nuclear Information System (INIS)

Henson, Sarah E.; Tsai, Shih-Chang; Malone, Cindy Sue; Soghomonian, Shahe V.; Ouyang, Yan; Wall, Randolph; Marahrens, York; Teitell, Michael A.

2006-01-01

Pir51, a protein of unknown function that interacts with Rad51, was identified in a screen for genes that were highly expressed in aggressive mantle cell lymphoma (MCL) versus indolent small lymphocytic lymphoma (SLL) patient samples. We show that Pir51 is a nuclear protein expressed in a variety of cell types and that its expression is regulated during the cell cycle in a pattern nearly identical to Rad51. Also similar to Rad51, Pir51 levels did not change in response to a variety of DNA damaging agents. siRNA depletion of Pir51 did not reduce homologous recombination repair (HRR), but sensitized cells to mitomycin C (MMC)-induced DNA crosslinking and resulted in elevated levels of double-strand breaks (DSBs) in metaphase chromosome spreads and reduced colony formation. Therefore, Pir51 maintains genomic integrity and potentially connects the early response to DNA crosslinks, orchestrated by the ATR kinase and Fanconi Anemia (FA) proteins, to later stages of Rad51-dependent repair. Our results provide the first example of a Rad51-binding protein that influences DNA crosslink repair without affecting homologous recombination repair

A novel interation of nucleolin with Rad51

International Nuclear Information System (INIS)

De, Ananya; Donahue, Sarah L.; Tabah, Azah; Castro, Nancy E.; Mraz, Naomi; Cruise, Jennifer L.; Campbell, Colin

2006-01-01

Nucleolin associates with various DNA repair, recombination, and replication proteins, and possesses DNA helicase, strand annealing, and strand pairing activities. Examination of nuclear protein extracts from human somatic cells revealed that nucleolin and Rad51 co-immunoprecipitate. Furthermore, purified recombinant Rad51 associates with in vitro transcribed and translated nucleolin. Electroporation-mediated introduction of anti-nucleolin antibody resulted in a 10- to 20-fold reduction in intra-plasmid homologous recombination activity in human fibrosarcoma cells. Additionally, introduction of anti-nucleolin antibody sensitized cells to death induced by the topoisomerase II inhibitor, amsacrine. Introduction of anti-Rad51 antibody also reduced intra-plasmid homologous recombination activity and induced hypersensitivity to amsacrine-induced cell death. Co-introduction of anti-nucleolin and anti-Rad51 antibodies did not produce additive effects on homologous recombination or on cellular sensitivity to amsacrine. The association of the two proteins raises the intriguing possibility that nucleolin binding to Rad51 may function to regulate homologous recombinational repair of chromosomal DNA

Comparative expression profiling of AtRAD5B and AtNDL1: Hints towards a role in G protein mediated signaling.

Science.gov (United States)

Khatri, Nisha; Singh, Swati; Hakim, Nasmeen; Mudgil, Yashwanti

2017-11-01

Arabidopsis AtRAD5B encodes for a putative helicase of the class SWItch/Sucrose Non-Fermentable (SWI/SNF) ATPases. We identified AtRAD5B as an interactor of N-MYC DOWNREGULATED-LIKE1 (AtNDL1) in a yeast two-hybrid screen. AtNDL1 is a G protein signaling component which regulates auxin transport and gradients together with GTP binding protein beta 1 (AGB1). Auxin gradients are known to recruit SWI/SNF remodeling complexes to the chromatin and regulate expression of genes involved in flower and leaf formation. In current study, a comparative spatial and temporal co-expression/localization analysis of AtNDL1, AGB1 with AtRAD5B was carried out in order to explore the possibility of their coexistence in a common signaling network. Translational fusion (GUS) of AtNDL1 and AtRAD5B in seedlings and reproductive organs revealed that both shared similar expression patterns with the highest expression observed in male reproductive organs. Moreover, they shared similar domains of localization in roots, suggesting their potential functioning together in reproductive and root development processes. This study predicts the existence of a signaling network involving AtNDL1, AGB1 with AtRAD5B. Copyright © 2017 Elsevier B.V. All rights reserved.

Down-regulation of Rad51 activity during meiosis in yeast prevents competition with Dmc1 for repair of double-strand breaks.

Directory of Open Access Journals (Sweden)

Yan Liu

2014-01-01

Full Text Available Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs.

The C-terminal region of Rad52 is essential for Rad52 nuclear and nucleolar localization, and accumulation at DNA damage sites immediately after irradiation

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Koike, Manabu, E-mail: m_koike@nirs.go.jp [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Yutoku, Yasutomo [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Graduate School of Science, Chiba University, Yayoicho, Inage-ku, Chiba 263-8522 (Japan); Koike, Aki [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

2013-05-31

Highlights: •Rad52 might play a key role in the repair of DSB immediately after irradiation. •EYFP-Rad52 accumulates rapidly at DSB sites and colocalizes with Ku80. •Accumulation of Rad52 at DSB sites is independent of the core NHEJ factors. •Localization and recruitment of Rad52 to DSB sites are dependent on the Rad52 CTR. •Basic amino acids in Rad52 CTR are highly conserved among vertebrate species. -- Abstract: Rad52 plays essential roles in homologous recombination (HR) and repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae. However, in vertebrates, knockouts of the Rad52 gene show no hypersensitivity to agents that induce DSBs. Rad52 localizes in the nucleus and forms foci at a late stage following irradiation. Ku70 and Ku80, which play an essential role in nonhomologous DNA-end-joining (NHEJ), are essential for the accumulation of other core NHEJ factors, e.g., XRCC4, and a HR-related factor, e.g., BRCA1. Here, we show that the subcellular localization of EYFP-Rad52(1–418) changes dynamically during the cell cycle. In addition, EYFP-Rad52(1–418) accumulates rapidly at microirradiated sites and colocalizes with the DSB sensor protein Ku80. Moreover, the accumulation of EYFP-Rad52(1–418) at DSB sites is independent of the core NHEJ factors, i.e., Ku80 and XRCC4. Furthermore, we observed that EYFP-Rad52(1–418) localizes in nucleoli in CHO-K1 cells and XRCC4-deficient cells, but not in Ku80-deficient cells. We also found that Rad52 nuclear localization, nucleolar localization, and accumulation at DSB sites are dependent on eight amino acids (411–418) at the end of the C-terminal region of Rad52 (Rad52 CTR). Furthermore, basic amino acids on Rad52 CTR are highly conserved among mammalian, avian, and fish homologues, suggesting that Rad52 CTR is important for the regulation and function of Rad52 in vertebrates. These findings also suggest that the mechanism underlying the regulation of subcellular localization of Rad52 is

Functional roles for Rad9 in prostate cancer

International Nuclear Information System (INIS)

Lieberman, H.B.; Broustas, C.G.

2012-01-01

The goal of this work is to understand the mechanistic relationship between high levels of Rad9 protein and prostate cancer. The study is based on several findings suggesting a role for Rad9 in this disease. Rad9 has all the hallmark features of an oncogene or tumor suppressor. It regulates genomic stability, multiple cell cycle checkpoints, apoptosis and DNA repair. In addition, it can transactivate downstream target genes via direct interaction with promoter DNA sequences. We found Rad9 protein levels were very high in prostate cancer cell lines. Furthermore, we examined 52 primary normal prostate and 339 prostate cancer specimens for Rad9 protein by immunohistochemical staining. Statistical significance for Rad9 positive staining versus cancer, and stain intensity versus Stage were tested. We get a p-value of <0.001 when comparing percentage positive by cancer Stage, or stain intensity by cancer Stage. Based on these data, we sought to define the nature of the relationship between Rad9 and prostate cancer. We demonstrate that Rad9 acts as an oncogene in prostate cancer by playing a critical role in tumor formation in a mouse xenograph model. We also show that Rad9 is important for cellular phenotypes essential for metastasis, including tumor cell migration, invasion and resistance to programmed cell death after detachment from extracellular matrix. Therefore, Rad9 is critical for several aspects of prostate tumor progression, and could serve as a novel target for anti-cancer therapy

The recombination protein RAD52 cooperates with the excision repair protein OGG1 for the repair of oxidative lesions in mammalian cells

DEFF Research Database (Denmark)

de Souza-Pinto, Nadja C; Maynard, Scott; Hashiguchi, Kazunari

2009-01-01

number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic...... knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1...... to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities....

RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates

International Nuclear Information System (INIS)

Reynolds, P.; Weber, S.; Prakash, L.

1985-01-01

The RAD6 gene of Saccharomyces cerevisiae is required for postreplication repair of UV-damaged DNA, for induced mutagenesis, and for sporulation. The authors have mapped the transcripts and determined the nucleotide sequence of the cloned RAD6 gene. The RAD6 gene encodes two transcripts of 0.98 and 0.86 kilobases which differ only in their 3' termini. The transcribed region contains an open reading frame of 516 nucleotides. The rad6-1 and rad6-3 mutant alleles, which the authors have cloned and sequenced, introduce amber and ochre nonsense mutations, respectively into the open reading frame, proving that it encodes the RAD6 protein. The RAD6 protein predicted by the nucleotide sequence is 172 amino acids long, has a molecular weight of 19,704, and contains 23.3% acidic and 11.6% basic residues. Its most striking feature is the highly acidic carboxyl terminus: 20 of the 23 terminal amino acids are acidic, including 13 consecutive aspartates. RAD6 protein thus resembles high mobility group proteins HMG-1 and HMG-2, which each contain a carboxyl-proximal tract of acidic amino acids. 48 references, 6 figures

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

International Nuclear Information System (INIS)

Glassner, B.J.; Mortimer, R.K.

1994-01-01

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

MRI for the assessment of malignancy in BI-RADS 4 mammographic microcalcifications.

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Barbara Bennani-Baiti

Full Text Available Assess the performance of breast MRI to diagnose breast cancer in BI-RADS 4 microcalcifications detected by mammography.This retrospective, IRB-approved study included 248 consecutive contrast-enhanced breast MRI (1.5T, protocol in accordance with EUSOBI recommendations performed to further diagnose BI-RADS 4 microcalcifications detected at mammography during a 3-year period. Standard of reference had to be established by histopathology. Routine consensus reading results by two radiologists were dichotomized as positive or negative and compared with the reference standard (benign vs malignant to calculate diagnostic parameters.There were 107 malignant and 141 benign microcalcifications. Malignancy rates were 18.3% (23/126 BI-RADS 4a, 41.7% (25/60 BI-RADS 4b and 95% (59/62 BI-RADS 4c. There were 103 true-positive, 116 true-negative, 25 false-positive, and 4 false-negative (one invasive cancer, three DCIS; 2 BI-RADS 4c, 1 BI-RADS 4b on mammography breast MRI findings, effecting a sensitivity, specificity, PPV, and NPV of 96.3% (95%-CI 90.7-99.0%, 82.3% (95%-CI 75.0-88.2%, 80.5% (95%-CI 72.5-87.0% and 96.7% (95%-CI 91.7-99.1%, respectively.MRI is an accurate tool to further diagnose BI-RADS 4a and 4b microcalcifications and may be helpful to avoid unnecessary biopsies in BI-RADS 4a and 4b lesions. BI-RADS 4c microcalcifications should be biopsied irrespective of MRI findings.

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

International Nuclear Information System (INIS)

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

2005-01-01

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

Recovery of deficient homologous recombination in Brca2-depleted mouse cells by wild-type Rad51 expression.

Science.gov (United States)

Lee, Shauna A; Roques, Céline; Magwood, Alissa C; Masson, Jean-Yves; Baker, Mark D

2009-02-01

The BRCA2 tumor suppressor is important in maintaining genomic stability. BRCA2 is proposed to control the availability, cellular localization and DNA binding activity of the central homologous recombination protein, RAD51, with loss of BRCA2 resulting in defective homologous recombination. Nevertheless, the roles of BRCA2 in regulating RAD51 and how other proteins implicated in RAD51 regulation, such as RAD52 and RAD54 function relative to BRCA2 is not known. In this study, we tested whether defective homologous recombination in Brca2-depleted mouse hybridoma cells could be rectified by expression of mouse Rad51 or the Rad51-interacting mouse proteins, Rad52 and Rad54. In the Brca2-depleted cells, defective homologous recombination can be restored by over-expression of wild-type mouse Rad51, but not mouse Rad52 or Rad54. Correction of the homologous recombination defect requires Rad51 ATPase activity. A sizeable fraction ( approximately 50%) of over-expressed wild-type Rad51 is nuclear localized. The restoration of homologous recombination in the presence of a low (i.e., non-functional) level of Brca2 by wild-type Rad51 over-expression is unexpected. We suggest that Rad51 may access the nuclear compartment in a Brca2-independent manner and when Rad51 is over-expressed, the normal requirement for Brca2 control over Rad51 function in homologous recombination is dispensable. Our studies support loss of Rad51 function as a critical underlying factor in the homologous recombination defect in the Brca2-depleted cells.

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

International Nuclear Information System (INIS)

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

1998-01-01

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

RAD21L, a novel cohesin subunit implicated in linking homologous chromosomes in mammalian meiosis.

Science.gov (United States)

Lee, Jibak; Hirano, Tatsuya

2011-01-24

Cohesins are multi-subunit protein complexes that regulate sister chromatid cohesion during mitosis and meiosis. Here we identified a novel kleisin subunit of cohesins, RAD21L, which is conserved among vertebrates. In mice, RAD21L is expressed exclusively in early meiosis: it apparently replaces RAD21 in premeiotic S phase, becomes detectable on the axial elements in leptotene, and stays on the axial/lateral elements until mid pachytene. RAD21L then disappears, and is replaced with RAD21. This behavior of RAD21L is unique and distinct from that of REC8, another meiosis-specific kleisin subunit. Remarkably, the disappearance of RAD21L at mid pachytene correlates with the completion of DNA double-strand break repair and the formation of crossovers as judged by colabeling with molecular markers, γ-H2AX, MSH4, and MLH1. RAD21L associates with SMC3, STAG3, and either SMC1α or SMC1β. Our results suggest that cohesin complexes containing RAD21L may be involved in synapsis initiation and crossover recombination between homologous chromosomes.

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

Energy Technology Data Exchange (ETDEWEB)

Wiese, Claudia; Dray, Eloise; Groesser, Torsten; San Filippo,Joseph; Shi, Idina; Collins, David W.; Tsai, Miaw-Sheue; Williams,Gareth; Rydberg, Bjorn; Sung, Patrick; Schild, David

2007-04-11

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds dsDNA and RAD51, and it greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide the first evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions

International Nuclear Information System (INIS)

Sung, P.; Prakash, S.; Prakash, L.

1990-01-01

The RAD6 gene of Saccharomyces cerevisiae is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. RAD6 protein is a ubiquitin-conjugating enzyme (E2) that has been shown to attach multiple molecules of ubiquitin to histones H2A and H2B. We have now examined whether the E2 activity of RAD6 is involved in its various biological functions. Since the formation of a thioester adduct between E2 and ubiquitin is necessary for E2 activity, the single cysteine residue (Cys-88) present in RAD6 was changed to alanine or valine. The mutant proteins were overproduced in yeast cells and purified to near homogeneity. We show that the rad6 Ala-88 and rad6 Val-88 mutant proteins lack the capacity for thioester formation with ubiquitin and, as a consequence, are totally devoid of any E2 activity. The rad6 Ala-88 and rad6 Val-88 mutations confer a defect in DNA repair, mutagenesis, and sporulation equivalent to that in the rad6 null allele. We suggest that the biological functions of RAD6 require its E2 activity. (author)

Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy

KAUST Repository

Reymer, A.; Frykholm, K.; Morimatsu, K.; Takahashi, M.; Norden, B.

2009-01-01

for central and N-terminal parts of pure (uncomplexed) Rad51 protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA filaments in solution. The structure, validated by comparison

The Medicago truncatula GRAS protein RAD1 supports arbuscular mycorrhiza symbiosis and Phytophthora palmivora susceptibility.

Science.gov (United States)

Rey, Thomas; Bonhomme, Maxime; Chatterjee, Abhishek; Gavrin, Aleksandr; Toulotte, Justine; Yang, Weibing; André, Olivier; Jacquet, Christophe; Schornack, Sebastian

2017-12-16

The roots of most land plants are colonized by symbiotic arbuscular mycorrhiza (AM) fungi. To facilitate this symbiosis, plant genomes encode a set of genes required for microbial perception and accommodation. However, the extent to which infection by filamentous root pathogens also relies on some of these genes remains an open question. Here, we used genome-wide association mapping to identify genes contributing to colonization of Medicago truncatula roots by the pathogenic oomycete Phytophthora palmivora. Single-nucleotide polymorphism (SNP) markers most significantly associated with plant colonization response were identified upstream of RAD1, which encodes a GRAS transcription regulator first negatively implicated in root nodule symbiosis and recently identified as a positive regulator of AM symbiosis. RAD1 transcript levels are up-regulated both in response to AM fungus and, to a lower extent, in infected tissues by P. palmivora where its expression is restricted to root cortex cells proximal to pathogen hyphae. Reverse genetics showed that reduction of RAD1 transcript levels as well as a rad1 mutant are impaired in their full colonization by AM fungi as well as by P. palmivora. Thus, the importance of RAD1 extends beyond symbiotic interactions, suggesting a general involvement in M. truncatula microbe-induced root development and interactions with unrelated beneficial and detrimental filamentous microbes. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Schizosaccharomyces pombe Rad22A and Rad22B have similar biochemical properties and form multimeric structures

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Vries, Femke A.T. de [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden (Netherlands); Zonneveld, Jose B.M. [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden (Netherlands); Groot, Anton J. de [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden (Netherlands); Koning, Roman I. [Department of Molecular Cell Biology, Leiden University Medical Center, Leiden (Netherlands); Zeeland, Albert A. van [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden (Netherlands); Pastink, Albert [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden (Netherlands)]. E-mail: A.Pastink@lumc.nl

2007-02-03

The Saccharomyces cerevisiae Rad52 protein has a crucial role in the repair of DNA double-strand breaks by homologous recombination. In vitro, Rad52 displays DNA binding and strand annealing activities and promotes Rad51-mediated strand exchange. Schizosaccharomyces pombe has two Rad52 homologues, Rad22A and Rad22B. Whereas rad22A deficient strains exhibit severe defects in repair and recombination, rad22B mutants have a much less severe phenotype. To better understand the role of Rad22A and Rad22B in double-strand break repair, both proteins were purified to near homogeneity. Using gel retardation and filter binding assays, binding of Rad22A and Rad22B to short single-stranded DNAs was demonstrated. Binding of Rad22A to double-stranded oligonucleotides or linearized plasmid molecules containing blunt ends or short single-stranded overhangs could not be detected. Rad22B also does not bind efficiently to short duplex oligonucleotides but binds readily to DNA fragments containing 3'-overhangs. Rad22A as well as Rad22B efficiently promote annealing of complementary single-stranded DNAs. In the presence of Rad22A annealing of complementary DNAs is almost 90%. Whereas in reactions containing Rad22B the maximum level of annealing is 60%, most likely due to inhibition of the reaction by duplex DNA. Gel-filtration experiments and electron microscopic analyses indicate self-association of Rad22A and Rad22B and the formation of multimeric structures as has been observed for Rad52 in yeast and man.

Overexpressed of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

Energy Technology Data Exchange (ETDEWEB)

Schild, David; Wiese, Claudia

2009-10-15

RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the 'recombination mediators'. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as 'recombination co-mediators'. Defects in either recombination mediators or comediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic restabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51.

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair.

Science.gov (United States)

Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

2016-10-01

USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.

The KYxxL motif in Rad17 protein is essential for the interaction with the 9–1–1 complex

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Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp [Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan); Ikeuchi, Masayoshi; Nakayama, Yuji [Department of Biochemistry & Molecular Biology, Kyoto Pharmaceutical University, Kyoto 607-8414 (Japan); Yamaguchi, Naoto, E-mail: nyama@faculty.chiba-u.jp [Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan)

2016-09-02

ATR-dependent DNA damage checkpoint is the major DNA damage checkpoint against UV irradiation and DNA replication stress. The Rad17–RFC and Rad9–Rad1–Hus1 (9–1–1) complexes interact with each other to contribute to ATR signaling, however, the precise regulatory mechanism of the interaction has not been established. Here, we identified a conserved sequence motif, KYxxL, in the AAA+ domain of Rad17 protein, and demonstrated that this motif is essential for the interaction with the 9–1–1 complex. We also show that UV-induced Rad17 phosphorylation is increased in the Rad17 KYxxL mutants. These data indicate that the interaction with the 9–1–1 complex is not required for Rad17 protein to be an efficient substrate for the UV-induced phosphorylation. Our data also raise the possibility that the 9–1–1 complex plays a negative regulatory role in the Rad17 phosphorylation. We also show that the nucleotide-binding activity of Rad17 is required for its nuclear localization. - Highlights: • We have identified a conserved KYxxL motif in Rad17 protein. • The KYxxL motif is crucial for the interaction with the 9–1–1 complex. • The KYxxL motif is dispensable or inhibitory for UV-induced Rad17 phosphorylation. • Nucleotide binding of Rad17 is required for its nuclear localization.

Function of Rad51 paralogs in eukaryotic homologous recombinational repair

International Nuclear Information System (INIS)

Liu, N.; Skowronek, K.

2003-01-01

Full text: Homologous recombinational repair (HRR) is an important mechanism for maintaining genetic integrity and cancer prevention by accurately repair of DNA double strand breaks induced by environmental insults or occurred in DNA replication. A critical step in HRR is the polymerization of Rad51 on single stranded DNA to form nuclear protein filaments, the later conduct DNA strand paring and exchange between homologous strands. A number of proteins, including replication protein A (RPA), Rad52 and Rad51 paralogs, are suggested to modulate or facilitate the process of Rad51 filament formation. Five Rad51 paralogs, namely XRCC2, XRCC3, Rad51B, Rad51C and Rad51D have been identified in eucaryotic cells. These proteins show distant protein sequence identity to Rad51, to yeast Rad51 paralogs (Rad55 and Rad57) and to each other. Hamster or chicken mutants of Rad51 paralogs exhibit hypersensitivity to a variety of DNA damaging agents, especially cross-linking agents, and are defective in assembly of Rad51 onto HRR site after DNA damage. Recent data from our and other labs showed that Rad51 paralogs constitute two distinct complexes in cell extracts, one contains XRCC2, Rad51B, Rad51C and Rad51D, and the other contains Rad51C and XRCC3. Rad51C is involved in both complexes. Our results also showed that XRCC3-Rad51C complex interacts with Rad51 in vivo. Furthermore, overexpression of Rad52 can partially suppress the hypersensitivity of XRCC2 mutant irs1 to ionizing radiation and corrected the defects in Rad51 focus formation. These results suggest that XRCC2 and other Rad51 paralogs play a mediator function to Rad51 in the early stage of HRR

Roles of Rad51 protein in homologous recombination in mammalian cells: relation with repair, replication and cell cycle

International Nuclear Information System (INIS)

Lambert, S.

2001-01-01

Homologous recombination (HR) is a fundamental process, allowing a faithful repair. In mammalian, MmRAD51, which is the homologue of Saccharomyces cerevisiae ScRAD51 key protein for HR, is an essential gene. This work is based on the characterisation of viable hyper and hypo-recombinant cell lines specifically affected in the Rad51 pathway. By expressing wild type and dominant negative forms of MmRad51, we demonstrated that Rad51 pathway participates to the repair by HR to induced DNA damages. However, inhibition of the Rad 51 pathway does not affect cell viability, spontaneously or after irradiation, whereas, radiation induced HR is inhibited. In the presence of DNA damages during late S and G2/M phase, inhibition of Rad51 pathway induced chromosomal aberrations, leading to a transient arrest in mitosis. This arrest is associated with an increased of cell death. However, a fraction of cells can escape from this transient arrest by forming tetraploid cells, associated with an absence of chromalid separation. Thus, in response to impaired Rad51 pathway, mitotic checkpoints seems to play an essential role. In line with this, we showed that the essential function of Rad51 is p53-dependent, which is in agreement with the role of p53 in tetraploidy inhibition. Our results suggest that the Rad51 protein could participate to the control of mitotic checkpoints and thus to the maintenance of genetic stability. This function could involve other Rad51 partners such as the tumour suppressors BRCA1, BRCA2 and p53. (author) [fr

Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

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Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

2014-01-17

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

Rad9 Has a Functional Role in Human Prostate Carcinogenesis

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Zhu, Aiping; Zhang, Charles Xia; Lieberman, Howard B.

2013-01-01

Prostate cancer is currently the most common type of neoplasm found in American men, other than skin cancer, and is the second leading cause of cancer death in males. Because cell cycle checkpoint proteins stabilize the genome, the relationship of one such protein, Rad9, to prostate cancer was investigated. We found that four prostate cancer cell lines (CWR22, DU145, LNCaP, and PC-3), relative to PrEC normal prostate cells, have aberrantly high levels of Rad9 protein. The 3′-end region of intron 2 of Rad9 in DU145 cells is hypermethylated at CpG islands, and treatment with 5′-aza-2′-deoxycytidine restores near-normal levels of methylation and reduces Rad9 protein abundance. Southern blot analyses indicate that PC-3 cells contain an amplified Rad9 copy number. Therefore, we provide evidence that Rad9 levels are high in prostate cancer cells due at least in part to aberrant methylation or gene amplification. The effectiveness of small interfering RNA to lower Rad9 protein levels in CWR22, DU145, and PC-3 cells correlated with reduction of tumorigenicity in nude mice, indicating that Rad9 actively contributes to the disease. Rad9 protein levels were high in 153 of 339 human prostate tumor biopsy samples examined and detectable in only 2 of 52 noncancerous prostate tissues. There was a strong correlation between Rad9 protein abundance and cancer stage. Rad9 protein level can thus provide a biomarker for advanced prostate cancer and is causally related to the disease, suggesting the potential for developing novel diagnostic, prognostic, and therapeutic tools based on detection or manipulation of Rad9 protein abundance. PMID:18316588

Regulation of Rad51-Mediated Homologous Recombination by BRCA2, DSS1 and RAD52

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Rants, Louise Olthaver Juhl

Homologous recombination (HR) provides a mechanism to restore integrity and maintain stability of the genetic material. HR is a major pathway for repair of DNA double-strand breaks (DSB), recovery of broken replication forks and generation of meiotic crossovers. The defining step in HR is homolog......Homologous recombination (HR) provides a mechanism to restore integrity and maintain stability of the genetic material. HR is a major pathway for repair of DNA double-strand breaks (DSB), recovery of broken replication forks and generation of meiotic crossovers. The defining step in HR...... is homologous strand exchange directed by the RecA-related recombinase Rad51. BRCA2 participates in HR by mediating Rad51 homology-directed repair. Both BRCA2 and Rad51 are essential for HR, DNA repair, and the maintenance of genome stability. In the present study, we seek to understand the mechanism of BRCA2...... with RAD52-mediated repair at sites of CPT-induced DNA damage. The synthetic lethality approach using RAD52 small molecule inhibitors in brca-deficient cancers is a promising therapeutic strategy for cancer treatment....

Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy

KAUST Repository

Reymer, A.

2009-07-08

To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a filament structure of a human Rad51 protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleo-protein complex. An inhomogeneously stretched double-stranded DNA fitted into the filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-protein filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning protein monomers along the DNA strand upon filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.

Mimivirus reveals Mre11/Rad50 fusion proteins with a sporadic distribution in eukaryotes, bacteria, viruses and plasmids

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Ogata Hiroyuki

2011-09-01

Full Text Available Abstract Background The Mre11/Rad50 complex and the homologous SbcD/SbcC complex in bacteria play crucial roles in the metabolism of DNA double-strand breaks, including DNA repair, genome replication, homologous recombination and non-homologous end-joining in cellular life forms and viruses. Here we investigated the amino acid sequence of the Mimivirus R555 gene product, originally annotated as a Rad50 homolog, and later shown to have close homologs in marine microbial metagenomes. Results Our bioinformatics analysis revealed that R555 protein sequence is constituted from the fusion of an N-terminal Mre11-like domain with a C-terminal Rad50-like domain. A systematic database search revealed twelve additional cases of Mre11/Rad50 (or SbcD/SbcC fusions in a wide variety of unrelated organisms including unicellular and multicellular eukaryotes, the megaplasmid of a bacterium associated to deep-sea hydrothermal vents (Deferribacter desulfuricans and the plasmid of Clostridium kluyveri. We also showed that R555 homologs are abundant in the metagenomes from different aquatic environments and that they most likely belong to aquatic viruses. The observed phyletic distribution of these fusion proteins suggests their recurrent creation and lateral gene transfers across organisms. Conclusions The existence of the fused version of protein sequences is consistent with known functional interactions between Mre11 and Rad50, and the gene fusion probably enhanced the opportunity for lateral transfer. The abundance of the Mre11/Rad50 fusion genes in viral metagenomes and their sporadic phyletic distribution in cellular organisms suggest that viruses, plasmids and transposons played a crucial role in the formation of the fusion proteins and their propagation into cellular genomes.

Phosphorylation of Rad9 at serine 328 by cyclin A-Cdk2 triggers apoptosis via interfering Bcl-xL.

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Zhuo Zhan

Full Text Available Cyclin A-Cdk2, a cell cycle regulated Ser/Thr kinase, plays important roles in a variety of apoptoticprocesses. However, the mechanism of cyclin A-Cdk2 regulated apoptosis remains unclear. Here, we demonstrated that Rad9, a member of the BH3-only subfamily of Bcl-2 proteins, could be phosphorylated by cyclin A-Cdk2 in vitro and in vivo. Cyclin A-Cdk2 catalyzed the phosphorylation of Rad9 at serine 328 in HeLa cells during apoptosis induced by etoposide, an inhibitor of topoisomeraseII. The phosphorylation of Rad9 resulted in its translocation from the nucleus to the mitochondria and its interaction with Bcl-xL. The forced activation of cyclin A-Cdk2 in these cells by the overexpression of cyclin A,triggered Rad9 phosphorylation at serine 328 and thereby promoted the interaction of Rad9 with Bcl-xL and the subsequent initiation of the apoptotic program. The pro-apoptotic effects regulated by the cyclin A-Cdk2 complex were significantly lower in cells transfected with Rad9S328A, an expression vector that encodes a Rad9 mutant that is resistant to cyclin A-Cdk2 phosphorylation. These findings suggest that cyclin A-Cdk2 regulates apoptosis through a mechanism that involves Rad9phosphorylation.

Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1).

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Liu, Jianyu; Stevens, Payton D; Eshleman, Nichole E; Gao, Tianyan

2013-08-09

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.

RAD18 and associated proteins are immobilized in nuclear foci in human cells entering S-phase with ultraviolet light-induced damage

International Nuclear Information System (INIS)

Watson, Nicholas B.; Nelson, Eric; Digman, Michelle; Thornburg, Joshua A.; Alphenaar, Bruce W.; McGregor, W. Glenn

2008-01-01

Proteins required for translesion DNA synthesis localize in nuclear foci of cells with replication-blocking lesions. The dynamics of this process were examined in human cells with fluorescence-based biophysical techniques. Photobleaching recovery and raster image correlation spectroscopy experiments indicated that involvement in the nuclear foci reduced the movement of RAD18 from diffusion-controlled to virtual immobility. Examination of the mobility of REV1 indicated that it is similarly immobilized when it is observed in nuclear foci. Reducing the level of RAD18 greatly reduced the focal accumulation of REV1 and reduced UV mutagenesis to background frequencies. Fluorescence lifetime measurements indicated that RAD18 and RAD6A or polη only transferred resonance energy when these proteins colocalized in damage-induced nuclear foci, indicating a close physical association only within such foci. Our data support a model in which RAD18 within damage-induced nuclear foci is immobilized and is required for recruitment of Y-family DNA polymerases and subsequent mutagenesis. In the absence of damage these proteins are not physically associated within the nucleoplasm

Roles for the yeast RAD18 and RAD52 DNA repair genes in UV mutagenesis.

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Armstrong, J D; Chadee, D N; Kunz, B A

1994-11-01

Experimental evidence indicates that although the Saccharomyces cerevisiae RAD18 and RAD52 genes are not required for nucleotide excision repair, they function in the processing of UV-induced DNA damage in yeast. Conflicting statements regarding the UV mutability of strains deleted for RAD18 prompted us to re-examine the influence of RAD18, and RAD52, on UV mutagenesis. To do so, we characterized mutations induced by UV in SUP4-o, a yeast suppressor tRNA gene. SUP4-o was maintained on a plasmid in isogenic strains that either carried one of two different rad18 deletions (rad18 delta) or had RAD52 disrupted. Both rad18 deletions decreased the frequency of UV-induced SUP4-o mutations to levels close to those for spontaneous mutagenesis in the rad18 delta backgrounds, and prevented a net increase in mutant yield. A detailed analysis of mutations isolated after UV irradiation of one of the rad18 delta strains uncovered little evidence of the specificity features typical for UV mutagenesis in the isogenic repair-proficient (RAD) parent (e.g., predominance of G.C-->A.T transitions). Evidently, UV induction of SUP4-o mutations is highly dependent on the RAD18 gene. Compared to the RAD strain, disruption of RAD52 reduced the frequency and yield of UV mutagenesis by about two-thirds. Closer inspection revealed that 80% of this reduction was due to a decrease in the frequency of G.C-->A.T transitions. In addition, there were differences in the distributions and site specificities of single base-pair substitutions. Thus, RAD52 also participates in UV mutagenesis of a plasmid-borne gene in yeast, but to a lesser extent than RAD18.

Simple rules for ultrasonographic subcategorization of BI-RADS{sup ®}-US 4 breast masses

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Jales, Rodrigo Menezes, E-mail: rodrigoj@hotmail.com [Department of Obstetrics and Gynecology, Faculty of Medical Sciences, State University of Campinas – Unicamp, Campinas, São Paulo (Brazil); Sarian, Luís Otavio, E-mail: luis.sarian@gmail.com [Department of Obstetrics and Gynecology, Faculty of Medical Sciences, State University of Campinas – Unicamp, Campinas, São Paulo (Brazil); Torresan, Renato, E-mail: torresan@terra.com.br [Department of Obstetrics and Gynecology, Faculty of Medical Sciences, State University of Campinas – Unicamp, Campinas, São Paulo (Brazil); Marussi, Emílio Francisco, E-mail: efmarussi@uol.com.br [Department of Obstetrics and Gynecology, Faculty of Medical Sciences, State University of Campinas – Unicamp, Campinas, São Paulo (Brazil); Álvares, Beatriz Regina, E-mail: alvaresb@terra.com.br [Department of Radiology, Faculty of Medical Sciences, State University of Campinas – Unicamp, Campinas, São Paulo (Brazil); Derchain, Sophie, E-mail: derchain@fcm.unicamp.br [Department of Obstetrics and Gynecology, Faculty of Medical Sciences, State University of Campinas – Unicamp, Campinas, São Paulo (Brazil)

2013-08-15

Objectives: To evaluate an objective method for ultrasonographic (US) subcategorization of BI-RADS{sup ®}-US 4 breast masses based on clear and simple rules in order for woman to benefit from a more complete and homogeneous breast mass analysis. Methods: In this cross-sectional study, we selected 330 women, with 339 US breast masses, classified as BI-RADS{sup ®}-US 4. Three physicians experienced in breast imaging independently reviewed all US images, assessing mass shape, margins, orientation, echo texture and vascularity. These experts further subdivided the masses into subcategories 4a, 4b and 4c, according to simple US rules. Inter-observer agreement was calculated for US features categories and for final subcategory assessment. We also estimated the positive predictive value (PPV) for BI-RADS{sup ®}-US subcategories 4a, 4b and 4c assigned by each of the three observers. Results: Pathological examination of all masses confirmed 144 (42%) malignant and 195 (58%) benign tumors. Moderate agreement was obtained for mass shape, margins, vascularity and for final BI-RADS{sup ®}-US 4 subcategory. Substantial agreement was obtained for the description of mass orientation and echo texture. The PPV for subcategories 4a, 4b and 4c were, 17%, 45% and 85%, respectively, for the first observer and 20%, 38% and 79% and 17%, 40% and 85% for the other two observers. Conclusion: Standardization of a US subcategorization of BI-RADS{sup ®}-US 4 breast masses seems to be feasible, with substantial inter-observer agreement and progressive increase in the PPV in the subcategories 4a, 4b and 4c, provided that clear and simple classification rules are defined.

Differential RPA-1 and RAD-51 recruitment in vivo throughout the C. elegans germline, as revealed by laser microirradiation.

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Koury, Emily; Harrell, Kailey; Smolikove, Sarit

2018-01-25

Studies of the repair pathways associated with DNA double strand breaks (DSBs) are numerous, and provide evidence for cell-cycle specific regulation of homologous recombination (HR) by the regulation of its associated proteins. Laser microirradiation is a well-established method to examine in vitro kinetics of repair and allows for live-imaging of DSB repair from the moment of induction. Here we apply this method to whole, live organisms, introducing an effective system to analyze exogenous, microirradiation-induced breaks in the Caenorhabditis elegans germline. Through this method we observed the sequential kinetics of the recruitment of ssDNA binding proteins RPA-1 and RAD-51 in vivo. We analyze these kinetics throughout different regions of the germline, and thus throughout a range of developmental stages of mitotic and meiotic nuclei. Our analysis demonstrates a largely conserved timing of recruitment of ssDNA binding proteins to DSBs throughout the germline, with a delay of RAD-51 recruitment at mid-pachytene nuclei. Microirradiated nuclei are viable and undergo a slow kinetics of resolution. We observe RPA-1 and RAD-51 colocalization for hours post-microirradiation throughout the germline, suggesting that there are mixed RPA-1/RAD-51 filaments. Finally, through live imaging analysis we observed RAD-51 foci movement with low frequency of coalescence. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Myocardin-related transcription factor regulates Nox4 protein expression

DEFF Research Database (Denmark)

Rozycki, Matthew; Bialik, Janne Folke; Speight, Pam

2016-01-01

translocation of MRTF. Because the Nox4 promoter harbors a serum response factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF (and thus cytoskeleton organization) could regulate Nox4 expression. We show that Nox4 protein is robustly induced in kidney tubular cells exclusively by combined application...... TGFβ/contact disruption-provoked Nox4 protein and mRNA expression, Nox4 promoter activation, and reactive oxygen species production. Mutation of the CC(A/T)6GG box eliminates the synergistic activation of the Nox4 promoter. Jasplakinolide-induced actin polymerization synergizes with TGFβ to facilitate...... MRTF-dependent Nox4 mRNA expression/promoter activation. Moreover, MRTF inhibition prevents Nox4 expression during TGFβ-induced fibroblast-myofibroblast transition as well. Although necessary, MRTF is insufficient; Nox4 expression also requires TGFβ-activated Smad3 and TAZ/YAP, two contact...

Correlation of RAD51 and radiosensitization of methotrexate

International Nuclear Information System (INIS)

Du Liqing; Bai Jianqiang; Liu Qiang; Wang Yan; Zhao Peng; Chen Fenghua; Wang Hong; Fan Feiyue

2012-01-01

Objective: To evaluate the correlation between homologous recombination repair protein RAD51 and methotrexate-enhanced radiosensitivity. Methods: Western blot and RT-PCR assays were used to detect RAD51 expression in HOS osteosarcoma cells exposed to γ-ray irradiation alone and in combination with methotrexate. Colony formation assay was used to test the survival fraction of HOS cells exposed to γ-rays and methotrexate. Results: Methotrexate inhibited both protein and RNA expressions of RAD51, and the combination of radiation and methotrexate enhanced the inhibition of RAD51 expression. Moreover, transfection of cells with RAD51 gene decreased cellular sensitivity to methotrexate and γ-rays. The sensitizer enhancement ratios after irradiation in combination with methotrexate were 1.51 and 0.99, respectively. Methotrexate was a preferred radiosensitizer to HOS cell. Conclusions: RAD51 might be involved in the methotrexate-enhanced radiosensitivity. (authors)

Defective thymine dimer excision in radiation-sensitive mutants rad10 and rad16 of Saccharomyces cerevisiae

Energy Technology Data Exchange (ETDEWEB)

Prakash, L [Rochester Univ., N.Y. (USA). School of Medicine and Dentistry

1977-04-01

Two rad mutants of yeast, rad10 and rad16, are shown to be defective in the removal of UV-induced pyrimidine dimers since DNAs obtained from irradiated cells following a post-irradiation incubation in the dark still retain UV-endonuclease-sensitive sites. Both rad10 and rad16 mutants are in the same pathway of excision-repair as the rad1, rad2, rad3, and rad4 mutants.

Functional analysis of the RAD50/MRE11 protein complex through targeted disruption of the murine RAD50 genomic locus: implications for DNA double strand break repair. An astro research fellowship presentation

International Nuclear Information System (INIS)

Yao, Michelle S.; Bladl, Anthony R.; Petrini, John H.J.

1997-01-01

Purpose/Objective: The products of the S. cerevisiae genes ScRAD50 and ScMRE11 act in a protein complex and are required for non-homologous end-joining, the predominant mechanism of DNA double strand break (dsb) repair in mammalian cells. Mutation of these genes results in sensitivity to ionizing radiation (IR), a defect in initiation of meiosis, increased and error-prone recombination during mitosis, and overall genomic instability. This resultant phenotype is reminiscent of that seen in mammalian syndromes of genomic instability such as ataxia-telangiectasia and Bloom syndrome, hallmarks of which are radiation sensitivity and predisposition to malignancy. The murine homologues to ScRAD50 and ScMRE11 have recently been identified; both demonstrate impressive primary sequence conservation with their yeast counterparts, and are expected to mediate conserved functions. The roles of muRAD50 in genomic maintenance and in dsb repair will be examined in two parts. The first will include a determination of normal muRAD50 expression patterns. Second, the effects of disruption of the muRAD50 gene will be assessed. A specific targeting event has introduced a conditional murad50 null mutation into the genome of murine embryonic stem (ES) cells. These mutant ES cells are being used to create mutant mice, thus allowing functional characterization of muRAD50 on both the cellular and organismic levels. Such analyses will contribute to the delineation of the mammalian dsb repair pathway and to the cellular response to IR, and will serve as a mammalian model system for genomic instability. Materials and Methods: Wild-type tissue expression patterns and protein-protein interactions were determined by standard biochemical techniques, including immunoprecipitation, polyacrylamide gel electrophoresis, and Western blotting. Molecular cloning techniques were used to create the gene targeting vectors, which were designed to result in either a deletion of exon 1 (equivalent to a null

Minocycline enhances mitomycin C-induced cytotoxicity through down-regulating ERK1/2-mediated Rad51 expression in human non-small cell lung cancer cells.

Science.gov (United States)

Ko, Jen-Chung; Wang, Tai-Jing; Chang, Po-Yuan; Syu, Jhan-Jhang; Chen, Jyh-Cheng; Chen, Chien-Yu; Jian, Yun-Ting; Jian, Yi-Jun; Zheng, Hao-Yu; Chen, Wen-Ching; Lin, Yun-Wei

2015-10-01

Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC. Copyright © 2015 Elsevier Inc. All rights reserved.

RAD9, RAD17; RAD24, and RAD53 control one pathway of resistance to γ irradiation in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Koltovaya, N.A.; Nikulushkina, Yu.V.; Roshina, M.P.; Devin, A.B.

2009-01-01

Mechanisms for the genetic control of the cell cycle transition (checkpoint control) have been studied in more detail in yeast Saccharomyces cerevisiae. To clarify tho role of the RAD9, RAD17, RAD24, and RAD53 checkpoint genes in cell radioresistance, diploid double mutants were analyzed for cell sensitivity to ionizing radiation. All mutations in combination with rad9Δ were shown to manifest the epistatic type of interaction. Our results suggest that the RAD9, RAD17, RAD24, and RAD53 checkpoint genes belong to a single epistasis group called the RAD9 group and participate in the same pathway. RAD9 and RAD53 have a positive effect on sensitivity to γ irradiation, whereas RAD17 and RAD24 have a negative effect. For haploid interactions between mutations may differ in the case of γ or UV irradiation, mutations - for example, rad9Δ and rad24Δ - were shown to have an additive effect in the first case and epistatic - in the second. The analyzed genes can also participate in minor mechanisms of radioresistance that are relatively independent of the above major mechanism

Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

International Nuclear Information System (INIS)

Imura, Yoshiyuki; Molho, Melissa; Chuang, Chingkai; Nagy, Peter D.

2015-01-01

Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase

Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

Energy Technology Data Exchange (ETDEWEB)

Imura, Yoshiyuki, E-mail: imura@brs.nihon-u.ac.jp; Molho, Melissa; Chuang, Chingkai; Nagy, Peter D., E-mail: pdnagy2@uky.edu

2015-10-15

Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.

Interactions of checkpoint-genes RAD9, RAD17, RAD24 and RAD53 determining radioresistance of Yeast Saccharomyces Cerevisiae

International Nuclear Information System (INIS)

Koltovaya, N.A.; Nikulushkina, Yu.V.; Roshchina, M.P.; Devin, A.B.

2007-01-01

The mechanisms of genetic control of progress through the division cell cycle (checkpoint-control) in yeast Saccharomyces cerevisiae have been studied intensively. To investigate the role of checkpoint-genes RAD9, RAD17, RAD24, RAD53 in cell radioresistance we have investigated cell sensitivity of double mutants to γ-ray. Double mutants involving various combinations with rad9Δ show epistatic interactions, i.e. the sensitivity of the double mutants to γ-ray was no greater than that of more sensitive of the two single mutants. This suggests that all these genes govern the same pathway. This group of genes was named RAD9-epistasis group. It is interesting to note that the genes RAD9 and RAD53 have positive effect but RAD17 and RAD24 have negative effect on radiosensitivity of yeast cells. Interactions between mutations may differ depending on the agent γ-ray or UV-light, for example mutations rad9Δ and rad24Δ show additive effect for γ-ray and epistatic effect for UV-light

In Vivo Delivery of miR-34a Sensitizes Lung Tumors to Radiation Through RAD51 Regulation

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Maria Angelica Cortez

2015-01-01

Full Text Available MiR-34a, an important tumor-suppressing microRNA, is downregulated in several types of cancer; loss of its expression has been linked with unfavorable clinical outcomes in non-small-cell lung cancer (NSCLC, among others. MiR-34a represses several key oncogenic proteins, and a synthetic mimic of miR-34a is currently being tested in a cancer trial. However, little is known about the potential role of miR-34a in regulating DNA damage response and repair. Here, we demonstrate that miR-34a directly binds to the 3’ untranslated region of RAD51 and regulates homologous recombination, inhibiting double-strand-break repair in NSCLC cells. We further demonstrate the therapeutic potential of miR-34a delivery in combination with radiotherapy in mouse models of lung cancer. Collectively, our results suggest that administration of miR-34a in combination with radiotherapy may represent a novel strategy for treating NSCLC.

Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

DEFF Research Database (Denmark)

Rougier, Jean-Sébastien; van Bemmelen, Miguel X; Bruce, M Christine

2004-01-01

-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na(v)1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na(v) proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na(v)1.2 and Na...... that Nedd4-dependent ubiquitination of Na(v) channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane....

Molecular anatomy of the recombination mediator function of Saccharomyces cerevisiae Rad52

DEFF Research Database (Denmark)

Seong, C.; Sehorn, M.G.; Plate, Iben

2008-01-01

A helical filament of Rad51 on single-strand DNA (ssDNA), called the presynaptic filament, catalyzes DNA joint formation during homologous recombination. Rad52 facilitates presynaptic filament assembly, and this recombination mediator activity is thought to rely on the interactions of Rad52...... with Rad51, the ssDNA-binding protein RPA, and ssDNA. The N-terminal region of Rad52, which has DNA binding activity and an oligomeric structure, is thought to be crucial for mediator activity and recombination. Unexpectedly, we find that the C-terminal region of Rad52 also harbors a DNA binding function....... Importantly, the Rad52 C-terminal portion alone can promote Rad51 presynaptic filament assembly. The middle portion of Rad52 associates with DNA-bound RPA and contributes to the recombination mediator activity. Accordingly, expression of a protein species that harbors the middle and C-terminal regions of Rad...

Identification of cloned genes that complement the rad50-1, rad51-1, rad54-3 and rad55-3 mutations in yeast

International Nuclear Information System (INIS)

Calderon, I.L.; Contopoulou, C.R.; Mortimer, R.K.

1982-01-01

Plasmids that complement the rad50-1, rad51-1, rad54-3 and rad55-3 mutations in yeast, have been isolated. They were obtained by transforming strains, carrying the leu2-112 leu2-3 alleles and the particular rad mutation, with YEp13 plasmids containing near random yeast DNA inserts. Rad + clones were identified among the Leu + transformants. Integration by targeting into the RAD55 locus showed that the rad55-3 complementing plasmid contained the actual RAD55 gene. BamHI fragments from each of the plasmids that complement rad50-1, rad51-1 and rad54-3, all of which lacked Rad + activity, were subcloned into the integrating plasmid YIp5 and the hybrid plasmids were used to transform a Rad + Ura - strain to Ura + . By genetic mapping, the rad51 and rad54 subclones were shown to integrate at their respective loci. However, the rad50 subclones integrated at a site unlinked to the RAD50 locus. This suggests that no homology exists between this BamHI fragment and the RAD50 gene. Integration at the RAD54 locus of the rad54 subclone made the host cell Ura + but Rad - ; excision of the plasmid was shown to be x-ray inducible and to restore the Ura - Rad + phenotype. These results indicate that the BamHI fragment of the RAD54 plasmid is internal to the RAD54 gene. We can conclude also that the RAD54 gene is not essential as cells bearing a disrupted copy of this gene are able to survive. Additionally, a plasmid carrying an amber suppressor has been isolated and characterized

Food Irradiation Is Done in Grays, not Rads

International Nuclear Information System (INIS)

Strom, Daniel J.

2002-01-01

One federal agency has chosen to use exclusively modern SI units of radiation dose in its regulations: the FDA. While not exactly hot news, this bold move by a U.S. government agency on November 26, 1997, should be noted by those who wish to encourage the switch from curies, working level months, rads, rems, and roentgens to becquerels, joule hours per cubic meter, grays, sieverts, and coulombs per kilogram. The regulation is 21 CFR 179, Irradiation in the Production, Processing, and Handling of Food. Specifically, 21 CFR 179.26 (b) 8. permits meat irradiation up to 4.5 kGy for refrigerated meat and 7.0 kGy for frozen meat. Prior to the 1997 addition, radiation doses had been quoted in grays (kGy) with rad (Mrad) values in parentheses. In the 1997 addition, the Mrads disappeared

Structure of a hexameric form of RadA recombinase from Methanococcus voltae

International Nuclear Information System (INIS)

Du, Liqin; Luo, Yu

2012-01-01

Hexameric rings of RadA recombinase from M. voltae have been crystallized. Structural comparisons suggest that homologues of RadA tend to form double-ringed assemblies. Archaeal RadA proteins are close homologues of eukaryal Rad51 and DMC1 proteins and are remote homologues of bacterial RecA proteins. For the repair of double-stranded breaks in DNA, these recombinases promote a pivotal strand-exchange reaction between homologous single-stranded and double-stranded DNA substrates. This DNA-repair function also plays a key role in the resistance of cancer cells to chemotherapy and radiotherapy and in the resistance of bacterial cells to antibiotics. A hexameric form of a truncated Methanococcus voltae RadA protein devoid of its small N-terminal domain has been crystallized. The RadA hexamers further assemble into two-ringed assemblies. Similar assemblies can be observed in the crystals of Pyrococcus furiosus RadA and Homo sapiens DMC1. In all of these two-ringed assemblies the DNA-interacting L1 region of each protomer points inward towards the centre, creating a highly positively charged locus. The electrostatic characteristics of the central channels can be utilized in the design of novel recombinase inhibitors

Characterization of RAD4 gene required for ultraviolet-induced excision repair of Saccharomyces cerevisiae propagated in Escherichia coli without inactivation

International Nuclear Information System (INIS)

Choi, I.S.; Kim, J.B.; Lee, K.N.; Park, S.D.

1990-01-01

The previously isolated RAD4 gene designated as pPC1 from the genomic library of Saccharomyces cerevisiae appeared to propagate in Escherichia coli and yet retained its complementing activity of rad4 mutants without inactivation. The subcloned RAD4 gene was found to be localized within a 2.5 kb DNA fragment flanking Bg/II and BamHI sites in the insert DNA, and was shown to have the same restriction map as a yeast chromosomal DNA, as determined by Southern hybridization. Tetrad analysis and pulse-field chromosome mapping have revealed that the cloned RAD4 gene can be mapped and integrated into the yeast chromosome V, the actual site of this gene. DNA-tRNA hybridization has shown that the isolated RAD4 gene did not contain a suppressor tRNA gene. These results have indicated that the pPC1 is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplification in E. coli. (author)

Cellular Dynamics of Rad51 and Rad54 in Response to Postreplicative Stress and DNA Damage in HeLa Cells.

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Choi, Eui-Hwan; Yoon, Seobin; Hahn, Yoonsoo; Kim, Keun P

2017-02-01

Homologous recombination (HR) is necessary for maintenance of genomic integrity and prevention of various mutations in tumor suppressor genes and proto-oncogenes. Rad51 and Rad54 are key HR factors that cope with replication stress and DNA breaks in eukaryotes. Rad51 binds to single-stranded DNA (ssDNA) to form the presynaptic filament that promotes a homology search and DNA strand exchange, and Rad54 stimulates the strand-pairing function of Rad51. Here, we studied the molecular dynamics of Rad51 and Rad54 during the cell cycle of HeLa cells. These cells constitutively express Rad51 and Rad54 throughout the entire cell cycle, and the formation of foci immediately increased in response to various types of DNA damage and replication stress, except for caffeine, which suppressed the Rad51-dependent HR pathway. Depletion of Rad51 caused severe defects in response to postreplicative stress. Accordingly, HeLa cells were arrested at the G2-M transition although a small amount of Rad51 was steadily maintained in HeLa cells. Our results suggest that cell cycle progression and proliferation of HeLa cells can be tightly controlled by the abundance of HR proteins, which are essential for the rapid response to postreplicative stress and DNA damage stress.

Identification of Rad23-4 gene required for pollen development in ...

African Journals Online (AJOL)

雨林木风

2012-05-31

May 31, 2012 ... in ultraviolet (UV)-B–treated rad23-4 mutants. Compared with the wild type ... discovered in yeast (Guzder et al., 1998). Recent studies showed that ... UV-B irradiation can induce accumulations of anthocyanin in the plants.

Diagnostic value of coustic radiation force impulse for BI-RADS category 4 breast lesions of different sizes.

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Wu, Rong

2018-04-14

To determine the diagnostic value of combined conventional ultrasound (US) and acoustic radiation force impulse (ARFI) imaging for the differential diagnosis of BI-RADS category 4 breast lesions of different sizes. From April 2013 to January 2015, 283 patients (with a total of 292 breast lesions) who underwent US and ARFI examination were included in this retrospective study. The SWV for the lesion and adjacent normal breast tissue were measured and the SWV ratio was calculated. VTI grade was also assessed. The lesions were separated into three groups on the basis of size, and two combinations of ARFI parameters (SWV + VTI and SWV ratio + VTI) were applied to reassess the BI-RADS categories. Diagnoses were confirmed by pathological examination after biopsy or surgery. ROC analysis was performed to assess the diagnostic efficiency of each method. The Z test was used to compare the difference between AUC of the two methods. Significant improvement was seen in the diagnostic performance of US with the use of the ARFI parameters SWV + VTI (77/179 [43.0%] of BI-RADS category 4A breast lesions were downgraded) and SWV ratio + VTI (64/179 [35.8%] of BI-RADS category 4A breast lesions were downgraded, including two malignant cases that were misdiagnosed as benign) (P BI-RADS category 4 breast lesions. The combination of SWV ratio + VTI can improve BI-RADS classification of small lesions (<10 mm size).

Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA

International Nuclear Information System (INIS)

Guzder, S.N.; Sung, P.; Prakash, S.; Prakash, L.

1993-01-01

Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA. Of the seven XP complementation groups, A--G, group A represents a severe and frequent form of the disease. The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene. Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA. The authors have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14. As determined by atomic emission spectroscopy, RAD14 contains one zinc atom. They also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions. In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA. Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites. These findings indicate that RAD14 functions in damage recognition during excision repair. 37 refs., 4 figs

RAD18 mediates resistance to ionizing radiation in human glioma cells

International Nuclear Information System (INIS)

Xie, Chen; Wang, Hongwei; Cheng, Hongbin; Li, Jianhua; Wang, Zhi; Yue, Wu

2014-01-01

Highlights: • RAD18 is an important mediator of the IR-induced resistance in glioma cell lines. • RAD18 overexpression confers resistance to IR-mediated apoptosis. • The elevated expression of RAD18 is associated with recurrent GBM who underwent IR therapy. - Abstract: Radioresistance remains a major challenge in the treatment of glioblastoma multiforme (GBM). RAD18 a central regulator of translesion DNA synthesis (TLS), has been shown to play an important role in regulating genomic stability and DNA damage response. In the present study, we investigate the relationship between RAD18 and resistance to ionizing radiation (IR) and examined the expression levels of RAD18 in primary and recurrent GBM specimens. Our results showed that RAD18 is an important mediator of the IR-induced resistance in GBM. The expression level of RAD18 in glioma cells correlates with their resistance to IR. Ectopic expression of RAD18 in RAD18-low A172 glioma cells confers significant resistance to IR treatment. Conversely, depletion of endogenous RAD18 in RAD18-high glioma cells sensitized these cells to IR treatment. Moreover, RAD18 overexpression confers resistance to IR-mediated apoptosis in RAD18-low A172 glioma cells, whereas cells deficient in RAD18 exhibit increased apoptosis induced by IR. Furthermore, knockdown of RAD18 in RAD18-high glioma cells disrupts HR-mediated repair, resulting in increased accumulation of DSB. In addition, clinical data indicated that RAD18 was significantly higher in recurrent GBM samples that were exposed to IR compared with the corresponding primary GBM samples. Collectively, our findings reveal that RAD18 may serve as a key mediator of the IR response and may function as a potential target for circumventing IR resistance in human GBM

Role of p70S6K1-mediated phosphorylation of eIF4B and PDCD4 proteins in the regulation of protein synthesis.

Science.gov (United States)

Dennis, Michael D; Jefferson, Leonard S; Kimball, Scot R

2012-12-14

Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5'-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.

DMC1 functions in a Saccharomyces cerevisiae meiotic pathway that is largely independent of the RAD51 pathway

International Nuclear Information System (INIS)

Dresser, M.E.; Ewing, D.J.; Conrad, M.N.; Dominguez, A.M.; Barstead, R.; Jiang, H.; Kodadek, T.

1997-01-01

Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p. A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype. A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D. Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p. Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p. However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes. Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination. Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin. Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains. (author)

RAD51C germline mutations in breast and ovarian cancer cases from high-risk families.

Directory of Open Access Journals (Sweden)

Jessica Clague

Full Text Available BRCA1 and BRCA2 are the most well-known breast cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. One such gene, RAD51C, is essential for homologous recombination repair. Several likely pathogenic RAD51C mutations have been identified in BRCA1- and BRCA2-negative breast and ovarian cancer families. We performed complete sequencing of RAD51C in germline DNA of 286 female breast and/or ovarian cancer cases with a family history of breast and ovarian cancers, who had previously tested negative for mutations in BRCA1 and BRCA2. We screened 133 breast cancer cases, 119 ovarian cancer cases, and 34 with both breast and ovarian cancers. Fifteen DNA sequence variants were identified; including four intronic, one 5' UTR, one promoter, three synonymous, and six non-synonymous variants. None were truncating. The in-silico SIFT and Polyphen programs were used to predict possible pathogenicity of the six non-synonomous variants based on sequence conservation. G153D and T287A were predicted to be likely pathogenic. Two additional variants, A126T and R214C alter amino acids in important domains of the protein such that they could be pathogenic. Two-hybrid screening and immunoblot analyses were performed to assess the functionality of these four non-synonomous variants in yeast. The RAD51C-G153D protein displayed no detectable interaction with either XRCC3 or RAD51B, and RAD51C-R214C displayed significantly decreased interaction with both XRCC3 and RAD51B (p<0.001. Immunoblots of RAD51C-Gal4 activation domain fusion peptides showed protein levels of RAD51C-G153D and RAD51C-R214C that were 50% and 60% of the wild-type, respectively. Based on these data, the RAD51C-G153D variant is likely to be pathogenic, while the RAD51C- R214C variant is hypomorphic of uncertain pathogenicity. These results provide further support that RAD51C is a rare breast and ovarian cancer susceptibility gene.

A Rad53 independent function of Rad9 becomes crucial for genome maintenance in the absence of the Recq helicase Sgs1.

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Ida Nielsen

Full Text Available The conserved family of RecQ DNA helicases consists of caretaker tumour suppressors, that defend genome integrity by acting on several pathways of DNA repair that maintain genome stability. In budding yeast, Sgs1 is the sole RecQ helicase and it has been implicated in checkpoint responses, replisome stability and dissolution of double Holliday junctions during homologous recombination. In this study we investigate a possible genetic interaction between SGS1 and RAD9 in the cellular response to methyl methane sulphonate (MMS induced damage and compare this with the genetic interaction between SGS1 and RAD24. The Rad9 protein, an adaptor for effector kinase activation, plays well-characterized roles in the DNA damage checkpoint response, whereas Rad24 is characterized as a sensor protein also in the DNA damage checkpoint response. Here we unveil novel insights into the cellular response to MMS-induced damage. Specifically, we show a strong synergistic functionality between SGS1 and RAD9 for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for SGS1 and RAD24. Intriguingly, it is a Rad53 independent function of Rad9, which becomes crucial for genome maintenance in the absence of Sgs1. Despite this, our dissection of the MMS checkpoint response reveals parallel, but unequal pathways for Rad53 activation and highlights significant differences between MMS- and hydroxyurea (HU-induced checkpoint responses with relation to the requirement of the Sgs1 interacting partner Topoisomerase III (Top3. Thus, whereas earlier studies have documented a Top3-independent role of Sgs1 for an HU-induced checkpoint response, we show here that upon MMS treatment, Sgs1 and Top3 together define a minor but parallel pathway to that of Rad9.

Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

Science.gov (United States)

Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

2016-04-01

Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC. Copyright © 2016 Elsevier Inc. All rights reserved.

Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation

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Luisina De Tullio

2017-10-01

Full Text Available Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second in the 3′→5′ direction along ssDNA saturated with replication protein A (RPA. We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates.

The cardiac copper chaperone proteins Sco1 and CCS are up-regulated, but Cox 1 and Cox4 are down-regulated, by copper deficiency.

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Getz, Jean; Lin, Dingbo; Medeiros, Denis M

2011-10-01

Copper is ferried in a cell complexed to chaperone proteins, and in the heart much copper is required for cytochrome c oxidase (Cox). It is not completely understood how copper status affects the levels of these proteins. Here we determined if dietary copper deficiency could up- or down-regulate select copper chaperone proteins and Cox subunits 1 and 4 in cardiac tissue of rats. Sixteen weanling male Long-Evans rats were randomized into treatment groups, one group receiving a copper-deficient diet (CCS, Sco1, Ctr1, Cox17, Cox1, and Cox4 by SDS-PAGE and Western blotting. No changes were observed in the concentrations of CTR1 and Cox17 between copper-adequate and copper-deficient rats. CCS and Sco1 were up-regulated and Cox1 and Cox4 were both down-regulated as a result of copper deficiency. These data suggest that select chaperone proteins and may be up-regulated, and Cox1 and 4 down-regulated, by a dietary copper deficiency, whereas others appear not to be affected by copper status.

Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

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Rojnik, Matija; Jevnikar, Zala; Mirkovic, Bojana; Janes, Damjan; Zidar, Nace; Kikelj, Danijel; Kos, Janko

2011-01-01

Background Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. Materials and methods BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. Results In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Conclusions Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression. PMID:22933963

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament.

KAUST Repository

Fornander, Louise H

2013-12-03

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.

Human RAD50 makes a functional DNA-binding complex.

Science.gov (United States)

Kinoshita, Eri; van Rossum-Fikkert, Sari; Sanchez, Humberto; Kertokalio, Aryandi; Wyman, Claire

2015-06-01

The MRE11-RAD50-NBS1 (MRN) complex has several distinct functions in DNA repair including important roles in both non-homologous end-joining (NHEJ) and homologous recombination (HR). The biochemical activities of MR(N) have been well characterized implying specific functional roles for the components. The arrangement of proteins in the complex implies interdependence of their biochemical activities making it difficult to separate specific functions. We obtained purified human RAD50 and observed that it binds ATP, undergoes ATP-dependent conformational changes as well as having ATPase activity. Scanning force microscopy analysis clearly showed that RAD50 binds DNA although not as oligomers. RAD50 alone was not functional in tethering DNA molecules. ATP increased formation of RAD50 multimers which were however globular lacking extended coiled coils, in contrast to the MR complex where ATP induced oligomers have obvious coiled coils protruding from a central domain. These results suggest that MRE11 is important in maintaining the structural arrangement of RAD50 in the protein complex and perhaps has a role in reinforcing proper alignment of the coiled coils in the ATP-bound state. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Role of teh Rad52 Amino-terminal DNA Binding Activity in DNA Strand Capture in Homologous Recombination

DEFF Research Database (Denmark)

Shi, Idina; Hallwyl, Swee Chuang Lim; Seong, Changhyun

2009-01-01

Saccharomyces cerevisiae Rad52 protein promotes homologous recombination by nucleating the Rad51 recombinase onto replication protein A-coated single-stranded DNA strands and also by directly annealing such strands. We show that the purified rad52-R70A mutant protein, with a compromised amino-ter...

Rad51C deficiency destabilizes XRCC3, impairs recombination and radiosensitizes S/G2-phase cells

Energy Technology Data Exchange (ETDEWEB)

Lio, Yi-Ching; Schild, David; Brenneman, Mark A.; Redpath, J. Leslie; Chen, David J.

2004-05-01

The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, XRCC3) are expressed in mitotically growing cells, and are thought to play mediating roles in homologous recombination, though their precise functions remain unclear. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA cross-linking agent mitomycin C, and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G{sub 2}/M phases of the cell cycle but not in G{sub 1} phase. Together, these results provide direct cellular evidence for the importance of human Rad51C in homologous recombinational repair.

Rad52 multimerization is important for its nuclear localization in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Plate, Iben; Albertsen, Line; Lisby, Michael

2008-01-01

Rad52 is essential for all homologous recombination and DNA double strand break repair events in Saccharomyces cerevisiae. This protein is multifunctional and contains several domains that allow it to interact with DNA as well as with different repair proteins. However, it has been unclear how Rad...

Coordination of Rad1-Rad10 interactions with Msh2-Msh3, Saw1 and RPA is essential for functional 3' non-homologous tail removal.

Science.gov (United States)

Eichmiller, Robin; Medina-Rivera, Melisa; DeSanto, Rachel; Minca, Eugen; Kim, Christopher; Holland, Cory; Seol, Ja-Hwan; Schmit, Megan; Oramus, Diane; Smith, Jessica; Gallardo, Ignacio F; Finkelstein, Ilya J; Lee, Sang Eun; Surtees, Jennifer A

2018-04-06

Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3' non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1-Rad10 is the structure-specific endonuclease that cleaves the tails in 3' non-homologous tail removal (3' NHTR). Rad1-Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1-Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2-Msh3 and Saw1 recruit Rad1-Rad10 in 3' NHTR; Rad14 recruits Rad1-Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3' NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3' NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A-Rad10 to recombination intermediates is defective. Interactions among rad1R218A-Rad10 and Msh2-Msh3 and Saw1 are altered and rad1R218A-Rad10 interactions with RPA are compromised. We propose a model in which Rad1-Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1-Rad10 and Msh2-Msh3, Saw1 and Msh2-Msh3 and Rad1-Rad10 and RPA. When any of these interactions is altered, 3' NHTR is impaired.

Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation

Science.gov (United States)

Dobson, Rachel; Stockdale, Christopher; Lapsley, Craig; Wilkes, Jonathan; McCulloch, Richard

2011-01-01

Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue. PMID:21615552

Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation.

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De Tullio, Luisina; Kaniecki, Kyle; Kwon, Youngho; Crickard, J Brooks; Sung, Patrick; Greene, Eric C

2017-10-17

Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3'→5' direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

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Manfrini, Nicola; Trovesi, Camilla; Wery, Maxime; Martina, Marina; Cesena, Daniele; Descrimes, Marc; Morillon, Antonin; d'Adda di Fagagna, Fabrizio; Longhese, Maria Pia

2015-02-01

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. © 2014 The Authors.

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament.

KAUST Repository

Fornander, Louise H; Renodon-Corniè re, Axelle; Kuwabara, Naoyuki; Ito, Kentaro; Tsutsui, Yasuhiro; Shimizu, Toshiyuki; Iwasaki, Hiroshi; Nordé n, Bengt; Takahashi, Masayuki

2013-01-01

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure

Comparison of clinicopathological findings among patients whose mammography results were classified as category 4 subgroups of the BI-RADS.

Science.gov (United States)

Leblebici, Ihsan Metin; Bozkurt, Suleyman; Eren, Turgut Tunc; Ozemir, Ibrahim Ali; Sagiroglu, Julide; Alimoglu, Orhan

2014-01-01

Our aim is to compare mammographic, demographic and clinicopathological characteristics of patients whose mammographies were classified as subgroups of BI-RADS 4 category (Breast Imaging - Reporting and Data System). In total, 103 patients with mammography (Senographe 600t Senix HF; General Electric, Moulineaux, France) results classified as BI-RADS 4 were included in the study. Demographic data (age, menopause, and family history) were recorded. All data were compared among BI-RADS 4 subgroups. In all, 68.9% (71/103), 7.8% (8/103) and 23.3% (24/103) the patients were in groups BI-RADS 4A, 4B and 4C, respectively. The incidence of malignancy was higher in Groups 4B and 4C than in Group 4A (p0.05). Mean age was lower in Group 4B than in Groups 4A and 4C (p<0.05). A positive family history was more common in Group 4A than in Group 4B (p=0.025). The frequency of menopausal patients was greater in Groups 4A and 4C than in Group 4B (p=0.021, and 0.003, respectively). The rate of malignancy was higher in Groups 4B, and 4C than in Group 4A. A positive family history was more common in Group 4A than in Group 4C. Groups 4A, and 4C patients tended to be older and were more likely to be menopausal than Group 4B patients.

Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription.

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Goran Periz

2015-04-01

Full Text Available Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, and its failure is associated with many neurodegenerative disorders. Here, we identify two genes, ufd-2 and spr-5, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in Caenorhabditis elegans. Loss of human orthologs ubiquitination factor E4 B (UBE4B and lysine-specific demethylase 1 (LSD1, respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries. An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity. These studies identify a new protein quality control pathway via regulation of transcription factors and point to the augmentation of protein quality control as a wide-spectrum antiproteotoxicity strategy.

Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates.

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Ashley, T; Plug, A W; Xu, J; Solari, A J; Reddy, G; Golub, E I; Ward, D C

1995-10-01

Antibodies against human Rad51 protein were used to examine the distribution of Rad51 on meiotic chromatin in mouse spermatocytes and oocytes as well as chicken oocytes during sequential stages of meiosis. We observed the following dynamic changes in distribution of Rad51 during meiosis: (1) in early leptotene nuclei there are multiple, apparently randomly distributed, foci that by late leptonema become organized into tracks of foci. (2) These foci persist into zygonema, but most foci are now localized on Rad51-positive axes that correspond to lateral elements of the synaptonemal complex. As homologs synapse foci from homologous axes fuse. The distribution and involvement of Rad51 foci as contact points between homologs suggest that they may be components to early recombination nodules. (3) As pachynema progresses the number of foci drops dramatically; the temporal occurrence (mice) and physical and numerical distribution of foci on axes (chickens) suggest that they may be a component of late recombination nodules. (4) In early pachynema there are numerous Rad51 foci on the single axis of the X (mouse spermatocytes) or the Z (chicken oocytes) chromosomes that neither pair, nor recombine. (5) In late pachynema in mouse spermatocytes, but not oocytes, the Rad51 signal is preferentially enhanced at both ends of all the bivalents. As bivalents in spermatocytes, but not oocytes, begin to desynapse at diplonema they are often held together at these Rad51-positive termini. These observations parallel observations that recombination rates are exceptionally high near chromosome ends in male but not female eutherian mammals. (6) From diakinesis through metaphase I, Rad51 protein is detected as low-intensity fluorescent doublets that localize with CREST-specific antigens (kinetochores), suggesting that Rad51 participates, at least as a structural component of the materials involved, in sister kinetochore cohesiveness. Finally, the changes in Rad51 distribution during meiosis

TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity

DEFF Research Database (Denmark)

Moudry, Pavel; Watanabe, Kenji; Wolanin, Kamila M.

2016-01-01

to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase-mediated phosphorylation of RAD51...

Computational study on the inhibitor binding mode and allosteric regulation mechanism in hepatitis C virus NS3/4A protein.

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Weiwei Xue

Full Text Available HCV NS3/4A protein is an attractive therapeutic target responsible for harboring serine protease and RNA helicase activities during the viral replication. Small molecules binding at the interface between the protease and helicase domains can stabilize the closed conformation of the protein and thus block the catalytic function of HCV NS3/4A protein via an allosteric regulation mechanism. But the detailed mechanism remains elusive. Here, we aimed to provide some insight into the inhibitor binding mode and allosteric regulation mechanism of HCV NS3/4A protein by using computational methods. Four simulation systems were investigated. They include: apo state of HCV NS3/4A protein, HCV NS3/4A protein in complex with an allosteric inhibitor and the truncated form of the above two systems. The molecular dynamics simulation results indicate HCV NS3/4A protein in complex with the allosteric inhibitor 4VA adopts a closed conformation (inactive state, while the truncated apo protein adopts an open conformation (active state. Further residue interaction network analysis suggests the communication of the domain-domain interface play an important role in the transition from closed to open conformation of HCV NS3/4A protein. However, the inhibitor stabilizes the closed conformation through interaction with several key residues from both the protease and helicase domains, including His57, Asp79, Asp81, Asp168, Met485, Cys525 and Asp527, which blocks the information communication between the functional domains interface. Finally, a dynamic model about the allosteric regulation and conformational changes of HCV NS3/4A protein was proposed and could provide fundamental insights into the allosteric mechanism of HCV NS3/4A protein function regulation and design of new potent inhibitors.

The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination

DEFF Research Database (Denmark)

Eppink, Berina; Tafel, Agnieszka A; Hanada, Katsuhiro

2011-01-01

with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES...

Differential hRad17 expression by histologic subtype of ovarian cancer

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Young Jennifer L

2011-03-01

Full Text Available Abstract Background In the search for unique ovarian cancer biomarkers, ovarian specific cDNA microarray analysis identified hRad17, a cell cycle checkpoint protein, as over-expressed in ovarian cancer. The aim of this study was to validate this expression. Methods Immunohistochemistry was performed on 72 serous, 19 endometrioid, 10 clear cell, and 6 mucinous ovarian cancers, 9 benign ovarian tumors, and 6 normal ovarian tissue sections using an anti-hRad17 antibody. Western blot analysis and quantitative PCR were performed using cell lysates and total RNA prepared from 17 ovarian cancer cell lines and 6 normal ovarian epithelial cell cultures (HOSE. Results Antibody staining confirmed upregulation of hRad17 in 49.5% of ovarian cancer cases. Immunohistochemistry demonstrated that only 42% of serous and 47% of endometrioid subtypes showed overexpression compared to 80% of clear cell and 100% of mucinous cancers. Western blot confirmed overexpression of hRad17 in cancer cell lines compared to HOSE. Quantitative PCR demonstrated an upregulation of hRad17 RNA by 1.5-7 fold. hRad17 RNA expression differed by subtype. Conclusions hRad17 is over-expressed in ovarian cancer. This over-expression varies by subtype suggesting a role in the pathogenesis of these types. Functional studies are needed to determine the potential role of this protein in ovarian cancer.

Differential expression and requirements for Schizosaccharomyces pombe RAD52 homologs in DNA repair and recombination

OpenAIRE

van den Bosch, Michael; Zonneveld, José B. M.; Vreeken, Kees; de Vries, Femke A. T.; Lohman, Paul H. M.; Pastink, Albert

2002-01-01

In fission yeast two RAD52 homologs have been identified, rad22A+ and rad22B+. Two-hybrid experiments and GST pull-down assays revealed physical interaction between Rad22A and Rad22B, which is dependent on the N-terminal regions. Interaction with Rhp51 is dependent on the C-terminal parts of either protein. Both Rad22A and Rad22B also interact with RPA. The expression of rad22B+ in mitotically dividing cells is very low in comparison with rad22A+ but is strongly enhanced after induction of me...

p53 is involved in clearance of ionizing radiation-induced RAD51 foci in a human colon cancer cell line

International Nuclear Information System (INIS)

Orre, Lukas M.; Stenerloew, Bo; Dhar, Sumeer; Larsson, Rolf; Lewensohn, Rolf; Lehtioe, Janne

2006-01-01

We have investigated p53-related differences in cellular response to DNA damaging agents, focusing on p53s effects on RAD51 protein level and sub-cellular localization post exposure to ionizing radiation. In a human colon cancer cell line, HCT116 and its isogenic p53-/- subcell line we show here p53-independent RAD51 foci formation but interestingly the resolution of RAD51 foci showed clear p53 dependence. In p53 wt cells, but not in p53-/- cells, RAD51 protein level decreased 48 h post irradiation and fluorescence immunostaining showed resolution of RAD51 foci and relocalization of RAD51 to nucleoli at time points corresponding to the decrease in RAD51 protein level. Both cell lines rejoined DNA double strand breaks efficiently with similar kinetics and p53 status did not influence sensitivity to DNA damaging agents. We suggest that p53 has a role in RAD51 clearance post DSB repair and that nucleoli might be sites of RAD51 protein degradation

Characterisation of the novel deleterious RAD51C p.Arg312Trp variant and prioritisation criteria for functional analysis of RAD51C missense changes.

Science.gov (United States)

Gayarre, Javier; Martín-Gimeno, Paloma; Osorio, Ana; Paumard, Beatriz; Barroso, Alicia; Fernández, Victoria; de la Hoya, Miguel; Rojo, Alejandro; Caldés, Trinidad; Palacios, José; Urioste, Miguel; Benítez, Javier; García, María J

2017-09-26

Despite a high prevalence of deleterious missense variants, most studies of RAD51C ovarian cancer susceptibility gene only provide in silico pathogenicity predictions of missense changes. We identified a novel deleterious RAD51C missense variant (p.Arg312Trp) in a high-risk family, and propose a criteria to prioritise RAD51C missense changes qualifying for functional analysis. To evaluate pathogenicity of p.Arg312Trp variant we used sequence homology, loss of heterozygosity (LOH) and segregation analysis, and a comprehensive functional characterisation. To define a functional-analysis prioritisation criteria, we used outputs for the known functionally confirmed deleterious and benign RAD51C missense changes from nine pathogenicity prediction algorithms. The p.Arg312Trp variant failed to correct mitomycin and olaparib hypersensitivity and to complement abnormal RAD51C foci formation according to functional assays, which altogether with LOH and segregation data demonstrated deleteriousness. Prioritisation criteria were based on the number of predictors providing a deleterious output, with a minimum of 5 to qualify for testing and a PredictProtein score greater than 33 to assign high-priority indication. Our study points to a non-negligible number of RAD51C missense variants likely to impair protein function, provides a guideline to prioritise and encourage their selection for functional analysis and anticipates that reference laboratories should have available resources to conduct such assays.

Resolving RAD51C function in late stages of homologous recombination

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Kuznetsov Sergey G

2007-06-01

Full Text Available Abstract DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile Rad51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction.

DELLA proteins regulate expression of a subset of AM symbiosis-induced genes in Medicago truncatula.

Science.gov (United States)

Floss, Daniela S; Lévesque-Tremblay, Véronique; Park, Hee-Jin; Harrison, Maria J

2016-01-01

The majority of the vascular flowering plants form symbiotic associations with fungi from the phylum Glomeromycota through which both partners gain access to nutrients, either mineral nutrients in the case of the plant, or carbon, in the case of the fungus. (1) The association develops in the roots and requires substantial remodeling of the root cortical cells where branched fungal hyphae, called arbuscules, are housed in a new membrane-bound apoplastic compartment. (2) Nutrient exchange between the symbionts occurs over this interface and its development and maintenance is critical for symbiosis. Previously, we showed that DELLA proteins, which are well known as repressors of gibberellic acid signaling, also regulate development of AM symbiosis and are necessary to enable arbuscule development. (3) Furthermore, constitutive overexpression of a dominant DELLA protein (della1-Δ18) is sufficient to induce transcripts of several AM symbiosis-induced genes, even in the absence of the fungal symbiont. (4) Here we further extend this approach and identify AM symbiosis genes that respond transcriptionally to constitutive expression of a dominant DELLA protein and also genes that do respond to this treatment. Additionally, we demonstrate that DELLAs interact with REQUIRED FOR ARBUSCULE DEVELOPMENT 1 (RAD1) which further extends our knowledge of GRAS factor complexes that have the potential to regulate gene expression during AM symbiosis.

Histo-pathological correlation of BI-RADS 4 lesions on mammography with emphasis on microcalcification patterns.

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F Ismail

2008-03-01

Full Text Available A retrospective study of 20 patients with Breast Imaging Reporting and Data System (BI-RADS 4 lesions was undertaken. These patients were classified as BI-RADS 4 lesions due to presence of a mass (clinical or on mammography, architectural distortion and microcalcifications (MC. In some patients, the pattern of MC was benign but there were other features that were suspicious of malignancy. A comparison was made with the histological diagnosis in order to compare the radiological appearance of benign and malignant microcalcification patterns with the final histology. The study design included retrospective analysis of patients with MC on digital mammography who underwent biopsy. An analysis of the histology was then undertaken. Other factors in the history and physical examination were also considered. Results showed that although the study was not statistically significant due to limited study population, interesting trends are determined in assessing calcification patterns using the Breast Imaging Reporting and Data System (BI-RADS classification system, since some lesions that were thought to have benign calcification patterns were actually malignant and vice versa. Further study in this field is required.

Requirement of Sequences outside the Conserved Kinase Domain of Fission Yeast Rad3p for Checkpoint Control

Science.gov (United States)

Chapman, Carolyn Riley; Evans, Sarah Tyler; Carr, Antony M.; Enoch, Tamar

1999-01-01

The fission yeast Rad3p checkpoint protein is a member of the phosphatidylinositol 3-kinase-related family of protein kinases, which includes human ATMp. Mutation of the ATM gene is responsible for the disease ataxia-telangiectasia. The kinase domain of Rad3p has previously been shown to be essential for function. Here, we show that although this domain is necessary, it is not sufficient, because the isolated kinase domain does not have kinase activity in vitro and cannot complement a rad3 deletion strain. Using dominant negative alleles of rad3, we have identified two sites N-terminal to the conserved kinase domain that are essential for Rad3p function. One of these sites is the putative leucine zipper, which is conserved in other phosphatidylinositol 3-kinase-related family members. The other is a novel motif, which may also mediate Rad3p protein–protein interactions. PMID:10512862

Comparison of Visual Assessment of Breast Density in BI-RADS 4th and 5th Editions With Automated Volumetric Measurement.

Science.gov (United States)

Youk, Ji Hyun; Kim, So Jung; Son, Eun Ju; Gweon, Hye Mi; Kim, Jeong-Ah

2017-09-01

The purpose of this study was to compare visual assessments of mammographic breast density by radiologists using BI-RADS 4th and 5th editions in correlation with automated volumetric breast density measurements. A total of 337 consecutive full-field digital mammographic examinations with standard views were retrospectively assessed by two radiologists for mammographic breast density according to BI-RADS 4th and 5th editions. Fully automated measurement of the volume of fibroglandular tissue and total breast and percentage breast density was performed with a commercially available software program. Interobserver and intraobserver agreement was assessed with kappa statistics. The distributions of breast density categories for both editions of BI-RADS were compared and correlated with volumetric data. Interobserver agreement on breast density category was moderate to substantial (κ = 0.58-0.63) with use of BI-RADS 4th edition and substantial (κ = 0.63-0.66) with use of the 5th edition but without significant difference between the two editions. For intraobserver agreement between the two editions, the distributions of density category were significantly different (p density data, including percentage breast density, were significantly different among density categories (p density assessment had good correlation with visual assessment for both editions of BI-RADS.

Rad52 forms DMA repair and recombination centers during S phase

DEFF Research Database (Denmark)

Lisby, M.; Rothstein, R.; Mortensen, Uffe Hasbro

2001-01-01

fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by gamma -irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively...

SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation

Energy Technology Data Exchange (ETDEWEB)

Park, Seung Kuk; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

2016-02-05

Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNA was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms.

Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.

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Yoshiaki Okada

Full Text Available In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4 is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.

Acid or erythromycin stress significantly improves transformation efficiency through regulating expression of DNA binding proteins in Lactococcus lactis F44.

Science.gov (United States)

Wang, Binbin; Zhang, Huawei; Liang, Dongmei; Hao, Panlong; Li, Yanni; Qiao, Jianjun

2017-12-01

Lactococcus lactis is a gram-positive bacterium used extensively in the dairy industry and food fermentation, and its biological characteristics are usually improved through genetic manipulation. However, poor transformation efficiency was the main restriction factor for the construction of engineered strains. In this study, the transformation efficiency of L. lactis F44 showed a 56.1-fold increase in acid condition (pH 5.0); meanwhile, erythromycin stress (0.04 μg/mL) promoted the transformation efficiency more significantly (76.9-fold). Notably, the transformation efficiency of F44e (L. lactis F44 harboring empty pLEB124) increased up to 149.1-fold under the synergistic stresses of acid and erythromycin. In addition, the gene expression of some DNA binding proteins (DprA, RadA, RadC, RecA, RecQ, and SsbA) changed correspondingly. Especially for radA, 25.1-fold improvement was detected when F44e was exposed to pH 5.0. Overexpression of some DNA binding proteins could improve the transformation efficiency. The results suggested that acid or erythromycin stress could improve the transformation efficiency of L. lactis through regulating gene expression of DNA binding proteins. We have proposed a simple but promising strategy for improving the transformation efficiency of L. lactis and other hard-transformed microorganisms. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in mammals

International Nuclear Information System (INIS)

McKay, Michael J.; Spek, Peter van der; Kanaar, Roland; Smit, Bep; Bootsma, Dirk; Hoeijmakers, Jan H. J.

1996-01-01

Purpose/Objective: Genetic factors are likely to be major determinants of human cellular ionizing radiation sensitivity. DNA double strand breaks (dsbs) are significant ionizing radiation-induced lesions; cellular DNA dsb processing is also important in a number of other contexts. To further the understanding of DNA dsb processing in mammalian cells, we cloned and sequenced mammalian homologs of the rad21 Schizosaccharomyces pombe DNA dsb repair gene. Materials and Methods: The genes were cloned by evolutionary walking, exploiting sequence homology between the yeast and mammalian genes. Results: No major motifs indicative of a particular function were present in the predicted amino acid sequences of the mammalian genes. Alignment of the Rad21 amino acid sequence with its putative homologs showed that similarity was distributed across the length of the proteins, with more highly conserved regions at both termini. The mHR21 sp (mouse homolog ofR ad21, S. pombe) and hHR21 sp (humanh omolog of Rad21, S. pombe) predicted proteins were 96% identical, whereas the human and S. pombe proteins were 25% identical and 47% similar. RNA blot analysis showed that mHR21 sp mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus. In addition to a 3.1kb mRNA transcript in all tissues, an additional 2.2kb transcript was present at a high level in post-meiotic spermatids, white expression of the 3.1kb mRNA in testis was confined to the meiotic compartment. hHR21 sp mRNA was cell cycle regulated in human cells, increasing in late S phase to a peak in G2 phase. The level of hHR21 sp transcripts was not altered by exposure of normal diploid fibroblasts to 10 Gy ionizing radiation. In situ hybridization showed mHR21 sp resided on chromosome 15D3, whereashHR21 sp localized to the syntenic 8q24 region. Conclusion: Cloning these novel mammalian genes and characterization of their protein products should contribute to the understanding of cellular

A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression.

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Quitterie Venot

Full Text Available ABCB4/MDR3, a member of the ABC superfamily, is an ATP-dependent phosphatidylcholine translocator expressed at the canalicular membrane of hepatocytes. Defects in the ABCB4 gene are associated with rare biliary diseases. It is essential to understand the mechanisms of its canalicular membrane expression in particular for the development of new therapies. The stability of several ABC transporters is regulated through their binding to PDZ (PSD95/DglA/ZO-1 domain-containing proteins. ABCB4 protein ends by the sequence glutamine-asparagine-leucine (QNL, which shows some similarity to PDZ-binding motifs. The aim of our study was to assess the potential role of the QNL motif on the surface expression of ABCB4 and to determine if PDZ domain-containing proteins are involved. We found that truncation of the QNL motif decreased the stability of ABCB4 in HepG2-transfected cells. The deleted mutant ABCB4-ΔQNL also displayed accelerated endocytosis. EBP50, a PDZ protein highly expressed in the liver, strongly colocalized and coimmunoprecipitated with ABCB4, and this interaction required the QNL motif. Down-regulation of EBP50 by siRNA or by expression of an EBP50 dominant-negative mutant caused a significant decrease in the level of ABCB4 protein expression, and in the amount of ABCB4 localized at the canalicular membrane. Interaction of ABCB4 with EBP50 through its PDZ-like motif plays a critical role in the regulation of ABCB4 expression and stability at the canalicular plasma membrane.

Lymphoid irradiation in intractable rheumatoid arthritis. A double-blind, randomized study comparing 750-rad treatment with 2,000-rad treatment

International Nuclear Information System (INIS)

Hanly, J.G.; Hassan, J.; Moriarty, M.; Barry, C.; Molony, J.; Casey, E.; Whelan, A.; Feighery, C.; Bresnihan, B.

1986-01-01

Twenty patients with intractable rheumatoid arthritis were treated with 750-rad or 2,000-rad lymphoid irradiation in a randomized double-blind comparative study. Over a 12-month followup period, there was a significant improvement in 4 of 7 and 6 of 7 standard parameters of disease activity following treatment with 750 rads and 2,000 rads, respectively. Transient, short-term toxicity was less frequent with the lower dose. In both groups, there was a sustained peripheral blood lymphopenia, a selective depletion of T helper (Leu-3a+) lymphocytes, and reduced in vitro mitogen responses. These changes did not occur, however, in synovial fluid. These results suggest that 750-rad lymphoid irradiation is as effective as, but less toxic than, that with 2,000 rads in the management of patients with intractable rheumatoid arthritis

Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

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Clemens, Michael J; Elia, Androulla; Morley, Simon J

2013-01-01

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

Development of the Computer Code to Determine an Individual Radionuclides in the Rad-wastes Container for Ulchin Units 3 and 4

Energy Technology Data Exchange (ETDEWEB)

Kang, D.W.; Chi, J.H.; Goh, E.O. [Korea Electric Power Research Institute, Taejon (Korea)

2001-07-01

A computer program, RASSAY was developed to evaluate accurately the activities of various nuclides in the rad-waste container for Ulchin units 3 and 4. This is the final report of the project, {sup D}evelopment of the Computer Code to Determine an Individual Radionuclides in the Rad-wastes Container for Ulchin Units 3 and 4 and includes the followings; 1) Structure of the computer code, RASSAY 2) An example of surface dose calculation by computer simulation using MCNP code 3) Methods of sampling and activity measurement of various Rad-wastes. (author). 21 refs., 35 figs., 6 tabs.

VEGF 936C > T Polymorphism and Association of BI-RADS Score in Women with Suspected Breast Cancer

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M. Wehrschuetz

2009-01-01

Full Text Available Purpose Vascular endothelial growth factor (VEGF is a potent regulator of angiogenesis and thereby involved in the development and progression of solid tumors. A 936C> T polymorphism in the VEGF gene has been associated with reduced VEGF plasma levels. Purpose of the present study was to analyze the potential association between VEGF genotype and radiological appearance of breast lesions by mammography. Materials and Methods Fifty two women with 54 suspected breast lesions were analyzed by the use of mammography with the standard breast imaging reporting and data systems (BI-RADS. Germline VEGF genotype was determined in all subjects by allele-specific digestion of amplification products. An open biopsy was performed on all lesions. Results VEGF CC, CT and TT genotypes were found in 41 (79%, 9 (17% and 2 (4% patients. By mammography 26, 16 and 12 suspected breast lesions were classified as BI-RADS scores 3, 4 and 5, respectively. Both carriers of the TT genotype were classified as BI-RADS 5, whereas among CT or CC carriers, BI-RADS scores 3, 4 and 5 were found in 26, 16 and 10 subjects (P T polymorphism seems to be associated with a high BI-RADS score in women with suspicious breast lesions.

Genetic Interactions Between the Meiosis-Specific Cohesin Components, STAG3, REC8, and RAD21L.

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Ward, Ayobami; Hopkins, Jessica; Mckay, Matthew; Murray, Steve; Jordan, Philip W

2016-06-01

Cohesin is an essential structural component of chromosomes that ensures accurate chromosome segregation during mitosis and meiosis. Previous studies have shown that there are cohesin complexes specific to meiosis, required to mediate homologous chromosome pairing, synapsis, recombination, and segregation. Meiosis-specific cohesin complexes consist of two structural maintenance of chromosomes proteins (SMC1α/SMC1β and SMC3), an α-kleisin protein (RAD21, RAD21L, or REC8), and a stromal antigen protein (STAG1, 2, or 3). STAG3 is exclusively expressed during meiosis, and is the predominant STAG protein component of cohesin complexes in primary spermatocytes from mouse, interacting directly with each α-kleisin subunit. REC8 and RAD21L are also meiosis-specific cohesin components. Stag3 mutant spermatocytes arrest in early prophase ("zygotene-like" stage), displaying failed homolog synapsis and persistent DNA damage, as a result of unstable loading of cohesin onto the chromosome axes. Interestingly, Rec8, Rad21L double mutants resulted in an earlier "leptotene-like" arrest, accompanied by complete absence of STAG3 loading. To assess genetic interactions between STAG3 and α-kleisin subunits RAD21L and REC8, our lab generated Stag3, Rad21L, and Stag3, Rec8 double knockout mice, and compared them to the Rec8, Rad21L double mutant. These double mutants are phenotypically distinct from one another, and more severe than each single knockout mutant with regards to chromosome axis formation, cohesin loading, and sister chromatid cohesion. The Stag3, Rad21L, and Stag3, Rec8 double mutants both progress further into prophase I than the Rec8, Rad21L double mutant. Our genetic analysis demonstrates that cohesins containing STAG3 and REC8 are the main complex required for centromeric cohesion, and RAD21L cohesins are required for normal clustering of pericentromeric heterochromatin. Furthermore, the STAG3/REC8 and STAG3/RAD21L cohesins are the primary cohesins required for

RAD51 interconnects between DNA replication, DNA repair and immunity.

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Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

2017-05-05

RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

S phase entry causes homocysteine-induced death while ataxia telangiectasia and Rad3 related protein functions anti-apoptotically to protect neurons.

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Ye, Weizhen; Blain, Stacy W

2010-08-01

A major phenotype seen in neurodegenerative disorders is the selective loss of neurons due to apoptotic death and evidence suggests that inappropriate re-activation of cell cycle proteins in post-mitotic neurons may be responsible. To investigate whether reactivation of the G1 cell cycle proteins and S phase entry was linked with apoptosis, we examined homocysteine-induced neuronal cell death in a rat cortical neuron tissue culture system. Hyperhomocysteinaemia is a physiological risk factor for a variety of neurodegenerative diseases, including Alzheimer's disease. We found that in response to homocysteine treatment, cyclin D1, and cyclin-dependent kinases 4 and 2 translocated to the nucleus, and p27 levels decreased. Both cyclin-dependent kinases 4 and 2 regained catalytic activity, the G1 gatekeeper retinoblastoma protein was phosphorylated and DNA synthesis was detected, suggesting transit into S phase. Double-labelling immunofluorescence showed a 95% co-localization of anti-bromodeoxyuridine labelling with apoptotic markers, demonstrating that those cells that entered S phase eventually died. Neurons could be protected from homocysteine-induced death by methods that inhibited G1 phase progression, including down-regulation of cyclin D1 expression, inhibition of cyclin-dependent kinases 4 or 2 activity by small molecule inhibitors, or use of the c-Abl kinase inhibitor, Gleevec, which blocked cyclin D and cyclin-dependent kinase 4 nuclear translocation. However, blocking cell cycle progression post G1, using DNA replication inhibitors, did not prevent apoptosis, suggesting that death was not preventable post the G1-S phase checkpoint. While homocysteine treatment caused DNA damage and activated the DNA damage response, its mechanism of action was distinct from that of more traditional DNA damaging agents, such as camptothecin, as it was p53-independent. Likewise, inhibition of the DNA damage sensors, ataxia-telangiectasia mutant and ataxia telangiectasia and Rad

Glia protein aquaporin-4 regulates aversive motivation of spatial memory in Morris water maze.

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Zhang, Ji; Li, Ying; Chen, Zhong-Guo; Dang, Hui; Ding, Jian-Hua; Fan, Yi; Hu, Gang

2013-12-01

Although extensive investigation has revealed that an astrocyte-specific protein aquaporin-4 (AQP4) participates in regulating synaptic plasticity and memory, a functional relationship between AQP4 and learning processing has not been clearly established. This study was designed to test our hypothesis that AQP4 modulates the aversive motivation in Morris water maze (MWM). Using hidden platform training, we observed that AQP4 KO mice significantly decreased their swimming velocity compared with wild-type (WT) mice. To test for a relationship between velocities and escape motivation, we removed the platform and subjected a new group of mice similar to the session of hidden platform training. We found that KO mice exhibited a gradual reduction in swimming velocity, while WT mice did not alter their velocity. In the subsequent probe trial, KO mice after no platform training significantly decreased their mean velocity compared with those KO mice after hide platform training. However, all of KO mice were not impaired in their ability to locate a visible, cued escape platform. Our findings, along with a previous report that AQP4 regulates memory consolidation, implicate a novel role for this glial protein in modulating the aversive motivation in spatial learning paradigm. © 2013 John Wiley & Sons Ltd.

Studies of DNA repair in saccharomyces cerevisiae. I. Characterization of a new allele of RAD6. II. Investigation of events in the first cell cycle after DNA damage

International Nuclear Information System (INIS)

Douthwright-Fasse, J.A.

1979-01-01

Studies in two independent, but related, areas of DNA repair have been carried out in Saccharomyces cerevisiae; characterization of a new allele in the RAD6 gene which suggests that the gene is multifunctional, and utilization of photoreactivation as a probe of events occurring during the first cell cycle after DNA damage. Strains carrying the new allele, designated rad6-4, are as sensitive to uv and ionizing radiation as those carrying rad6-1 or rad6-3 but, unlike them, are capable of induced mutagenesis and sporulation. Although rad6-4 may well be a missense mutation, the evidence shows that it is unlikely that this phenotype is due to leakiness. Instead, the data suggest that the RAD6 gene is multifunctional. One function is necessary to recover from DNA damage in an error-free manner, and the other is concerned with mutagenic processes and sporulation. Rad6-1 and rad6-3 strains are deficient in both of these functions, while rad6-4 strains are deficient only in the error-free function. The loss of photoreversibility (LOP) of ultraviolet induced mutations to arginine independence in an excision defective strain carrying arg4-17 examines the events occurring in the first cell cycle after DNA damage. LOP is dependent upon de novo protein synthesis. LOP begins immediately after UV irradiation, before semiconservative DNA synthesis takes place, and is complete after four hours in growth medium.There is no evidence indicating whether the normal function of the protein is involved in excision repair, or in one of the two repair processes believed to be inducible; induced mutagenesis or recombinational repair

Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment.

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Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

2013-12-01

Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

Studies of DNA repair in Saccharomyces cerevisiae. I. Characterization of a new allele of RAD6. II. Investigation of events in the first cell cycle after DNA damage

International Nuclear Information System (INIS)

Dolthwright-Fasse, J.A.

1980-01-01

Studies in two independent, but related, areas of DNA repair have been carried out in the eucaryotic yeast, Saccharomyces cerevisiae. The first is the characterization of a new allele in the RAD6 gene suggesting that the gene is multifunctional. The second is the utilization of photoreactivation as a probe of events occurring during the first cell cycle after DNA damage. Strains carrying the new allele, designated rad6-4, of the RAD6 locus are about as sensitive to uv and ionizing radiation as those carrying rad6-1 or rad6-3. Although rad6-4 may well be a missense mutation, the data suggest that the RAD6 gene is multifunctional. One function is necessary to recover from DNA damage in an error-free manner, and the other is concerned with mutagenic processes and sporulation. The loss of photoreversibility (LOP) of ultraviolet induced mutations to arginine independence in an excision defective strain carrying arg4-17 examines the events occurring in the first cell cycle. The post uv protein synthesis causes pyrimidine dimmers to become inaccessible to the photoreactivating enzyme in some unknown manner. There is no evidence indicating whether the normal function of the protein is involved in excision repair, or in one of the two repair processes believed to be inducible; induced mutagenesis or recombinational repair

RAD9 deficiency enhances radiation induced bystander DNA damage and transcriptomal response

International Nuclear Information System (INIS)

Ghandhi, Shanaz A; Ponnaiya, Brian; Panigrahi, Sunil K; Hopkins, Kevin M; Cui, Qingping; Hei, Tom K; Amundson, Sally A; Lieberman, Howard B

2014-01-01

Radiation induced bystander effects are an important component of the overall response of cells to irradiation and are associated with human health risks. The mechanism responsible includes intra-cellular and inter-cellular signaling by which the bystander response is propagated. However, details of the signaling mechanism are not well defined. We measured the bystander response of Mrad9 +/+ and Mrad9 −/− mouse embryonic stem cells, as well as human H1299 cells with inherent or RNA interference-mediated reduced RAD9 levels after exposure to 1 Gy α particles, by scoring chromosomal aberrations and micronuclei formation, respectively. In addition, we used microarray gene expression analyses to profile the transcriptome of directly irradiated and bystander H1299 cells. We demonstrated that Mrad9 null enhances chromatid aberration frequency induced by radiation in bystander mouse embryonic stem cells. In addition, we found that H1299 cells with reduced RAD9 protein levels showed a higher frequency of radiation induced bystander micronuclei formation, compared with parental cells containing inherent levels of RAD9. The enhanced bystander response in human cells was associated with a unique transcriptomic profile. In unirradiated cells, RAD9 reduction broadly affected stress response pathways at the mRNA level; there was reduction in transcript levels corresponding to genes encoding multiple members of the UVA-MAPK and p38MAPK families, such as STAT1 and PARP1, suggesting that these signaling mechanisms may not function optimally when RAD9 is reduced. Using network analysis, we found that differential activation of the SP1 and NUPR1 transcriptional regulators was predicted in directly irradiated and bystander H1299 cells. Transcription factor prediction analysis also implied that HIF1α (Hypoxia induced factor 1 alpha) activation by protein stabilization in irradiated cells could be a negative predictor of the bystander response, suggesting that local hypoxic stress

Regulation of Homologous Recombination by SUMOylation

DEFF Research Database (Denmark)

Pinela da Silva, Sonia Cristina

factors such as the homologous recombination (HR) machinery. HR constitutes the main DSB repair pathway in Saccharomyces cerevisiae and despite being largely considered an error-free process and essential for genome stability, uncontrolled recombination can lead to loss of heterozygosity, translocations......, deletions, and genome rearrangements that can lead to cell death or cancer in humans. The post-translational modification by SUMO (small ubiquitinlike modifier) has proven to be an important regulator of HR and genome integrity, but the molecular mechanisms responsible for these roles are still unclear....... In this study I present new insights for the role of SUMOylation in regulating HR by dissecting the role of SUMO in the interaction between the central HR-mediator protein Rad52 and its paralogue Rad59 and the outcome of recombination. This data provides evidence for the importance of SUMO in promoting protein...

Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis.

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Gardner, Thomas W; Abcouwer, Steven F; Losiewicz, Mandy K; Fort, Patrice E

2015-09-15

Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1. Copyright © 2015 the American Physiological Society.

ACR BI-RADS Assessment Category 4 Subdivisions in Diagnostic Mammography: Utilization and Outcomes in the National Mammography Database.

Science.gov (United States)

Elezaby, Mai; Li, Geng; Bhargavan-Chatfield, Mythreyi; Burnside, Elizabeth S; DeMartini, Wendy B

2018-05-01

Purpose To determine the utilization and positive predictive value (PPV) of the American College of Radiology (ACR) Breast Imaging Data and Reporting System (BI-RADS) category 4 subdivisions in diagnostic mammography in the National Mammography Database (NMD). Materials and Methods This study involved retrospective review of diagnostic mammography data submitted to the NMD from January 1, 2008 to December 30, 2014. Utilization rates of BI-RADS category 4 subdivisions were compared by year, facility (type, location, census region), and examination (indication, finding type) characteristics. PPV3 (positive predictive value for biopsies performed) was calculated overall and according to category 4 subdivision. The χ 2 test was used to test for significant associations. Results Of 1 309 950 diagnostic mammograms, 125 447 (9.6%) were category 4, of which 33.3% (41 841 of 125 447) were subdivided. Subdivision utilization rates were higher (P use, subdivisions were utilized in the minority (33.3% [41 841 of 125 447]) of category 4 diagnostic mammograms, with variability based on facility and examination characteristics. When subdivisions were used, PPV3s were in BI-RADS-specified malignancy ranges. This analysis supports the use of subdivisions in broad practice and, given benefits for patient care, should motivate increased utilization. © RSNA, 2018 Online supplemental material is available for this article.

Hypoxic induction of the regulator of G-protein signalling 4 gene is mediated by the hypoxia-inducible factor pathway.

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Sam W Z Olechnowicz

Full Text Available The transcriptional response to hypoxia is largely dependent on the Hypoxia Inducible Factors (HIF-1 and HIF-2 in mammalian cells. Many target genes have been characterised for these heterodimeric transcription factors, yet there is evidence that the full range of HIF-regulated genes has not yet been described. We constructed a TetON overexpression system in the rat pheochromocytoma PC-12 cell line to search for novel HIF and hypoxia responsive genes. The Rgs4 gene encodes the Regulator of G-Protein Signalling 4 (RGS4 protein, an inhibitor of signalling from G-protein coupled receptors, and dysregulation of Rgs4 is linked to disease states such as schizophrenia and cardiomyopathy. Rgs4 was found to be responsive to HIF-2α overexpression, hypoxic treatment, and hypoxia mimetic drugs in PC-12 cells. Similar responses were observed in human neuroblastoma cell lines SK-N-SH and SK-N-BE(2C, but not in endothelial cells, where Rgs4 transcript is readily detected but does not respond to hypoxia. Furthermore, this regulation was found to be dependent on transcription, and occurs in a manner consistent with direct HIF transactivation of Rgs4 transcription. However, no HIF binding site was detectable within 32 kb of the human Rgs4 gene locus, leading to the possibility of regulation by long-distance genomic interactions. Further research into Rgs4 regulation by hypoxia and HIF may result in better understanding of disease states such as schizophrenia, and also shed light on the other roles of HIF yet to be discovered.

Hypoxic Induction of the Regulator of G-Protein Signalling 4 Gene Is Mediated by the Hypoxia-Inducible Factor Pathway

Science.gov (United States)

Olechnowicz, Sam W. Z.; Fedele, Anthony O.; Peet, Daniel J.

2012-01-01

The transcriptional response to hypoxia is largely dependent on the Hypoxia Inducible Factors (HIF-1 and HIF-2) in mammalian cells. Many target genes have been characterised for these heterodimeric transcription factors, yet there is evidence that the full range of HIF-regulated genes has not yet been described. We constructed a TetON overexpression system in the rat pheochromocytoma PC-12 cell line to search for novel HIF and hypoxia responsive genes. The Rgs4 gene encodes the Regulator of G-Protein Signalling 4 (RGS4) protein, an inhibitor of signalling from G-protein coupled receptors, and dysregulation of Rgs4 is linked to disease states such as schizophrenia and cardiomyopathy. Rgs4 was found to be responsive to HIF-2α overexpression, hypoxic treatment, and hypoxia mimetic drugs in PC-12 cells. Similar responses were observed in human neuroblastoma cell lines SK-N-SH and SK-N-BE(2)C, but not in endothelial cells, where Rgs4 transcript is readily detected but does not respond to hypoxia. Furthermore, this regulation was found to be dependent on transcription, and occurs in a manner consistent with direct HIF transactivation of Rgs4 transcription. However, no HIF binding site was detectable within 32 kb of the human Rgs4 gene locus, leading to the possibility of regulation by long-distance genomic interactions. Further research into Rgs4 regulation by hypoxia and HIF may result in better understanding of disease states such as schizophrenia, and also shed light on the other roles of HIF yet to be discovered. PMID:22970249

Targeting Rad50 sensitizes human nasopharyngeal carcinoma cells to radiotherapy

International Nuclear Information System (INIS)

Chang, Lihong; Huang, Jiancong; Wang, Kai; Li, Jingjia; Yan, Ruicheng; Zhu, Ling; Ye, Jin; Wu, Xifu; Zhuang, Shimin; Li, Daqing; Zhang, Gehua

2016-01-01

The Mre11-Rad50-Nbs1 (MRN) complex is well known for its crucial role in initiating DNA double strand breaks (DSBs) repair pathways to resistant irradiation (IR) injury and thus facilitating radioresistance which severely reduces radiocurability of nasopharyngeal cancer (NPC). Targeting native cellular MRN function would sensitize NPC cells to IR. A recombinant adenovirus containing a mutant Rad50 gene (Ad-RAD50) expressing Rad50 zinc hook domain but lacking the ATPase domain and the Mre11 interaction domain was constructed to disrupt native cellular MRN functions. The effects of Ad-RAD50 on the MRN functions were assessed in NPC cells lines using western blot, co-immunoprecipitation and confocal microscopy analyses. The increased radiosensitivity of transient Ad-RAD50 to IR was examined in NPC cells, including MTT assay, colony formation. The molecular mechanisms of radiosensitization were confirmed by neutral comet assay and western bolts. Nude mice subcutaneous injection, tumor growth curve and TUNEL assay were used to evaluate tumor regression and apoptosis in vivo. Rad50 is remarkably upregulated in NPC cells after IR, implying the critical role of Rad50 in MRN functions. The transient expression of this mutant Rad50 decreased the levels of native cellular Rad50, Mre11 and Nbs1, weakened the interactions among these proteins, abrogated the G2/M arrest induced by DSBs and reduced the DNA repair ability in NPC cells. A combination of IR and mutant RAD50 therapy produced significant tumor cytotoxicity in vitro, with a corresponding increase in DNA damage, prevented proliferation and cell viability. Furthermore, Ad-RAD50 sensitized NPC cells to IR by causing dramatic tumor regression and inducing apoptosis in vivo. Our findings define a novel therapeutic approach to NPC radiosensitization via targeted native cellular Rad50 disruption. The online version of this article (doi:10.1186/s12885-016-2190-8) contains supplementary material, which is available to

Subcategorization of Suspicious Breast Lesions (BI-RADS Category 4) According to MRI Criteria: Role of Dynamic Contrast-Enhanced and Diffusion-Weighted Imaging.

Science.gov (United States)

Maltez de Almeida, João Ricardo; Gomes, André Boechat; Barros, Thomas Pitangueira; Fahel, Paulo Eduardo; de Seixas Rocha, Mário

2015-07-01

The purposes of this study were to investigate whether dynamic contrast-enhanced MRI is adequate for subcategorization of suspicious lesions (BI-RADS category 4) and to evaluate whether use of DWI improves diagnostic performance. The study group was composed of 103 suspicious lesions found in 83 subjects. Patient ages and lesion sizes were compiled, and two radiologists reanalyzed the images; subcategorized the findings as BI-RADS 4A, 4B, or 4C; and calculated apparent diffusion coefficient (ADC) values. The stratified variables were tested by univariate analysis and inserted in two multivariate predictive models, which were used to generate ROC curves and compare AUCs. Positive predictive values (PPVs) for each subcategory and ADC level were calculated, and interobserver agreement was tested. Forty-four (42.7%) suspicious findings proved malignant. Except for age (p = 0.08), all stratified predictor variables were significant in univariate analyses (p BI-RADS 4 subcategory (4A, 0.15; 4B, 0.37; 4C, 0.84). ADC values of 1.10 × 10(-3) mm(2)/s or less had the second highest PPV (0.77). Interobserver agreement was substantial at a kappa value of 0.80 (95% CI, 0.70-0.90; p BI-RADS category 4) can be satisfactorily performed with DCE-MRI and slightly improved when DWI is introduced.

Post-translational regulation of Oct4 transcriptional activity.

Directory of Open Access Journals (Sweden)

Jonathan P Saxe

Full Text Available Oct4 is a key component of the molecular circuitry which regulates embryonic stem cell proliferation and differentiation. It is essential for maintenance of undifferentiated, pluripotent cell populations, and accomplishes these tasks by binding DNA in multiple heterodimer and homodimer configurations. Very little is known about how formation of these complexes is regulated, or the mechanisms through which Oct4 proteins respond to complex extracellular stimuli which regulate pluripotency. Here, we provide evidence for a phosphorylation-based mechanism which regulates specific Oct4 homodimer conformations. Point mutations of a putative phosphorylation site can specifically abrogate transcriptional activity of a specific homodimer assembly, with little effect on other configurations. Moreover, we performed bioinformatic predictions to identify a subset of Oct4 target genes which may be regulated by this specific assembly, and show that altering Oct4 protein levels affects transcription of Oct4 target genes which are regulated by this assembly but not others. Finally, we identified several signaling pathways which may mediate this phosphorylation and act in combination to regulate Oct4 transcriptional activity and protein stability. These results provide a mechanism for rapid and reversible alteration of Oct4 transactivation potential in response to extracellular signals.

Characterization of the interaction between the cohesin subunits Rad21 and SA1/2.

Directory of Open Access Journals (Sweden)

Nenggang Zhang

Full Text Available The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits, namely Rad21/Mcd1/Scc1, Smc1, Smc3, and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is generated during DNA replication and maintained until the onset of anaphase. Among the many proposed models of the cohesin complex, the 'core' cohesin subunits Smc1, Smc3, and Rad21 are almost universally displayed as tripartite ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core subunit SA1/SA2. To gain deeper insight into the function of SA1/SA2 in the cohesin complex, we have mapped the interactive regions of SA2 and Rad21 in vitro and ex vivo. Whereas SA2 interacts with Rad21 through a broad region (301-750 aa, Rad21 binds to SA proteins through two SA-binding motifs on Rad21, namely N-terminal (NT and middle part (MP SA-binding motif, located at 60-81 aa of the N-terminus and 383-392 aa of the MP of Rad21, respectively. The MP SA-binding motif is a 10 amino acid, α-helical motif. Deletion of these 10 amino acids or mutation of three conserved amino acids (L(385, F(389, and T(390 in this α-helical motif significantly hinders Rad21 from physically interacting with SA1/2. Besides the MP SA-binding motif, the NT SA-binding motif is also important for SA1/2 interaction. Although mutations on both SA-binding motifs disrupt Rad21-SA1/2 interaction, they had no apparent effect on the Smc1-Smc3-Rad21 interaction. However, the Rad21-Rad21 dimerization was reduced by the mutations, indicating potential involvement of the two SA-binding motifs in the formation of the two-ring handcuff for chromosomal cohesion. Furthermore, mutant Rad21 proteins failed to significantly rescue precocious chromosome separation caused by depletion of endogenous Rad21 in mitotic cells, further indicating the physiological significance of the two SA-binding motifs of Rad21.

Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

KAUST Repository

Nomme, Julian; Renodon-Corniè re, Axelle; Asanomi, Yuya; Sakaguchi, Kazuyasu; Stasiak, Alicja Z; Stasiak, Andrzej; Norden, Bengt; Tran, Vinh; Takahashi, Masayuki

2010-01-01

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

KAUST Repository

Nomme, Julian

2010-08-01

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

RGS6, but not RGS4, is the dominant regulator of G protein signaling (RGS) modulator of the parasympathetic regulation of mouse heart rate.

Science.gov (United States)

Wydeven, Nicole; Posokhova, Ekaterina; Xia, Zhilian; Martemyanov, Kirill A; Wickman, Kevin

2014-01-24

Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K(+) channel IKACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4(-/-), Rgs6(-/-), and Rgs4(-/-):Rgs6(-/-) mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M2R-IKACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M2R-IKACh signaling pathway in SAN cells to carbachol and a significant slowing of M2R-IKACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6(-/-) mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6(-/-) and Gβ5(-/-) mice, suggest that the partial rescue of phenotypes in Rgs4(-/-):Rgs6(-/-) mice is attributable to another R7 RGS protein whose influence on M2R-IKACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M2R-IKACh signaling in mice.

Rad10 exhibits lesion-dependent genetic requirements for recruitment to DNA double-strand breaks in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Moore, Destaye M; Karlin, Justin; González-Barrera, Sergio

2009-01-01

In the yeast Saccharomyces cerevisiae, the Rad1-Rad10 protein complex participates in nucleotide excision repair (NER) and homologous recombination (HR). During HR, the Rad1-Rad10 endonuclease cleaves 3' branches of DNA and aberrant 3' DNA ends that are refractory to other 3' processing enzymes. ...

Interdependence of the rad50 hook and globular domain functions.

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Hohl, Marcel; Kochańczyk, Tomasz; Tous, Cristina; Aguilera, Andrés; Krężel, Artur; Petrini, John H J

2015-02-05

Rad50 contains a conserved Zn(2+) coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here, we focused on rad50 mutations flanking the Zn(2+)-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double-strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end joining, and DNA double-strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled-coil and globular ATPase domains, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular Basis for Enhancement of the Meiotic DMCI Recombinase by RAD51AP1

Energy Technology Data Exchange (ETDEWEB)

Dray, Eloise; Dunlop, Myun Hwa; Kauppi, Liisa; San Filippo, Joseph San; Wiese, Claudia; Tsai, Miaw-Sheue; Begovic, Sead; Schild, David; Jasin, Maria; Keeney, Scott; Sung, Patrick

2010-11-05

Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 Associated Protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional co-operation is dependent on complex formation between DMC1 and RAD51AP1, and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci co-localize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.

Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo

DEFF Research Database (Denmark)

Burgess, Rebecca C; Lisby, Michael; Altmannova, Veronika

2009-01-01

, and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2......Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 "anti-recombinase" restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we...... removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR....

G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor.

Science.gov (United States)

Morita, Tsuyoshi; Hayashi, Ken'ichiro

2013-08-02

Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin-MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF-SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin-MRTFs interaction. Copyright © 2013 Elsevier Inc. All rights reserved.

PP32 and SET/TAF-Iβ proteins regulate the acetylation of newly synthesized histone H4.

Science.gov (United States)

Saavedra, Francisco; Rivera, Carlos; Rivas, Elizabeth; Merino, Paola; Garrido, Daniel; Hernández, Sergio; Forné, Ignasi; Vassias, Isabelle; Gurard-Levin, Zachary A; Alfaro, Iván E; Imhof, Axel; Almouzni, Geneviève; Loyola, Alejandra

2017-11-16

Newly synthesized histones H3 and H4 undergo a cascade of maturation steps to achieve proper folding and to establish post-translational modifications prior to chromatin deposition. Acetylation of H4 on lysines 5 and 12 by the HAT1 acetyltransferase is observed late in the histone maturation cascade. A key question is to understand how to establish and regulate the distinct timing of sequential modifications and their biological significance. Here, we perform proteomic analysis of the newly synthesized histone H4 complex at the earliest time point in the cascade. In addition to known binding partners Hsp90 and Hsp70, we also identify for the first time two subunits of the histone acetyltransferase inhibitor complex (INHAT): PP32 and SET/TAF-Iβ. We show that both proteins function to prevent HAT1-mediated H4 acetylation in vitro. When PP32 and SET/TAF-Iβ protein levels are down-regulated in vivo, we detect hyperacetylation on lysines 5 and 12 and other H4 lysine residues. Notably, aberrantly acetylated H4 is less stable and this reduces the interaction with Hsp90. As a consequence, PP32 and SET/TAF-Iβ depleted cells show an S-phase arrest. Our data demonstrate a novel function of PP32 and SET/TAF-Iβ and provide new insight into the mechanisms regulating acetylation of newly synthesized histone H4. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Dosimetric properties of the pocket alarm dosimeter type Alnor RAD 21L, RAD 21H, RAD 22

International Nuclear Information System (INIS)

Hauser, M.; Burgkhardt, B.; Piesch, E.

1981-02-01

In personnel monitoring pocket dosimeters with build-in alarm devices are increasingly in use. The report presents results of a test performed at Karlsruhe for the pocket dose and alarm meter type Alnor RAD 21L, RAD 21H, RAD 22. The properties investigated are above all linearity and reproducibility of the dose reading as well as of the acoustic alarm indication, dependence of the dose reading on the photon energy, the direction of the radiation incidence, the dose rate, the temperature, operational characteristic of the batteries. (orig.) [de

Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons.

Science.gov (United States)

Bohnsack, John Peyton; Carlson, Stephen L; Morrow, A Leslie

2016-06-01

The GABAA α4 subunit exists in two distinct populations of GABAA receptors. Synaptic GABAA α4 receptors are localized at the synapse and mediate phasic inhibitory neurotransmission, while extrasynaptic GABAA receptors are located outside of the synapse and mediate tonic inhibitory transmission. These receptors have distinct pharmacological and biophysical properties that contribute to interest in how these different subtypes are regulated under physiological and pathological states. We utilized subcellular fractionation procedures to separate these populations of receptors in order to investigate their regulation by protein kinases in cortical cultured neurons. Protein kinase A (PKA) activation decreases synaptic α4 expression while protein kinase C (PKC) activation increases α4 subunit expression, and these effects are associated with increased β3 S408/409 or γ2 S327 phosphorylation respectively. In contrast, PKA activation increases extrasynaptic α4 and δ subunit expression, while PKC activation has no effect. Our findings suggest synaptic and extrasynaptic GABAA α4 subunit expression can be modulated by PKA to inform the development of more specific therapeutics for neurological diseases that involve deficits in GABAergic transmission. Copyright © 2016 Elsevier Ltd. All rights reserved.

RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells.

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King, Harry O; Brend, Tim; Payne, Helen L; Wright, Alexander; Ward, Thomas A; Patel, Karan; Egnuni, Teklu; Stead, Lucy F; Patel, Anjana; Wurdak, Heiko; Short, Susan C

2017-01-10

Patients with glioblastoma die from local relapse despite surgery and high-dose radiotherapy. Resistance to radiotherapy is thought to be due to efficient DNA double-strand break (DSB) repair in stem-like cells able to survive DNA damage and repopulate the tumor. We used clinical samples and patient-derived glioblastoma stem cells (GSCs) to confirm that the DSB repair protein RAD51 is highly expressed in GSCs, which are reliant on RAD51-dependent DSB repair after radiation. RAD51 expression and RAD51 foci numbers fall when these cells move toward astrocytic differentiation. In GSCs, the small-molecule RAD51 inhibitors RI-1 and B02 prevent RAD51 focus formation, reduce DNA DSB repair, and cause significant radiosensitization. We further demonstrate that treatment with these agents combined with radiation promotes loss of stem cells defined by SOX2 expression. This indicates that RAD51-dependent repair represents an effective and specific target in GSCs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Regulators of Slc4 bicarbonate transporter activity

Directory of Open Access Journals (Sweden)

Ian M. Thornell

2015-06-01

Full Text Available The Slc4 family of transporters is comprised of anion exchangers (AE1-4, Na-coupled bicarbonate transporters (NCBTs including electrogenic Na/bicarbonate cotransporters (NBCe1 and NBCe2, electroneutral Na/bicarbonate cotransporters (NBCn1 and NBCn2, and the electroneutral Na-driven Cl-bicarbonate exchanger (NDCBE, as well as a borate transporter (BTR1. These transporters regulate intracellular pH (pHi and contribute to steady-state pHi, but are also involved in other physiological processes including CO2 carriage by red blood cells and solute secretion/reabsorption across epithelia. Acid-base transporters function as either acid extruders or acid loaders, with the Slc4 proteins moving HCO3– either into or out of cells. According to results from both molecular and functional studies, multiple Slc4 proteins and/or associated splice variants with similar expected effects on pHi are often found in the same tissue or cell. Such apparent redundancy is likely to be physiologically important. In addition to regulating pHi, a HCO3– transporter contributes to a cell’s ability to fine tune the intracellular regulation of the cotransported/exchanged ion(s (e.g., Na+ or Cl–. In addition, functionally similar transporters or splice variants with different regulatory profiles will optimize pH physiology and solute transport under various conditions or within subcellular domains. Such optimization will depend on activated signaling pathways and transporter expression profiles. In this review, we will summarize and discuss both classical and more recently identified regulators of the Slc4 proteins. Some of these regulators include traditional second messengers, lipids, binding proteins, autoregulatory domains, and less conventional regulators. The material presented will provide insight into the diversity and physiological significance of multiple members within the Slc4 gene family.

Structured reporting platform improves CAD-RADS assessment.

Science.gov (United States)

Szilveszter, Bálint; Kolossváry, Márton; Karády, Júlia; Jermendy, Ádám L; Károlyi, Mihály; Panajotu, Alexisz; Bagyura, Zsolt; Vecsey-Nagy, Milán; Cury, Ricardo C; Leipsic, Jonathon A; Merkely, Béla; Maurovich-Horvat, Pál

2017-11-01

Structured reporting in cardiac imaging is strongly encouraged to improve quality through consistency. The Coronary Artery Disease - Reporting and Data System (CAD-RADS) was recently introduced to facilitate interdisciplinary communication of coronary CT angiography (CTA) results. We aimed to assess the agreement between manual and automated CAD-RADS classification using a structured reporting platform. Five readers prospectively interpreted 500 coronary CT angiographies using a structured reporting platform that automatically calculates the CAD-RADS score based on stenosis and plaque parameters manually entered by the reader. In addition, all readers manually assessed CAD-RADS blinded to the automatically derived results, which was used as the reference standard. We evaluated factors influencing reader performance including CAD-RADS training, clinical load, time of the day and level of expertise. Total agreement between manual and automated classification was 80.2%. Agreement in stenosis categories was 86.7%, whereas the agreement in modifiers was 95.8% for "N", 96.8% for "S", 95.6% for "V" and 99.4% for "G". Agreement for V improved after CAD-RADS training (p = 0.047). Time of the day and clinical load did not influence reader performance (p > 0.05 both). Less experienced readers had a higher total agreement as compared to more experienced readers (87.0% vs 78.0%, respectively; p = 0.011). Even though automated CAD-RADS classification uses data filled in by the readers, it outperforms manual classification by preventing human errors. Structured reporting platforms with automated calculation of the CAD-RADS score might improve data quality and support standardization of clinical decision making. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Functions and structures of eukaryotic recombination proteins

International Nuclear Information System (INIS)

Ogawa, Tomoko

1994-01-01

We have found that Rad51 and RecA Proteins form strikingly similar structures together with dsDNA and ATP. Their right handed helical nucleoprotein filaments extend the B-form DNA double helixes to 1.5 times in length and wind the helix. The similarity and uniqueness of their structures must reflect functional homologies between these proteins. Therefore, it is highly probable that similar recombination proteins are present in various organisms of different evolutional states. We have succeeded to clone RAD51 genes from human, mouse, chicken and fission yeast genes, and found that the homologues are widely distributed in eukaryotes. The HsRad51 and MmRad51 or ChRad51 proteins consist of 339 amino acids differing only by 4 or 12 amino acids, respectively, and highly homologous to both yeast proteins, but less so to Dmcl. All of these proteins are homologous to the region from residues 33 to 240 of RecA which was named ''homologous core. The homologous core is likely to be responsible for functions common for all of them, such as the formation of helical nucleoprotein filament that is considered to be involved in homologous pairing in the recombination reaction. The mouse gene is transcribed at a high level in thymus, spleen, testis, and ovary, at lower level in brain and at a further lower level in some other tissues. It is transcribed efficiently in recombination active tissues. A clear functional difference of Rad51 homologues from RecA was suggested by the failure of heterologous genes to complement the deficiency of Scrad51 mutants. This failure seems to reflect the absence of a compatible partner, such as ScRad52 protein in the case of ScRad51 protein, between different species. Thus, these discoveries play a role of the starting point to understand the fundamental gene targeting in mammalian cells and in gene therapy. (J.P.N.)

Claudins, dietary milk proteins, and intestinal barrier regulation.

Science.gov (United States)

Kotler, Belinda M; Kerstetter, Jane E; Insogna, Karl L

2013-01-01

The family of claudin proteins plays an important role in regulating the intestinal barrier by modulating the permeability of tight junctions. The impact of dietary protein on claudin biology has not been studied extensively. Whey proteins have been reported to improve intestinal barrier function, but their mechanism of action is not clear. Recent studies, however, have demonstrated increased intestinal claudin expression in response to milk protein components. Reviewed here are new findings suggesting that whey-protein-derived transforming growth factor β transcriptionally upregulates claudin-4 expression via a Smad-4-dependent pathway. These and other data, including limited clinical studies, are summarized below and, in the aggregate, suggest a therapeutic role for whey protein in diseases of intestinal barrier dysfunction, perhaps, in part, by regulating claudin expression. © 2013 International Life Sciences Institute.

G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor

International Nuclear Information System (INIS)

Morita, Tsuyoshi; Hayashi, Ken’ichiro

2013-01-01

Highlights: •Tβ4 competed with MRTF-A for G-actin binding. •Tβ4 activated the MRTF–SRF signaling pathway. •Tβ4 increased the endogenous expression of SRF-dependent genes. -- Abstract: Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin–MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF–SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin–MRTFs interaction

G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor

Energy Technology Data Exchange (ETDEWEB)

Morita, Tsuyoshi, E-mail: tsuyo@nbiochem.med.osaka-u.ac.jp; Hayashi, Ken’ichiro

2013-08-02

Highlights: •Tβ4 competed with MRTF-A for G-actin binding. •Tβ4 activated the MRTF–SRF signaling pathway. •Tβ4 increased the endogenous expression of SRF-dependent genes. -- Abstract: Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin–MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF–SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin–MRTFs interaction.

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

Science.gov (United States)

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

2017-06-19

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

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

Science.gov (United States)

Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

2012-01-01

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

Regulation of protein translation initiation in response to ionizing radiation

International Nuclear Information System (INIS)

Trivigno, Donatella; Bornes, Laura; Huber, Stephan M; Rudner, Justine

2013-01-01

Proliferating tumor cells require continuous protein synthesis. De novo synthesis of most proteins is regulated through cap-dependent translation. Cellular stress such as ionizing radiation (IR) blocks cap-dependent translation resulting in shut-down of global protein translation which saves resources and energy needed for the stress response. At the same time, levels of proteins required for stress response are maintained or even increased. The study aimed to analyze the regulation of signaling pathways controlling protein translation in response to IR and the impact on Mcl-1, an anti-apoptotic and radioprotective protein, which levels rapidly decline upon IR. Protein levels and processing were analyzed by Western blot. The assembly of the translational pre-initiation complex was examined by Immunoprecipitation and pull-down experiments with 7-methyl GTP agarose. To analyze IR-induced cell death, dissipation of the mitochondrial membrane potential and DNA fragmentation were determined by flow cytometry. Protein levels of the different initiation factors were down-regulated using RNA interference approach. IR induced caspase-dependent cleavage of the translational initiation factors eIF4G1, eIF3A, and eIF4B resulting in disassembly of the cap-dependent initiation complex. In addition, DAP5-dependent initiation complex that regulates IRES-dependent translation was disassembled in response to IR. Moreover, IR resulted in dephosphorylation of 4EBP1, an inhibitor of cap-dependent translation upstream of caspase activation. However, knock-down of eIF4G1, eIF4B, DAP5, or 4EBP1 did not affect IR-induced decline of the anti-apoptotic protein Mcl-1. Our data shows that cap-dependent translation is regulated at several levels in response to IR. However, the experiments indicate that IR-induced Mcl-1 decline is not a consequence of translational inhibition in Jurkat cells

Regulation of protein translation initiation in response to ionizing radiation

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Trivigno Donatella

2013-02-01

Full Text Available Abstract Background Proliferating tumor cells require continuous protein synthesis. De novo synthesis of most proteins is regulated through cap-dependent translation. Cellular stress such as ionizing radiation (IR blocks cap-dependent translation resulting in shut-down of global protein translation which saves resources and energy needed for the stress response. At the same time, levels of proteins required for stress response are maintained or even increased. The study aimed to analyze the regulation of signaling pathways controlling protein translation in response to IR and the impact on Mcl-1, an anti-apoptotic and radioprotective protein, which levels rapidly decline upon IR. Methods Protein levels and processing were analyzed by Western blot. The assembly of the translational pre-initiation complex was examined by Immunoprecipitation and pull-down experiments with 7-methyl GTP agarose. To analyze IR-induced cell death, dissipation of the mitochondrial membrane potential and DNA fragmentation were determined by flow cytometry. Protein levels of the different initiation factors were down-regulated using RNA interference approach. Results IR induced caspase-dependent cleavage of the translational initiation factors eIF4G1, eIF3A, and eIF4B resulting in disassembly of the cap-dependent initiation complex. In addition, DAP5-dependent initiation complex that regulates IRES-dependent translation was disassembled in response to IR. Moreover, IR resulted in dephosphorylation of 4EBP1, an inhibitor of cap-dependent translation upstream of caspase activation. However, knock-down of eIF4G1, eIF4B, DAP5, or 4EBP1 did not affect IR-induced decline of the anti-apoptotic protein Mcl-1. Conclusion Our data shows that cap-dependent translation is regulated at several levels in response to IR. However, the experiments indicate that IR-induced Mcl-1 decline is not a consequence of translational inhibition in Jurkat cells.

Regulator of G protein signaling 2 (RGS2 and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1 in live cells.

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Sungho Ghil

Full Text Available Protease-activated receptor 1 (PAR1 is a G-protein coupled receptor (GPCR that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET, we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven and either RGS2-Luciferase (RGS2-Luc or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.

Frank-ter Haar syndrome protein Tks4 regulates epidermal growth factor-dependent cell migration.

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Bögel, Gábor; Gujdár, Annamária; Geiszt, Miklós; Lányi, Árpád; Fekete, Anna; Sipeki, Szabolcs; Downward, Julian; Buday, László

2012-09-07

Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.

Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS 2, 3 and 4 in Astrocyte-Like Cells.

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Till Nicolas Eusemann

Full Text Available The "regulators of g-protein signalling" (RGS comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.

Comparison of Danish dichotomous and BI-RADS classifications of mammographic density.

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Hodge, Rebecca; Hellmann, Sophie Sell; von Euler-Chelpin, My; Vejborg, Ilse; Andersen, Zorana Jovanovic

2014-06-01

In the Copenhagen mammography screening program from 1991 to 2001, mammographic density was classified either as fatty or mixed/dense. This dichotomous mammographic density classification system is unique internationally, and has not been validated before. To compare the Danish dichotomous mammographic density classification system from 1991 to 2001 with the density BI-RADS classifications, in an attempt to validate the Danish classification system. The study sample consisted of 120 mammograms taken in Copenhagen in 1991-2001, which tested false positive, and which were in 2012 re-assessed and classified according to the BI-RADS classification system. We calculated inter-rater agreement between the Danish dichotomous mammographic classification as fatty or mixed/dense and the four-level BI-RADS classification by the linear weighted Kappa statistic. Of the 120 women, 32 (26.7%) were classified as having fatty and 88 (73.3%) as mixed/dense mammographic density, according to Danish dichotomous classification. According to BI-RADS density classification, 12 (10.0%) women were classified as having predominantly fatty (BI-RADS code 1), 46 (38.3%) as having scattered fibroglandular (BI-RADS code 2), 57 (47.5%) as having heterogeneously dense (BI-RADS 3), and five (4.2%) as having extremely dense (BI-RADS code 4) mammographic density. The inter-rater variability assessed by weighted kappa statistic showed a substantial agreement (0.75). The dichotomous mammographic density classification system utilized in early years of Copenhagen's mammographic screening program (1991-2001) agreed well with the BI-RADS density classification system.

Down-regulation of Transducin-Like Enhancer of Split protein 4 in hepatocellular carcinoma promotes cell proliferation and epithelial-Mesenchymal-Transition

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Wu, Xiao-cai; Xiao, Cui-cui; Li, Hua [Department of Hepatic Surgery, 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou (China); Guangdong Provincial Key Laboratory of Liver Disease Research, Guangzhou (China); Tai, Yan; Zhang, Qi [Guangdong Provincial Key Laboratory of Liver Disease Research, Guangzhou (China); Yang, Yang, E-mail: yysysu2@163.com [Department of Hepatic Surgery, 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou (China)

2016-08-19

Background: Transducin-Like Enhancer of Split protein 4 (TLE4) has been reported to be involved in some subsets of acute myeloid leukemia and colorectal cancer. In the present study, we aimed to explore the role of TLE4 in tumorigenesis and cancer progression in hepatocellular carcinoma (HCC). Methods: The expression pattern of TLE4 in HCC was determined by Western-blot and qRT-PCR, gain-of-function and loss-of-function was used to explore the biological role of TLE4 in HCC cells. A xenograft model was established to confirm its effects on proliferation. Results: The protein expression levels of TLE4 were significantly down-regulated in HCC tissues compared to matched adjacent normal liver tissues. In vitro, down-regulation of TLE4 in Huh7 or SMMC-7721 promoted cell proliferation and ectopical expression of TLE4 in Hep3B or Bel-7404 suppressed cell proliferation. In addition, the cell colony formation ability was enhanced after down-regulation of TLE4 expression in Huh-7 but suppressed after over-expression in Hep3B. Furthermore, down-regulation of TLE4 increased the cell invasion ability, as well as increased the expression level of Vimentin and decreased that of E-cadherin, indicating a phenotype of epithelial-mesenchymal transition (EMT) in HCC cells. On the contrary, ectopical expression of TLE4 in HCC cells decreased the cell invasion ability and inhibited EMT. In vivo, compared to control group, xenograft tumor volumes were significantly decreased in TLE4 overexpression group. Conclusions: These results demonstrated that TLE4 might play important regulatory roles in cellular proliferation and EMT process in HCC. - Highlights: • TLE4 is significantly down-regulated in HCC samples. • Down regulated of TLE4 in HCC cells promotes cell proliferation. • Down regulated of TLE4 in HCC cells promotes epithelial-to-mesenchymal transition.

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

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Inano, Shojiro; Sato, Koichi; Katsuki, Yoko; Kobayashi, Wataru; Tanaka, Hiroki; Nakajima, Kazuhiro; Nakada, Shinichiro; Miyoshi, Hiroyuki; Knies, Kerstin; Takaori-Kondo, Akifumi; Schindler, Detlev; Ishiai, Masamichi; Kurumizaka, Hitoshi; Takata, Minoru

2017-06-01

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of a breast cancer family double heterozygote for RAD51C and BRCA2 gene mutations

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Ahlborn, Lise B; Steffensen, Ane Y; Jønson, Lars

2015-01-01

for mutations in the RAD51C and BRCA2 genes. The RAD51C missense mutation p.Arg258His has previously been identified in a homozygous state in a patient with Fanconi anemia. This mutation is known to affect the DNA repair function of the RAD51C protein. The BRCA2 p.Leu3216Leu synonymous mutation has not been...

Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51.

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van Heemst, D; Swart, K; Holub, E F; van Dijk, R; Offenberg, H H; Goosen, T; van den Broek, H W; Heyting, C

1997-05-01

We have cloned the uvsC gene of Aspergillus nidulans by complementation of the A. nidulans uvsC114 mutant. The predicted protein UVSC shows 67.4% sequence identity to the Saccharomyces cerevisiae Rad51 protein and 27.4% sequence identity to the Escherichia coli RecA protein. Transcription of uvsC is induced by methyl-methane sulphonate (MMS), as is transcription of RAD51 of yeast. Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant. The coding sequence of the uvsC114 allele has a deletion of 6 bp, which results in deletion of two amino acids and replacement of one amino acid in the translation product. In order to gain more insight into the biological function of the uvsC gene, a uvsC null mutant was constructed, in which the entire uvsC coding sequence was replaced by a selectable marker gene. Meiotic and mitotic phenotypes of a uvsC+ strain, the uvsC114 mutant and the uvsC null mutant were compared. The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant. The uvsC114 mutant arrested in meiotic prophase-I. The uvsC null mutant arrested at an earlier stage, before the onset of meiosis. One possible interpretation of these meiotic phenotypes is that the A. nidulans homologue of Rad51 of yeast has a role both in the specialized processes preceding meiosis and in meiotic prophase I.

Predictive values of Bi-Rads categories 3, 4 and 5 in non-palpable breast masses evaluated by mammography, ultrasound and magnetic resonance imaging; Valores preditivos das categorias 3, 4 e 5 do sistema Bi-Rads em lesoes mamarias nodulares nao-palpaveis avaliadas por mamografia, ultra-sonografia e ressonancia magnetica

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Roveda Junior, Decio; Fleury, Eduardo de Castro Faria [Santa Casa de Misericordia de Sao Paulo, SP (Brazil). Faculdade de Ciencias Medicas. Servico de Diagnostico por Imagem]. E-mail: decio.jr@uol.com.br; Piato, Sebastiao [Santa Casa de Misericordia de Sao Paulo, SP (Brazil). Dept. de Obstetricia e Ginecologia. Clinica Ginecologica; Oliveira, Vilmar Marques de [Santa Casa de Misericordia de Sao Paulo, SP (Brazil). Dept. de Obstetricia e Ginecologia. Ginecologia Geral; Rinaldi, Jose Francisco [Santa Casa de Misericordia de Sao Paulo, SP (Brazil). Dept. de Obstetricia e Ginecologia. Clinica de Mastologia; Ferreira, Carlos Alberto Pecci [Santa Casa de Misericordia de Sao Paulo, SP (Brazil). Faculdade de Ciencias Medicas. Dept. de Clinica Medica. Servico de Imagenologia Mamaria

2007-03-15

Objective: To evaluate the predictive value of BI-RADS{sup TM} categories 3, 4 and 5 in non-palpable breast masses assessed by mammography, ultrasound and magnetic resonance imaging. Materials And Methods: Twenty-nine patients with BI-RADS categories 3, 4 and 5 non-palpable breast masses identified by mammograms were submitted to complementary ultrasound and magnetic resonance imaging studies, besides excisional biopsy. In total, 30 biopsies were performed. The lesions as well as their respective BI-RADS classification into 3, 4 and 5 were correlated with the histopathological results. The predictive values calculation was made by means of specific mathematical equations. Results: Negative predictive values for category 3 were: mammography, 69.23%; ultrasound, 70.58%; and magnetic resonance imaging, 100%. Positive predictive values for category 4 were: mammography, 63.63%; ultrasound, 50%; and magnetic resonance imaging, 30.76%. For category 5, positive predictive values were: mammography and ultrasound, 100%; and magnetic resonance imaging, 92.85%. Conclusion: For category 3, the negative predictive value of magnetic resonance imaging was high, and for categories 4 and 5, the positive predictive values of the three modalities were moderate. (author)

Identification and functional characterisation of Complement Regulator Acquiring Surface Protein-1 of serum resistant Borrelia garinii OspA serotype 4

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Zipfel Peter F

2010-02-01

Full Text Available Abstract Background B. burgdorferi sensu lato (sl is the etiological agent of Lyme borreliosis in humans. Spirochetes have adapted themselves to the human immune system in many distinct ways. One important immune escape mechanism for evading complement activation is the binding of complement regulators Factor H (CFH or Factor H-like protein1 (FHL-1 to Complement Regulator-Acquiring Surface Proteins (CRASPs. Results We demonstrate that B. garinii OspA serotype 4 (ST4 PBi resist complement-mediated killing by binding of FHL-1. To identify the primary ligands of FHL-1 four CspA orthologs from B. garinii ST4 PBi were cloned and tested for binding to human CFH and FHL-1. Orthologs BGA66 and BGA71 were found to be able to bind both complement regulators but with different intensities. In addition, all CspA orthologs were tested for binding to mammalian and avian CFH. Distinct orthologs were able to bind to CFH of different animal origins. Conclusions B. garinii ST4 PBi is able to evade complement killing and it can bind FHL-1 to membrane expressed proteins. Recombinant proteins BGA66 can bind FHL-1 and human CFH, while BGA71 can bind only FHL-1. All recombinant CspA orthologs from B. garinii ST4 PBi can bind CFH from different animal origins. This partly explains the wide variety of animals that can be infected by B. garinii.

Kinase-Mediated Regulation of P4-ATPases

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Frøsig, Merethe Mørch

designed a fast and efficient screening strategy to identify novel regulator proteins of P4-ATPases. The system is based on heterologous expression in a specially designed yeast strain, and regulatory proteins can be identified via change in activity of the P4-ATPase of interest. Hereby the first steps...

Human RAD18 interacts with ubiquitylated chromatin components and facilitates RAD9 recruitment to DNA double strand breaks.

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Akiko Inagaki

Full Text Available RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY and female (XX cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2.

RadConEd: A Graphical Data Editor for the Radiological Consequences Model, RadCon

International Nuclear Information System (INIS)

Crawford, J.; Domel, R.U.

2000-05-01

This document describes the application, RadConEd, which has been designed and implemented to enable users of the RadCon system to update these parameter files. The RadCon system is written in the Java programming language, and as such provides portability across computer platforms. The software described in this report was developed in line with the portability requirements of RadCon, thus providing a uniform user interface across computer platforms and bypassing the need of using system editors. In addition a number of data integrity measures were implemented

A miR-590/Acvr2a/Rad51b Axis Regulates DNA Damage Repair during mESC Proliferation

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Qidong Liu

2014-12-01

Full Text Available Embryonic stem cells (ESCs enable rapid proliferation that also causes DNA damage. To maintain genomic stabilization during rapid proliferation, ESCs must have an efficient system to repress genotoxic stress. Here, we show that withdrawal of leukemia inhibitory factor (LIF, which maintains the self-renewal capability of mouse ESCs (mESCs, significantly inhibits the cell proliferation and DNA damage of mESCs and upregulates the expression of miR-590. miR-590 promotes single-strand break (SSB and double-strand break (DSB damage repair, thus slowing proliferation of mESCs without influencing stemness. miR-590 directly targets Activin receptor type 2a (Acvr2a to mediate Activin signaling. We identified the homologous recombination-mediated repair (HRR gene, Rad51b, as a downstream molecule of the miR-590/Acvr2a pathway regulating the SSB and DSB damage repair and cell cycle. Our study shows that a miR-590/Acvr2a/Rad51b signaling axis ensures the stabilization of mESCs by balancing DNA damage repair and rapid proliferation during self-renewal.

DNA repair genes RAD52 and SRS2, a cell wall synthesis regulator gene SMI1, and the membrane sterol synthesis scaffold gene ERG28 are important in efficient Agrobacterium-mediated yeast transformation with chromosomal T-DNA.

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Ohmine, Yuta; Satoh, Yukari; Kiyokawa, Kazuya; Yamamoto, Shinji; Moriguchi, Kazuki; Suzuki, Katsunori

2016-04-02

Plant pathogenic Agrobacterium strains can transfer T-DNA regions of their Ti plasmids to a broad range of eukaryotic hosts, including fungi, in vitro. In the recent decade, the yeast Saccharomyces cerevisiae is used as a model host to reveal important host proteins for the Agrobacterium-mediated transformation (AMT). Further investigation is required to understand the fundamental mechanism of AMT, including interaction at the cell surface, to expand the host range, and to develop new tools. In this study, we screened a yeast mutant library for low AMT mutant strains by advantage of a chromosome type T-DNA, which transfer is efficient and independent on integration into host chromosome. By the mutant screening, we identified four mutant strains (srs2Δ, rad52Δ, smi1Δ and erg28Δ), which showed considerably low AMT efficiency. Structural analysis of T-DNA product replicons in AMT colonies of mutants lacking each of the two DNA repair genes, SRS2 and RAD52, suggested that the genes act soon after T-DNA entry for modification of the chromosomal T-DNA to stably maintain them as linear replicons and to circularize certain T-DNA simultaneously. The cell wall synthesis regulator SMI1 might have a role in the cell surface interaction between the donor and recipient cells, but the smi1Δ mutant exhibited pleiotropic effect, i.e. low effector protein transport as well as low AMT for the chromosomal T-DNA, but relatively high AMT for integrative T-DNAs. The ergosterol synthesis regulator/enzyme-scaffold gene ERG28 probably contributes by sensing a congested environment, because growth of erg28Δ strain was unaffected by the presence of donor bacterial cells, while the growth of the wild-type and other mutant yeast strains was suppressed by their presence. RAD52 and the DNA helicase/anti-recombinase gene SRS2 are necessary to form and maintain artificial chromosomes through the AMT of chromosomal T-DNA. A sterol synthesis scaffold gene ERG28 is important in the high

Post-transcriptional regulation of MRE11 expression in muscle-invasive bladder tumours.

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Martin, Rebecca M; Kerr, Martin; Teo, Mark T W; Jevons, Sarah J; Koritzinsky, Marianne; Wouters, Bradly G; Bhattarai, Selina; Kiltie, Anne E

2014-02-28

Predictive assays are needed to help optimise treatment in muscle-invasive bladder cancer, where patients can be treated by either cystectomy or radical radiotherapy. Our finding that low tumour MRE11 expression is predictive of poor response to radiotherapy but not cystectomy was recently independently validated. Here we investigated further the mechanism underlying low MRE11 expression seen in poorly-responding patients. MRE11 RNA and protein levels were measured in 88 bladder tumour patient samples, by real-time PCR and immunohistochemistry respectively, and a panel of eight bladder cancer cell lines was screened for MRE11, RAD50 and NBS1 mRNA and protein expression. There was no correlation between bladder tumour MRE11 protein and RNA scores (Spearman's rho 0.064, p=0.65), suggesting MRE11 is controlled post-transcriptionally, a pattern confirmed in eight bladder cancer cell lines. In contrast, NBS1 and RAD50 mRNA and protein levels were correlated (p=0.01 and p=0.03, respectively), suggesting primary regulation at the level of transcription. MRE11 protein levels were correlated with NBS1 and RAD50 mRNA and protein levels, implicating MRN complex formation as an important determinant of MRE11 expression, driven by RAD50 and NBS1 expression. Our findings of the post-transcriptional nature of the control of MRE11 imply that any predictive assays used in patients need to be performed at the protein level rather than the mRNA level.

Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

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Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

2013-11-01

Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons. © 2013 International Society for Neurochemistry.

Regulators of G-protein signaling 4: modulation of 5-HT1A-mediated neurotransmitter release in vivo.

Science.gov (United States)

Beyer, Chad E; Ghavami, Afshin; Lin, Qian; Sung, Amy; Rhodes, Kenneth J; Dawson, Lee A; Schechter, Lee E; Young, Kathleen H

2004-10-01

Regulators of G-protein signaling (RGS) play a key role in the signal transduction of G-protein-coupled receptors (GPCRs). Specifically, RGS proteins function as GTPase accelerating proteins (GAPs) to dampen or "negatively regulate" GPCR-mediated signaling. Our group recently showed that RGS4 effectively GAPs Galpha(i)-mediated signaling in CHO cells expressing the serotonin-1A (5-HT(1A)) receptor. However, whether a similar relationship exists in vivo has yet to be identified. In present studies, a replication-deficient herpes simplex virus (HSV) was used to elevate RGS4 mRNA in the rat dorsal raphe nuclei (DRN) while extracellular levels of 5-HT in the striatum were monitored by in vivo microdialysis. Initial experiments conducted with noninfected rats showed that acute administration of 8-OH-DPAT (0.01-0.3 mg/kg, subcutaneous [s.c.]) dose dependently decreased striatal levels of 5-HT, an effect postulated to result from activation of somatodendritic 5-HT(1A) autoreceptors in the DRN. In control rats receiving a single intra-DRN infusion of HSV-LacZ, 8-OH-DPAT (0.03 mg/kg, s.c.) decreased 5-HT levels to an extent similar to that observed in noninfected animals. Conversely, rats infected with HSV-RGS4 in the DRN showed a blunted neurochemical response to 8-OH-DPAT (0.03 mg/kg, s.c.); however, increasing the dose to 0.3 mg/kg reversed this effect. Together, these findings represent the first in vivo evidence demonstrating that RGS4 functions to GAP Galpha(i)-coupled receptors and suggest that drug discovery efforts targeting RGS proteins may represent a novel mechanism to manipulate 5-HT(1A)-mediated neurotransmitter release.

LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

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Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

2010-12-03

LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation

Energy Technology Data Exchange (ETDEWEB)

Kotnik Halavaty, Katarina; Regan, Jennifer; Mehta, Kavi; Laimins, Laimonis, E-mail: l-laimins@northwestern.edu

2014-03-15

Human papillomaviruses (HPV) infect stratified epithelia and link their life cycles to epithelial differentiation. The HPV E5 protein plays a role in the productive phase of the HPV life cycle but its mechanism of action is still unclear. We identify a new binding partner of E5, A4, using a membrane-associated yeast-two hybrid system. The A4 protein co-localizes with HPV 31 E5 in perinuclear regions and forms complexes with E5 and Bap31. In normal keratinocytes, A4 is found primarily in basal cells while in HPV positive cells high levels of A4 are seen in both undifferentiated and differentiated cells. Reduction of A4 expression by shRNAs, enhanced HPV genome amplification and increased cell proliferation ability following differentiation but this was not seen in cells lacking E5. Our studies suggest that the A4 protein is an important E5 binding partner that plays a role in regulating cell proliferation ability upon differentiation. - Highlights: • A4 associates with HPV 31 E5 proteins. • A4 is localized to endoplasmic reticulum. • HPV proteins induce A4 expression in suprabasal layers of stratified epithelium. • E5 is important for proliferation ability of differentiating HPV positive cells.

Positive Predictive Value of BI-RADS Categorization in an Asian Population

Directory of Open Access Journals (Sweden)

Yah-Yuen Tan

2004-07-01

Full Text Available The Breast Imaging Reporting And Data System (BI-RADS categorization of mammograms is useful in estimating the risk of malignancy, thereby guiding management decisions. However, in Asian women, in whom breast density is increased, the sensitivity of mammography is correspondingly lower. We sought to determine the positive predictive value of BI-RADS categorization for malignancy in our Asian population and, hence, its value in helping us to choose between the various modalities for breast biopsy. We retrospectively reviewed all patients with occult breast lesions detected on mammography or ultrasound who underwent needle-localization open breast biopsy (NLOB in our institution over a 6-year period. There were 470 biopsies in 427 patients; 16% of lesions were malignant. The positive predictive value of BI-RADS 4 and 5 lesions for cancer was 0.27 and 0.84, respectively. While most BI-RADS 5 mass lesions were invasive cancers, the majority of calcifications in this category were in situ carcinomas. We conclude that BI-RADS remains useful in aiding decision-making for biopsy in our Asian population. Based on positive predictive values, we recommend percutaneous breast biopsy for initial evaluation of lesions categorized as BI-RADS 4 or less. For BI-RADS 5 lesions with microcalcifications, open surgical biopsy as a diagnostic and therapeutic procedure may be more appropriate. In the case of a BI-RADS 5 lesion associated with a mass, initial percutaneous biopsy may be useful for diagnosis, followed by a planned single-stage surgical procedure as necessary.

Analysis list: RAD21 [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ncedbc.jp/kyushu-u/hg19/target/RAD21.1.tsv http://dbarchive.biosciencedbc.jp/kyushu...-u/hg19/target/RAD21.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/RAD21.10.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/RAD21.Blood.tsv,http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/RAD21.Breast.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/RAD21.Dige...stive_tract.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/RAD21.Liver.tsv,http://dbarchive.bioscience

The RadAssessor manual

Energy Technology Data Exchange (ETDEWEB)

Seitz, Sharon L.

2007-02-01

THIS manual will describe the functions and capabilities that are available from the RadAssessor database and will demonstrate how to retrieve and view its information. You’ll learn how to start the database application, how to log in, how to use the common commands, and how to use the online help if you have a question or need extra guidance. RadAssessor can be viewed from any standard web browser. Therefore, you will not need to install any special software before using RadAssessor.

Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing.

Directory of Open Access Journals (Sweden)

Nilesh V Khade

Full Text Available Yeast Rad52 (yRad52 has two important functions at homologous DNA recombination (HR; annealing complementary single-strand DNA (ssDNA molecules and recruiting Rad51 recombinase onto ssDNA (recombination mediator activity. Its human homolog (hRAD52 has a lesser role in HR, and apparently lacks mediator activity. Here we show that yRad52 can load human Rad51 (hRAD51 onto ssDNA complexed with yeast RPA in vitro. This is biochemically equivalent to mediator activity because it depends on the C-terminal Rad51-binding region of yRad52 and on functional Rad52-RPA interaction. It has been reported that the N-terminal two thirds of both yRad52 and hRAD52 is essential for binding to and annealing ssDNA. Although a second DNA binding region has been found in the C-terminal region of yRad52, its role in ssDNA annealing is not clear. In this paper, we also show that the C-terminal region of yRad52, but not of hRAD52, is involved in ssDNA annealing. This suggests that the second DNA binding site is required for the efficient ssDNA annealing by yRad52. We propose an updated model of Rad52-mediated ssDNA annealing.

Dynamic organization of genetic recombination proteins and chromosomes

International Nuclear Information System (INIS)

Essers, J.; Van Cappellen, G.; Van Drunen, E.; Theil, A.; Jaspers, N.N.G.J.; Houtsmuller, A.B.; Vermeulen, W.; Kanaar, R.

2003-01-01

Homologous recombination requires the co-ordinated action of the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DSB induction. We probed the nature of the DNA damage-induced foci in living cells with the use of photobleaching techniques. These foci are not static assemblies of DNA repair proteins. Instead, they are dynamic structures of which Rad51 is a stable core component, while Rad52 and Rad54 reversibly interact with the structure. Furthermore, even though the RAD52 group proteins colocalize in the DNA damage-induced foci, the majority of the proteins are not part of the same multi-protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi-protein complexes, rather than stable holo-complexes, allows greater flexibility during the transaction. In case of DNA repair, for example, it allows cross talk between different DNA repair pathways and coupling to other DNA transactions, such as replication. In addition to the behavior of proteins in living cells, we have tracked chromosomes during cell division. Our results suggest that the relative position of chromosomes in the mother cell is conserved in its daughter cells

Predictive values of Bi-Rads categories 3, 4 and 5 in non-palpable breast masses evaluated by mammography, ultrasound and magnetic resonance imaging

International Nuclear Information System (INIS)

Roveda Junior, Decio; Fleury, Eduardo de Castro Faria; Piato, Sebastiao; Oliveira, Vilmar Marques de; Rinaldi, Jose Francisco; Ferreira, Carlos Alberto Pecci

2007-01-01

Objective: To evaluate the predictive value of BI-RADS TM categories 3, 4 and 5 in non-palpable breast masses assessed by mammography, ultrasound and magnetic resonance imaging. Materials And Methods: Twenty-nine patients with BI-RADS categories 3, 4 and 5 non-palpable breast masses identified by mammograms were submitted to complementary ultrasound and magnetic resonance imaging studies, besides excisional biopsy. In total, 30 biopsies were performed. The lesions as well as their respective BI-RADS classification into 3, 4 and 5 were correlated with the histopathological results. The predictive values calculation was made by means of specific mathematical equations. Results: Negative predictive values for category 3 were: mammography, 69.23%; ultrasound, 70.58%; and magnetic resonance imaging, 100%. Positive predictive values for category 4 were: mammography, 63.63%; ultrasound, 50%; and magnetic resonance imaging, 30.76%. For category 5, positive predictive values were: mammography and ultrasound, 100%; and magnetic resonance imaging, 92.85%. Conclusion: For category 3, the negative predictive value of magnetic resonance imaging was high, and for categories 4 and 5, the positive predictive values of the three modalities were moderate. (author)

Adenovirus Protein E4-ORF1 activation of PI3 kinase reveals differential regulation of downstream effector pathways in adipocytes

OpenAIRE

Chaudhary, Natasha; Gonzalez, Eva; Chang, Sung-Hee; Geng, Fuqiang; Rafii, Shahin; Altorki, Nasser K.; McGraw, Timothy E.

2016-01-01

Insulin activation of phosphatidylinositol 3-kinase (PI3K) regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but...

Regeneration of CFUs in the marrow of mice exposed to 300 rads after having recovered from 950 rads

International Nuclear Information System (INIS)

Kedo, A.; Barone, J.; Fried, W.

1976-01-01

Exposure to 950 rads 60 Co radiation has been reported to cause long-lasting damage to the hematopoietic stroma (HS), although the size of the CFUs population recovers to pre-irradiation levels. In these studies HS damage was detected only after subcutaneously implanting the femurs of the irradiated mice into syngeneic hosts. To exclude the possibility that what was considered to be HS damage was merely caused by artifacts due to the process of implantation in a new host, the rate of regeneration of CFUs in mice which had recovered from 950 rads prior to receiving 300 rads 60 Co radiation (950 + 300 rads group) was compared with that of mice which received only 300 rads (0 + 300 rads group). The CFUs population in the 950 + 300 rads group grew exponentially for 2 weeks at a rate which did not differ significantly from that of CFUs in the 0 + 300 rads group. However, the rate of CFUs growth reached a plateau before full recovery was achieved in contrast to that in the 0 + 300 rads mice. It was therefore concluded that the incomplete regeneration of CFUs in the marrows of 950 + 300 rads mice was most likely caused by X-irradiation-induced damage to the HS rather than damage to the inherent repopulation potential of the CFUs per se. (author)

C. elegans ring finger protein RNF-113 is involved in interstrand DNA crosslink repair and interacts with a RAD51C homolog.

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Hyojin Lee

Full Text Available The Fanconi anemia (FA pathway recognizes interstrand DNA crosslinks (ICLs and contributes to their conversion into double-strand DNA breaks, which can be repaired by homologous recombination. Seven orthologs of the 15 proteins associated with Fanconi anemia are functionally conserved in the model organism C. elegans. Here we report that RNF-113, a ubiquitin ligase, is required for RAD-51 focus formation after inducing ICLs in C. elegans. However, the formation of foci of RPA-1 or FCD-2/FANCD2 in the FA pathway was not affected by depletion of RNF-113. Nevertheless, the RPA-1 foci formed did not disappear with time in the depleted worms, implying serious defects in ICL repair. As a result, RNF-113 depletion increased embryonic lethality after ICL treatment in wild-type worms, but it did not increase the ICL-induced lethality of rfs-1/rad51C mutants. In addition, the persistence of RPA-1 foci was suppressed in doubly-deficient rnf-113;rfs-1 worms, suggesting that there is an epistatic interaction between the two genes. These results lead us to suggest that RNF-113 and RFS-1 interact to promote the displacement of RPA-1 by RAD-51 on single-stranded DNA derived from ICLs.

Rad and Mubad in Shahnameh of Ferdowsi

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z Delpazir

2011-09-01

However, the important points overlooked by explicators are the relationship between Rad and Mubad (Zoroastrian priest and the reason why these two words have co-occurred so frequently in Shahnameh, the most famous Persian national epic. It seems that Rad in Shahnameh, based on Avesta and Pahlavi texts, is often construed as Sadane or Dastoor that was a high position in ancient Iran’s religious hierarchy. Thus, Rads and Mubads were both considered members of religious communities. This study tries to investigate the role and position of Rads and Mubads and their relationship with one another, based on Shahnameh of Ferdowsi, in three chapters: The etymology of Rad Rad in Shahnameh The relationship between Rads and Mubads.

The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus

DEFF Research Database (Denmark)

Torres-Rosell, Jordi; Sunjevaric, Ivana; De Piccoli, Giacomo

2007-01-01

at an extranucleolar site. The nucleolar exclusion of Rad52 recombination foci entails Mre11 and Smc5-Smc6 complexes and depends on Rad52 SUMO (small ubiquitin-related modifier) modification. Remarkably, mutations that abrogate these activities result in the formation of Rad52 foci within the nucleolus and cause r...

Predicted RNA Binding Proteins Pes4 and Mip6 Regulate mRNA Levels, Translation, and Localization during Sporulation in Budding Yeast.

Science.gov (United States)

Jin, Liang; Zhang, Kai; Sternglanz, Rolf; Neiman, Aaron M

2017-05-01

In response to starvation, diploid cells of Saccharomyces cerevisiae undergo meiosis and form haploid spores, a process collectively referred to as sporulation. The differentiation into spores requires extensive changes in gene expression. The transcriptional activator Ndt80 is a central regulator of this process, which controls many genes essential for sporulation. Ndt80 induces ∼300 genes coordinately during meiotic prophase, but different mRNAs within the NDT80 regulon are translated at different times during sporulation. The protein kinase Ime2 and RNA binding protein Rim4 are general regulators of meiotic translational delay, but how differential timing of individual transcripts is achieved was not known. This report describes the characterization of two related NDT80 -induced genes, PES4 and MIP6 , encoding predicted RNA binding proteins. These genes are necessary to regulate the steady-state expression, translational timing, and localization of a set of mRNAs that are transcribed by NDT80 but not translated until the end of meiosis II. Mutations in the predicted RNA binding domains within PES4 alter the stability of target mRNAs. PES4 and MIP6 affect only a small portion of the NDT80 regulon, indicating that they act as modulators of the general Ime2/Rim4 pathway for specific transcripts. Copyright © 2017 American Society for Microbiology.

O-GlcNAc regulates NEDD4-1 stability via caspase-mediated pathway

International Nuclear Information System (INIS)

Jiang, Kuan; Bai, Bingyang; Ta, Yajie; Zhang, Tingling; Xiao, Zikang; Wang, Peng George; Zhang, Lianwen

2016-01-01

O-GlcNAc modification of cytosolic and nuclear proteins regulates essential cellular processes such as stress responses, transcription, translation, and protein degradation. Emerging evidence indicates O-GlcNAcylation has a dynamic interplay with ubiquitination in cellular regulation. Here, we report that O-GlcNAc indirectly targets a vital E3 ubiquitin ligase enzyme of NEDD4-1. The protein level of NEDD4-1 is accordingly decreased following an increase of overall O-GlcNAc level upon PUGNAc or glucosamine stimulation. O-GlcNAc transferase (OGT) knockdown, overexpression and mutation results confirm that the stability of NEDD4-1 is negatively regulated by cellular O-GlcNAc. Moreover, the NEDD4-1 degradation induced by PUGNAc or GlcN is significantly inhibited by the caspase inhibitor. Our study reveals a regulation mechanism of NEDD4-1 stability by O-GlcNAcylation. - Highlights: • Reduced NEDD4-1 correlates with increased overall O-GlcNAc level. • OGT negatively regulates NEDD4-1 stability. • O-GlcNAc regulates NEDD4-1 through caspase-mediated pathway.

Accuracy of CESM versus conventional mammography and ultrasound in evaluation of BI-RADS 3 and 4 breast lesions with pathological correlation

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Maha Helal

2017-09-01

Full Text Available Aim: Assess accuracy of contrast enhanced spectral mammography (CESM versus conventional mammography and ultrasound in evaluation of BI-RADS 3 and 4 breast lesions with pathological correlation. Patients and methods: Thirty female patients with 35 breast lesions diagnosed by conventional imaging as BI-RADS 3 and 4, presented to Women’s Imaging Unit of Radiology Department between January and December 2015, age ranged from 23 to 70 years. All patients underwent conventional mammography and ultrasound then CESM. Results: Patients divided into two groups, benign and malignant lesions group according to histological analysis. Mammography results that malignant lesions detected in 18/35 (51.4% while benign lesions 17/35 (48.6%. Ultrasound revealed 27/35 (77.1% lesions were malignant and 8/35 (22.9% lesions benign. But CESM, revealed 25/35 (71.4% lesions were malignant & 10/35 (28.6% lesions benign. Among 7 patients with multifocal/ multi-centric histologically proven malignant lesions, all detected by CESM 7/7 cases (100% versus 2/7 cases (28.6% and 6/7 cases (85.7% detected by mammography and ultrasound respectively. Based on, CESM had 95.2% sensitivity and 82.9% diagnostic accuracy. Conclusion: CESM has better diagnostic accuracy than mammography alone and mammography plus ultrasound. CESM has 82.9% diagnostic accuracy in comparison to 51.4% for mammography and 77.1% for ultrasound. Keywords: Breast lesions, CESM, BI-RADS lexicon

The role of RAD51 in etoposide (VP16) resistance in small cell lung cancer

DEFF Research Database (Denmark)

Hansen, Lasse Tengbjerg; Lundin, Cecilia; Spang-Thomsen, Mogens

2003-01-01

Etoposide (VP16) is a potent inducer of DNA double-strand breaks (DSBs) and is efficiently used in small cell lung cancer (SCLC) therapy. However, acquired VP16 resistance remains an important barrier to effective treatment. To understand the underlying mechanisms for VP16 resistance in SCLC, we...... investigated DSB repair and cellular VP16 sensitivity of SCLC cells. VP16 sensitivity and RAD51, DNA-PK(cs), topoisomerase IIalpha and P-glycoprotein protein levels were determined in 17 SCLC cell lines. In order to unravel the role of RAD51 in VP16 resistance, we cloned the human RAD51 gene, transfected SCLC...... cells with RAD51 sense or antisense constructs and measured the VP16 resistance. Finally, we measured VP16-induced DSBs in the 17 SCLC cell lines. Two cell lines exhibited a multidrug-resistant phenotype. In the other SCLC cell lines, the cellular VP16 resistance was positively correlated with the RAD51...

Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks.

Science.gov (United States)

Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia; Evangelou, Konstantinos; Da-Ré, Caterina; Huber, Florian; Padayachy, Laura; Tardy, Sebastien; Nicati, Noemie L; Barriot, Samia; Ochs, Fena; Lukas, Claudia; Lukas, Jiri; Gorgoulis, Vassilis G; Scapozza, Leonardo; Halazonetis, Thanos D

2016-12-15

Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Cohesin Rad21 Mediates Loss of Heterozygosity and Is Upregulated via Wnt Promoting Transcriptional Dysregulation in Gastrointestinal Tumors

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Huiling Xu

2014-12-01

Full Text Available Summary: Loss of heterozygosity (LOH of the adenomatous polyposis coli (APC gene triggers a series of molecular events leading to intestinal adenomagenesis. Haploinsufficiency of the cohesin Rad21 influences multiple initiating events in colorectal cancer (CRC. We identify Rad21 as a gatekeeper of LOH and a β-catenin target gene and provide evidence that Wnt pathway activation drives RAD21 expression in human CRC. Genome-wide analyses identified Rad21 as a key transcriptional regulator of critical CRC genes and long interspersed element (LINE-1 or L1 retrotransposons. Elevated RAD21 expression tracks with reactivation of L1 expression in human sporadic CRC, implicating cohesin-mediated L1 expression in global genomic instability and gene dysregulation in cancer. : Rad21 holds the cohesin complex together as part of its role in chromosome partitioning and DNA repair. Xu et al. identify Rad21 as a key mediator of Apc gene heterozygous loss, the event initiating intestinal tumorigenesis. The subsequent activation of the Wnt pathway further induces Rad21, global gene dysregulation, chromosome instability, and pervasive retrotransposon activation.

RAD51 and RTEL1 compensate telomere loss in the absence of telomerase.

Science.gov (United States)

Olivier, Margaux; Charbonnel, Cyril; Amiard, Simon; White, Charles I; Gallego, Maria E

2018-03-16

Replicative erosion of telomeres is naturally compensated by telomerase and studies in yeast and vertebrates show that homologous recombination can compensate for the absence of telomerase. We show that RAD51 protein, which catalyzes the key strand-invasion step of homologous recombination, is localized at Arabidopsis telomeres in absence of telomerase. Blocking the strand-transfer activity of the RAD51 in telomerase mutant plants results in a strikingly earlier onset of developmental defects, accompanied by increased numbers of end-to-end chromosome fusions. Imposing replication stress through knockout of RNaseH2 increases numbers of chromosome fusions and reduces the survival of these plants deficient for telomerase and homologous recombination. This finding suggests that RAD51-dependent homologous recombination acts as an essential backup to the telomerase for compensation of replicative telomere loss to ensure genome stability. Furthermore, we show that this positive role of RAD51 in telomere stability is dependent on the RTEL1 helicase. We propose that a RAD51 dependent break-induced replication process is activated in cells lacking telomerase activity, with RTEL1 responsible for D-loop dissolution after telomere replication.

Regulation of microtubule-based transport by MAP4

Science.gov (United States)

Semenova, Irina; Ikeda, Kazuho; Resaul, Karim; Kraikivski, Pavel; Aguiar, Mike; Gygi, Steven; Zaliapin, Ilya; Cowan, Ann; Rodionov, Vladimir

2014-01-01

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2–dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2–based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2–dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect. PMID:25143402

Brca2 C-terminus interacts with Rad51 and contributes to nuclear forcus formation in double-strand break repair of DNA

International Nuclear Information System (INIS)

Ochiai, Kazuhiko; Morimatsu, Masami; Yoshikawa, Yasunaga; Syuto, Bunei; Hashizume, Kazuyoshi

2004-01-01

In humans and mice, the interaction between the breast cancer susceptibility protein, Brca2, and Rad51 recombinase is essential for DNA repair by homologous recombination, the failure of this process can predispose to cancer. Cells with mutated Brca2 are hypersensitive to ionizing radiation (IR) and exhibit defective DNA repair. Using yeast and mammalian two-hybrid assays, we demonstrate that canine Rad51 protein interacts specifically with the C-terminus of canine Brca2. In support of the biological significance of this interaction, we found that radiation-induced focus formation of Rad51 in COS-7 cells was compromised by forced expression of the C-terminus of canine Brca2. A similar result was obtained for the murine C-terminus. These data suggest that the C-terminal domain of canine Brca2 functions to bind Rad51 and that this domain contributes to the IR-induced assembly of the Rad51 complex in vivo. (author)

Influence of different inhibitors on the activity of the RAD54 dependent step of DNA repair in Saccharomyces cerevisiae

Energy Technology Data Exchange (ETDEWEB)

Siede, W.; Obermaier, S.; Eckhardt, F.

1985-01-01

The recombinagenic pathway of DNA repair in yeast was characterized by the effect of different inhibitors on the temperature-dependent survival after ..gamma..-irradiation in haploid cells of the thermoconditional mutant rad54-3. Blocking protein synthesis with cycloheximide in replicating cells caused partial inhibition of the RAD54 dependent function but some repair activity remained detectable. This indicates that ..gamma..-rays can induce RAD54 activity above some constitutive level of function. Inhibition of DNA replication by hydroxyurea efficiently blocked the RAD54 dependent function in stationary-phase cells but not in logarithmic-phase cells. In logarithmic-phase cells, the authors found a strong inhibitory effect of caffeine on the RAD54 mediated repair process.

Early Postoperative Low Expression of RAD50 in Rectal Cancer Patients Associates with Disease-Free Survival

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Vincent Ho

2017-11-01

Full Text Available Background: Molecular biomarkers have the potential to predict response to the treatment of rectal cancer. In this study, we aimed to evaluate the prognostic and clinicopathological implication of RAD50 (DNA repair protein RAD50 homolog expression in rectal cancer. Methods: A total of 266 rectal cancer patients who underwent surgery and received chemo- and radiotherapy between 2000 and 2011 were involved in the study. Postoperative RAD50 expression was determined by immunohistochemistry in surgical samples (n = 266. Results: Using Kaplan–Meier survival analysis, we found that low RAD50 expression in postoperative samples was associated with worse disease free survival (p = 0.001 and overall survival (p < 0.001 in early stage/low-grade tumors. In a comparison of patients with low vs. high RAD50 expression, we found that low levels of postoperative RAD50 expression in rectal cancer tissues were significantly associated with perineural invasion (p = 0.002. Conclusion: Expression of RAD50 in rectal cancer may serve as a prognostic biomarker for long-term survival of patients with perineural invasion-positive tumors and for potential use in early stage and low-grade rectal cancer assessment.

FBH1 helicase disrupts RAD51 filaments in vitro and modulates homologous recombination in mammalian cells

DEFF Research Database (Denmark)

Simandlova, Jitka; Zagelbaum, Jennifer; Payne, Miranda J

2013-01-01

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD......51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences...... filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under...

Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

NARCIS (Netherlands)

Koppelman, S.J.

1995-01-01

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

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

International Nuclear Information System (INIS)

Schiestl, R.H.; Prakash, S.; Prakash, L.

1990-01-01

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 (rad6Δ) mutations and show that they also suppress the γ-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 γ-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 rad6Δ is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6Δ 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

Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily.

Directory of Open Access Journals (Sweden)

Aleksander F Sikorski

2007-01-01

Full Text Available The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type, 4.1B (brain type, and 4.1N (neuron type, and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK, non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.

The F-box protein Cdc4/Fbxw7 is a novel regulator of neural crest development in Xenopus laevis

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Hartley Rebecca S

2010-01-01

Full Text Available Abstract Background The neural crest is a unique population of cells that arise in the vertebrate ectoderm at the neural plate border after which they migrate extensively throughout the embryo, giving rise to a wide range of derivatives. A number of proteins involved in neural crest development have dynamic expression patterns, and it is becoming clear that ubiquitin-mediated protein degradation is partly responsible for this. Results Here we demonstrate a novel role for the F-box protein Cdc4/Fbxw7 in neural crest development. Two isoforms of Xenopus laevis Cdc4 were identified, and designated xCdc4α and xCdc4β. These are highly conserved with vertebrate Cdc4 orthologs, and the Xenopus proteins are functionally equivalent in terms of their ability to degrade Cyclin E, an established vertebrate Cdc4 target. Blocking xCdc4 function specifically inhibited neural crest development at an early stage, prior to expression of c-Myc, Snail2 and Snail. Conclusions We demonstrate that Cdc4, an ubiquitin E3 ligase subunit previously identified as targeting primarily cell cycle regulators for proteolysis, has additional roles in control of formation of the neural crest. Hence, we identify Cdc4 as a protein with separable but complementary functions in control of cell proliferation and differentiation.

FRPR-4 Is a G-Protein Coupled Neuropeptide Receptor That Regulates Behavioral Quiescence and Posture in Caenorhabditis elegans.

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Matthew D Nelson

Full Text Available Neuropeptides signal through G-protein coupled receptors (GPCRs to regulate a broad array of animal behaviors and physiological processes. The Caenorhabditis elegans genome encodes approximately 100 predicted neuropeptide receptor GPCRs, but in vivo roles for only a few have been identified. We describe here a role for the GPCR FRPR-4 in the regulation of behavioral quiescence and locomotive posture. FRPR-4 is activated in cell culture by several neuropeptides with an amidated isoleucine-arginine-phenylalanine (IRF motif or an amidated valine-arginine-phenylalanine (VRF motif at their carboxy termini, including those encoded by the gene flp-13. Loss of frpr-4 function results in a minor feeding quiescence defect after heat-induced cellular stress. Overexpression of frpr-4 induces quiescence of locomotion and feeding as well as an exaggerated body bend posture. The exaggerated body bend posture requires the gene flp-13. While frpr-4 is expressed broadly, selective overexpression of frpr-4 in the proprioceptive DVA neurons results in exaggerated body bends that require flp-13 in the ALA neuron. Our results suggest that FLP-13 and other neuropeptides signal through FRPR-4 and other receptors to regulate locomotion posture and behavioral quiescence.

Compensatory role for Rad52 during recombinational repair in Ustilago maydis

DEFF Research Database (Denmark)

Kojic, Milorad; Mao, Ninghui; Zhou, Qingwen

2008-01-01

A single Rad52-related protein is evident by blast analysis of the Ustilago maydis genome database. Mutants created by disruption of the structural gene exhibited few discernible defects in resistance to UV, ionizing radiation, chemical alkylating or cross-linking agents. No deficiency was noted...

UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast.

Science.gov (United States)

Lawrence, C W; O'Brien, T; Bond, J

1984-01-01

The UV-induced reversion of two his4 frameshift alleles was much reduced in rad6 mutants of Saccharomyces cerevisiae, an observation that is consistent with the hypothesis that RAD6 function is required for the induction of all types of genetic alteration in misrepair mutagenesis. The reversion of these his4 alleles, together with two others of the same type, was also reduced in rev1 and rev3 mutant strains; in these, however, the extent of the reduction varied considerably with test allele used, in a manner analogous to the results in these strains for base repair substitution test alleles. The general features of UV-induced frameshift and substitution mutagenesis therefore appear quite similar, indicating that they may depend on related processes. If this conclusion is correct, greater attention must be given to integrating models which account for the production of nucleotide additions and deletions into those concerning misrepair mutagenesis.

Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

KAUST Repository

Fornander, Louise H; Frykholm, Karolin; Reymer, Anna; Renodon-Corniè re, Axelle; Takahashi, Masayuki; Nordé n, Bengt

2012-01-01

Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated

Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats.

Science.gov (United States)

Zargar, Sana; Moreira, Tracy S; Samimi-Seisan, Helena; Jeganathan, Senthure; Kakade, Dhanshri; Islam, Nushaba; Campbell, Jonathan; Adegoke, Olasunkanmi A J

2011-06-01

Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.

Recruitment kinetics of DNA repair proteins Mdc1 and Rad52 but not 53BP1 depend on damage complexity.

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Volker Hable

Full Text Available The recruitment kinetics of double-strand break (DSB signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/µm to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET = 2.6 keV/µm to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T(0 after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ(1. Mdc1 accumulates faster (T(0 = 17 ± 2 s, τ(1 = 98 ± 11 s than 53BP1 (T(0 = 77 ± 7 s, τ(1 = 310 ± 60 s after high LET irradiation. However, recruitment of Mdc1 slows down (T(0 = 73 ± 16 s, τ(1 = 1050 ± 270 s after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ(1 of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies.

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

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Jeong-Min Park

2016-11-01

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

The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4α

International Nuclear Information System (INIS)

Klapper, Maja; Boehme, Mike; Nitz, Inke; Doering, Frank

2007-01-01

The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4α (HNF-4α), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4α binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4α by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4α, that are both candidate genes for diabetes type 2, may be a powerful approach

The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4{alpha}

Energy Technology Data Exchange (ETDEWEB)

Klapper, Maja [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Boehme, Mike [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Nitz, Inke [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Doering, Frank [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany)

2007-04-27

The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4{alpha} binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4{alpha} by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4{alpha}, that are both candidate genes for diabetes type 2, may be a powerful approach.

Human pentraxin 3 binds to the complement regulator c4b-binding protein.

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Anne Braunschweig

Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

RadCat 2.0 User Guide.

Energy Technology Data Exchange (ETDEWEB)

Osborn, Douglas.; Weiner, Ruth F.; Mills, George Scott; Hamp, Steve C.; O' Donnell, Brandon, M.; Orcutt, David J.; Heames, Terence J.; Hinojosa, Daniel

2005-01-01

This document provides a detailed discussion and a guide for the use of the RadCat 2.0 Graphical User Interface input file generator for the RADTRAN 5.5 code. The differences between RadCat 2.0 and RadCat 1.0 can be attributed to the differences between RADTRAN 5 and RADTRAN 5.5 as well as clarification for some of the input parameters. 3

The neuronal PAS domain protein 4 (Npas4 is required for new and reactivated fear memories.

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Jonathan E Ploski

Full Text Available The Neuronal PAS domain protein 4 (Npas4 is a neuronal activity-dependent immediate early gene that has recently been identified as a transcription factor which regulates the transcription of genes that control inhibitory synapse development and synaptic plasticity. The role Npas4 in learning and memory, however, is currently unknown. Here, we systematically examine the role of Npas4 in auditory Pavlovian fear conditioning, an amygdala-dependent form of emotional learning. In our first series of experiments, we show that Npas4 mRNA and protein are regulated in the rat lateral nucleus of the amygdala (LA in a learning-dependent manner. Further, knockdown of Npas4 protein in the LA via adeno-associated viral (AAV mediated gene delivery of RNAi was observed to impair fear memory formation, while innate fear and the expression of fear memory were not affected. In our second series of experiments, we show that Npas4 protein is regulated in the LA by retrieval of an auditory fear memory and that knockdown of Npas4 in the LA impairs retention of a reactivated, but not a non-reactivated, fear memory. Collectively, our findings provide the first comprehensive look at the functional role of Npas4 in learning and memory.

Diagnostic value of commercially available shear-wave elastography for breast cancers: integration into BI-RADS classification with subcategories of category 4.

Science.gov (United States)

Youk, Ji Hyun; Gweon, Hye Mi; Son, Eun Ju; Han, Kyung Hwa; Kim, Jeong-Ah

2013-10-01

To evaluate the diagnostic performance of shear-wave elastography (SWE) for breast cancer and to determine whether the integration of SWE into BI-RADS with subcategories of category 4 improves the diagnostic performance. A total of 389 breast masses (malignant 120, benign 269) in 324 women who underwent SWE before ultrasound-guided core biopsy or surgery were included. The qualitative SWE feature was assessed using a four-colour overlay pattern. Quantitative elasticity values including the lesion-to-fat elasticity ratio (Eratio) were measured. Diagnostic performance of B-mode ultrasound, SWE, or their combined studies was compared using the area under the ROC curve (AUC). AUC of Eratio (0.952) was the highest among elasticity values (mean, maximum, and minimum elasticity, 0.949, 0.939, and 0.928; P = 0.04) and AUC of colour pattern was 0.947. AUC of combined studies was significantly higher than for a single study (P Shear-wave elastography showed a good diagnostic performance. Adding SWE features to BI-RADS improved the diagnostic performance and may be helpful to stratify category 4 lesions. • Quantitative and qualitative shear-wave elastography provides further diagnostic information during breast ultrasound. • The elasticity ratio (E ratio ) showed the best diagnostic performance in SWE. • E ratio and four-colour overlay pattern significantly differed between benign and malignant lesions. • SWE features allowed further stratification of BI-RADS category 4 lesions.

Diagnostic value of breast ultrasound in mammography BI-RADS 0 and clinically indeterminate or suspicious of malignancy breast lesions

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Dobrosavljević Aleksandar

2016-01-01

Full Text Available Background/Aim. Not only that ultrasound makes the difference between cystic and solid changes in breast tissue, as it was the case at the beginning of its use, but it also makes the differential diagnosis in terms of benign-malignant. The aim of this study was to assess the role of sonography in the diagnosis of palpable breast masses according to the American College of Radiology Ultrasonographic Breast Imaging Reporting and Data System (BI-RADS and to correlate the BI-RADS 4 and BI-RADS 5 category with pathohistological findings. Methods. A retrospective study was conducted with the breast sonograms of 30 women presented with palpable breast masses found to be mammography category BI-RADS 0 and ultrasonographic BI-RADS categories 4 and 5. The sonographic categories were correlated with pathohistological findings. Results. Surgical biopsy in 30 masses revealed: malignancy (56.7%, fibroadenoma (26.7%, fibrocystic dysplasia with/without atypia (10%, lipoma (3.3% and intramammary lymph node (3.3%. Correlation between BI-RADS categories and pathohistological findings was found (p < 0.05. All BI-RADS 5 masses were malignant, while in BI-RADS 4A category fibroadenomas dominated. A total of 53.8% of all benign lesions were found in women 49 years of age or younger as compared with 35.3% of all malignancies in this group (p < 0.05. Conclusion. Ultrasonography BI-RADS improved classification of breast masses. The ultrasound BI-RADS 4 (A, B, C and BI-RADS 5 lesions should be worked-up with biopsy.

Adenovirus Protein E4-ORF1 Activation of PI3 Kinase Reveals Differential Regulation of Downstream Effector Pathways in Adipocytes.

Science.gov (United States)

Chaudhary, Natasha; Gonzalez, Eva; Chang, Sung-Hee; Geng, Fuqiang; Rafii, Shahin; Altorki, Nasser K; McGraw, Timothy E

2016-12-20

Insulin activation of phosphatidylinositol 3-kinase (PI3K) regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but not regulation of Glut4. This uncoupling of PI3K effects occurs despite E4-ORF1 activating PI3K and downstream signaling to levels achieved by insulin. Although E4-ORF1 does not fully recapitulate insulin's effects on Glut4, it enhances insulin-stimulated insertion of Glut4-containing vesicles to the plasma membrane independent of Rab10, a key regulator of Glut4 trafficking. E4-ORF1 also stimulates plasma membrane translocation of ubiquitously expressed Glut1 glucose transporter, an effect that is likely essential for E4-ORF1 to promote an anabolic metabolism in a broad range of cell types. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Adenovirus Protein E4-ORF1 Activation of PI3 Kinase Reveals Differential Regulation of Downstream Effector Pathways in Adipocytes

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Natasha Chaudhary

2016-12-01

Full Text Available Insulin activation of phosphatidylinositol 3-kinase (PI3K regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but not regulation of Glut4. This uncoupling of PI3K effects occurs despite E4-ORF1 activating PI3K and downstream signaling to levels achieved by insulin. Although E4-ORF1 does not fully recapitulate insulin’s effects on Glut4, it enhances insulin-stimulated insertion of Glut4-containing vesicles to the plasma membrane independent of Rab10, a key regulator of Glut4 trafficking. E4-ORF1 also stimulates plasma membrane translocation of ubiquitously expressed Glut1 glucose transporter, an effect that is likely essential for E4-ORF1 to promote an anabolic metabolism in a broad range of cell types.

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5′-DNA end

Science.gov (United States)

Sato, Koichi; Shimomuki, Mayo; Katsuki, Yoko; Takahashi, Daisuke; Kobayashi, Wataru; Ishiai, Masamichi; Miyoshi, Hiroyuki; Takata, Minoru; Kurumizaka, Hitoshi

2016-01-01

The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork. PMID:27694619

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs

OpenAIRE

Rong, Liwei; Livingstone, Mark; Sukarieh, Rami; Petroulakis, Emmanuel; Gingras, Anne-Claude; Crosby, Katherine; Smith, Bradley; Polakiewicz, Roberto D.; Pelletier, Jerry; Ferraiuolo, Maria A.; Sonenberg, Nahum

2008-01-01

Eukaryotic initiation factor (eIF) 4E, the mRNA 5′-cap-binding protein, mediates the association of eIF4F with the mRNA 5′-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported...

RadCon: A Radiological Consequences Model

International Nuclear Information System (INIS)

Crawford, J.; Domel, R.U.

2000-05-01

RadCon estimates the dose received by user selected groups in the population from an accidental release of radionuclides to the environment. The exposure pathways considered are external exposure from the cloud and ground and internal exposure from inhalation and ingestion of contaminated food. Atmospheric dispersion modelling is carried out externally to RadCon.Given a two dimensional time varying air and ground concentration of radioactive elements, RadCon allows the user to: view the air and ground concentration over the affected area, select optional parameters and calculate the dose to people,display the results to the user, and change the parameter values. RadCon offers two user interfaces: 1) the standard graphical user interface which is started using Java DoseApp at the command line, or by setting up a shortcut to this command (particularly when RadCon is installed on a PC) and 2) the text based interface used to generate information for the model inter-comparison exercise . This is initiated using Java BIOMASS at the command line, or an equivalent shortcut. The text based interface was developed for research purposes and is not generally available. Appendices A, B and C provide a summary of instructions on setting up RadCon. This will generally be carried out by the computer support personnel

Curcumin enhances the mitomycin C-induced cytotoxicity via downregulation of MKK1/2-ERK1/2-mediated Rad51 expression in non-small cell lung cancer cells

International Nuclear Information System (INIS)

Ko, Jen-Chung; Tsai, Min-Shao; Weng, Shao-Hsing; Kuo, Ya-Hsun; Chiu, Yu-Fan; Lin, Yun-Wei

2011-01-01

Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been reported to suppress the proliferation of a wide variety of tumor cells. Rad51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. A high expression of Rad51 has been reported in chemo- or radio-resistant carcinomas. Therefore, in the current study, we will examine whether curcumin could enhance the effects of mitomycin C (MMC), a DNA interstrand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression. Exposure of two human non-small lung cancer (NSCLC) cell lines (A549 and H1975) to curcumin could suppress MMC-induced MKK1/2-ERK1/2 signal activation and Rad51 protein expression. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased Rad51 protein levels in curcumin and MMC co-treated human lung cells. Moreover, the synergistic cytotoxic effect induced by curcumin combined with MMC was decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. In contrast, MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through downregulation of ERK1/2 activation and Rad51 expression. Depletion of endogenous Rad51 expression by siRad51 RNA transfection significantly enhanced MMC and/or curcumin induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. We concluded that Rad51 inhibition may be an additional action mechanism for enhancing the chemosensitization of MMC by curcumin in NSCLC. - Highlights: → Curcumin downregulates MKK-ERK-mediated Rad51 expression. → Curcumin enhances mitomycin C-induced cytotoxicity. → Rad51 protects cells from cytotoxic effects induced by curcumin and mitomycin C. → Rad51 inhibition enhances the chemosensitization of

Rad51 expression levels predict synthetic lethality and metastatic potential in high grade breast cancers

International Nuclear Information System (INIS)

Wiegmans, A.P.; Al-Ejeh, F.; Khanna, K.K.

2012-01-01

Among women with breast cancer, 30-40% will develop metastatic disease and only achieve an overall survival of less than 5 years. Despite new-targeted therapy, breast tumors that harbour similar histology or molecular phenotype differ in their response to treatment. To uncover potential new therapeutic targets and improve outcome, we performed data mining of cancer micro array databases. We found that high expression of the homologous recombination protein, RAD51, was significantly associated with high-grade breast cancer, aggressive subtypes and increased risk of metastasis. We confirmed using immunohistochemistry that RAD5 1 was highly expressed in metastatic tumours and high-grade triple negative, HER2+ and luminal-B tumours. This provided a rationale for targeting RAD5 1 in high-grade, therapy-resistant breast cancers. Here, we report for the first time preclinical evaluation of RAD5 1 as a therapeutic target. We found that, in-vitro high RAD5 expressing cell lines were resistant to PARP inhibitor while knockdown reversed this resistance. In-vivo, knockdown of RAD5 1 inhibited metastatic progression using a syngeneic breast cancer model and the seeding of human xenografts to distant sites, including brain and lung. Concurrent PARP inhibition reduced primary tumor growth and delayed metastasis supporting synthetic lethality in-vivo. Together these insights provide pre-clinical data demonstrating RAD5 1 as a new biomarker and potential therapeutic target against aggressive metastatic breast cancer. (author)

Rad50S alleles of the Mre11 complex: questions answered and questions raised.

Science.gov (United States)

Usui, Takehiko; Petrini, John H J; Morales, Monica

2006-08-15

We find that Rad50S mutations in yeast and mammals exhibit constitutive PIKK (PI3-kinase like kinase)-dependent signaling [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7 (2001) 1255-1266.; M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-4354.]. The signaling depends on Mre11 complex functions, consistent with its role as a DNA damage sensor. Rad50S is distinct from hypomorphic mutations of Mre11 and Nbs1 in mammals [M. Morales, J.W. Theunissen, C.F. Kim, R. Kitagawa, M.B. Kastan, J.H. Petrini, The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor. Genes Dev. 19 (2005) 3043-3054.; J.P. Carney, R.S. Maser, H. Olivares, E.M. Davis, Le M. Beau, J.R. Yates, III, L. Hays, W.F. Morgan, J.H. Petrini, The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93 (1998) 477-486.; G.S. Stewart, R.S. Maser, T. Stankovic, D.A. Bressan, M.I. Kaplan, N.G. Jaspers, A. Raams, P.J. Byrd, J.H. Petrini, A.M. Taylor, The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell 99 (1999) 577-587.; B.R. Williams, O.K. Mirzoeva, W.F. Morgan, J. Lin, W. Dunnick, J.H. Petrini, A murine model of nijmegen breakage syndrome. Curr. Biol. 12 (2002) 648-653.; J.W. Theunissen, M.I. Kaplan, P.A. Hunt, B.R. Williams, D.O. Ferguson, F.W. Alt, J.H. Petrini, Checkpoint failure and chromosomal instability without lymphomagenesis in Mre11(ATLD1/ATLD1) mice. Mol. Cell 12 (2003) 1511-1523.] and the Mre11 complex deficiency in yeast [T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response

PI-RADS v2: Current standing and future outlook.

Science.gov (United States)

Smith, Clayton P; Türkbey, Barış

2018-05-01

The Prostate Imaging-Reporting and Data System (PI-RADS) was created in 2012 to establish standardization in prostate multiparametric magnetic resonance imaging (mpMRI) acquisition, interpretation, and reporting. In hopes of improving upon some of the PI-RADS v1 shortcomings, the PI-RADS Steering Committee released PI-RADS v2 in 2015. This paper reviews the accuracy, interobserver agreement, and clinical outcomes of PI-RADS v2 and comments on the limitations of the current literature. Overall, PI-RADS v2 shows improved sensitivity and similar specificity compared to PI-RADS v1. However, concerns exist regarding interobserver agreement and the heterogeneity of the study methodology.

A novel RAD21 variant associated with intrafamilial phenotypic variation in Cornelia de Lange syndrome - review of the literature

DEFF Research Database (Denmark)

Boyle, M I; Jespersgaard, C; Nazaryan-Petersen, Lusine

2017-01-01

In a patient with CdLS (IV.16) we identifed a novel single basepair deletion (c.704delG) in RAD21, which encodes a cohesin pathway protein. The variant is predicted to result in a premature stop codon [p.(Ser235Ilefs*19)] and hereby would have a deleterious effect. RAD21 variants have previously ...

RadVel: The Radial Velocity Modeling Toolkit

Science.gov (United States)

Fulton, Benjamin J.; Petigura, Erik A.; Blunt, Sarah; Sinukoff, Evan

2018-04-01

RadVel is an open-source Python package for modeling Keplerian orbits in radial velocity (RV) timeseries. RadVel provides a convenient framework to fit RVs using maximum a posteriori optimization and to compute robust confidence intervals by sampling the posterior probability density via Markov Chain Monte Carlo (MCMC). RadVel allows users to float or fix parameters, impose priors, and perform Bayesian model comparison. We have implemented real-time MCMC convergence tests to ensure adequate sampling of the posterior. RadVel can output a number of publication-quality plots and tables. Users may interface with RadVel through a convenient command-line interface or directly from Python. The code is object-oriented and thus naturally extensible. We encourage contributions from the community. Documentation is available at http://radvel.readthedocs.io.

Long Term RadNet Quality Data

Data.gov (United States)

U.S. Environmental Protection Agency — This RadNet Quality Data Asset includes all data since initiation and when ERAMS was expanded to become RadNet, name changed to reflect new mission. This includes...

Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale

Science.gov (United States)

Yu, Xiong; Egelman, Edward H.

2010-01-01

Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single stranded DNA with ∼ 9 subunits per turn in contrast to the filaments formed on double stranded DNA with ∼ 6.4 subunits per turn, and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy (STEM) that the Dmc1 filament formed on single stranded DNA has a mass per unit length expected from ∼ 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions, and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or STEM to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated. PMID:20600108

Simulation of the GCR spectrum in the Mars curiosity rover's RAD detector using MCNP6

Science.gov (United States)

Ratliff, Hunter N.; Smith, Michael B. R.; Heilbronn, Lawrence

2017-08-01

The paper presents results from MCNP6 simulations of galactic cosmic ray (GCR) propagation down through the Martian atmosphere to the surface and comparison with RAD measurements made there. This effort is part of a collaborative modeling workshop for space radiation hosted by Southwest Research Institute (SwRI). All modeling teams were tasked with simulating the galactic cosmic ray (GCR) spectrum through the Martian atmosphere and the Radiation Assessment Detector (RAD) on-board the Curiosity rover. The detector had two separate particle acceptance angles, 4π and 30 ° off zenith. All ions with Z = 1 through Z = 28 were tracked in both scenarios while some additional secondary particles were only tracked in the 4π cases. The MCNP6 4π absorbed dose rate was 307.3 ± 1.3 μGy/day while RAD measured 233 μGy/day. Using the ICRP-60 dose equivalent conversion factors built into MCNP6, the simulated 4π dose equivalent rate was found to be 473.1 ± 2.4 μSv/day while RAD reported 710 μSv/day.

The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs.

Science.gov (United States)

Yamagishi, Ryota; Tsusaka, Takeshi; Mitsunaga, Hiroko; Maehata, Takaharu; Hoshino, Shin-ichi

2016-04-07

Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27(kip1)and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27(kip1)mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells. © The Author(s) 2016. Published by Oxford

RadGenomics project

Energy Technology Data Exchange (ETDEWEB)

Iwakawa, Mayumi; Imai, Takashi; Harada, Yoshinobu [National Inst. of Radiological Sciences, Chiba (Japan). Frontier Research Center] [and others

2002-06-01

Human health is determined by a complex interplay of factors, predominantly between genetic susceptibility, environmental conditions and aging. The ultimate aim of the RadGenomics (Radiation Genomics) project is to understand the implications of heterogeneity in responses to ionizing radiation arising from genetic variation between individuals in the human population. The rapid progression of the human genome sequencing and the recent development of new technologies in molecular genetics are providing us with new opportunities to understand the genetic basis of individual differences in susceptibility to natural and/or artificial environmental factors, including radiation exposure. The RadGenomics project will inevitably lead to improved protocols for personalized radiotherapy and reductions in the potential side effects of such treatment. The project will contribute to future research into the molecular mechanisms of radiation sensitivity in humans and will stimulate the development of new high-throughput technologies for a broader application of biological and medical sciences. The staff members are specialists in a variety of fields, including genome science, radiation biology, medical science, molecular biology, and informatics, and have joined the RadGenomics project from various universities, companies, and research institutes. The project started in April 2001. (author)

RadGenomics project

International Nuclear Information System (INIS)

Iwakawa, Mayumi; Imai, Takashi; Harada, Yoshinobu

2002-01-01

Human health is determined by a complex interplay of factors, predominantly between genetic susceptibility, environmental conditions and aging. The ultimate aim of the RadGenomics (Radiation Genomics) project is to understand the implications of heterogeneity in responses to ionizing radiation arising from genetic variation between individuals in the human population. The rapid progression of the human genome sequencing and the recent development of new technologies in molecular genetics are providing us with new opportunities to understand the genetic basis of individual differences in susceptibility to natural and/or artificial environmental factors, including radiation exposure. The RadGenomics project will inevitably lead to improved protocols for personalized radiotherapy and reductions in the potential side effects of such treatment. The project will contribute to future research into the molecular mechanisms of radiation sensitivity in humans and will stimulate the development of new high-throughput technologies for a broader application of biological and medical sciences. The staff members are specialists in a variety of fields, including genome science, radiation biology, medical science, molecular biology, and informatics, and have joined the RadGenomics project from various universities, companies, and research institutes. The project started in April 2001. (author)

Hybridization Capture Using RAD Probes (hyRAD, a New Tool for Performing Genomic Analyses on Collection Specimens.

Directory of Open Access Journals (Sweden)

Tomasz Suchan

Full Text Available In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD or performing size selection of the resulting fragments (in the case of single-digest RAD. Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD. In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites

BAR domain proteins regulate Rho GTPase signaling.

Science.gov (United States)

Aspenström, Pontus

2014-01-01

BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis.

Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

KAUST Repository

Fornander, Louise H

2012-02-22

Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

Oak Ridge Reservation Environmental Protection Rad Neshaps Radionuclide Inventory Web Database and Rad Neshaps Source and Dose Database.

Science.gov (United States)

Scofield, Patricia A; Smith, Linda L; Johnson, David N

2017-07-01

The U.S. Environmental Protection Agency promulgated national emission standards for emissions of radionuclides other than radon from US Department of Energy facilities in Chapter 40 of the Code of Federal Regulations (CFR) 61, Subpart H. This regulatory standard limits the annual effective dose that any member of the public can receive from Department of Energy facilities to 0.1 mSv. As defined in the preamble of the final rule, all of the facilities on the Oak Ridge Reservation, i.e., the Y-12 National Security Complex, Oak Ridge National Laboratory, East Tennessee Technology Park, and any other U.S. Department of Energy operations on Oak Ridge Reservation, combined, must meet the annual dose limit of 0.1 mSv. At Oak Ridge National Laboratory, there are monitored sources and numerous unmonitored sources. To maintain radiological source and inventory information for these unmonitored sources, e.g., laboratory hoods, equipment exhausts, and room exhausts not currently venting to monitored stacks on the Oak Ridge National Laboratory campus, the Environmental Protection Rad NESHAPs Inventory Web Database was developed. This database is updated annually and is used to compile emissions data for the annual Radionuclide National Emission Standards for Hazardous Air Pollutants (Rad NESHAPs) report required by 40 CFR 61.94. It also provides supporting documentation for facility compliance audits. In addition, a Rad NESHAPs source and dose database was developed to import the source and dose summary data from Clean Air Act Assessment Package-1988 computer model files. This database provides Oak Ridge Reservation and facility-specific source inventory; doses associated with each source and facility; and total doses for the Oak Ridge Reservation dose.

Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI).

Science.gov (United States)

Fukuda, Tomoyuki; Ohya, Yoshikazu

2006-02-01

During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.

IFN-γ Induces Mimic Extracellular Trap Cell Death in Lung Epithelial Cells Through Autophagy-Regulated DNA Damage.

Science.gov (United States)

Lin, Chiou-Feng; Chien, Shun-Yi; Chen, Chia-Ling; Hsieh, Chia-Yuan; Tseng, Po-Chun; Wang, Yu-Chih

2016-02-01

Treatment of interferon-γ (IFN-γ) causes cell growth inhibition and cytotoxicity in lung epithelial malignancies. Regarding the induction of autophagy related to IFN-γ signaling, this study investigated the link between autophagy and IFN-γ cytotoxicity. In A549 human lung cancer cells, IFN-γ treatment induced concurrent apoptotic and nonapoptotic events. Unexpectedly, the nonapoptotic cells present mimic extracellular trap cell death (ETosis), which was regulated by caspase-3 and by autophagy induction through immunity-related GTPase family M protein 1 and activating transcription factor 6. Furthermore, IFN-γ signaling controlled mimic ETosis through a mechanism involving an autophagy- and Fas-associated protein with death domain-controlled caspase-8/-3 activation. Following caspase-mediated lamin degradation, IFN-γ caused DNA damage-associated ataxia telangiectasia and Rad3-related protein (ATR)/ataxia telangiectasia mutated (ATM)-regulated mimic ETosis. Upon ATR/ATM signaling, peptidyl arginine deiminase 4 (PAD4)-mediated histone 3 citrullination promoted mimic ETosis. Such IFN-γ-induced effects were defective in PC14PE6/AS2 human lung cancer cells, which were unsusceptible to IFN-γ-induced autophagy. Due to autophagy-based caspase cascade activation, IFN-γ triggers unconventional caspase-mediated DNA damage, followed by ATR/ATM-regulated PAD4-mediated histone citrullination during mimic ETosis in lung epithelial malignancy.

Germline variants in MRE11/RAD50/NBN complex genes in childhood leukemia

International Nuclear Information System (INIS)

Mosor, Maria; Ziółkowska-Suchanek, Iwona; Nowicka, Karina; Dzikiewicz-Krawczyk, Agnieszka; Januszkiewicz–Lewandowska, Danuta; Nowak, Jerzy

2013-01-01

The MRE11, RAD50, and NBN genes encode proteins of the MRE11-RAD50-NBN (MRN) complex involved in cellular response to DNA damage and the maintenance of genome stability. In our previous study we showed that the germline p.I171V mutation in NBN may be considered as a risk factor in the development of childhood acute lymphoblastic leukemia (ALL) and some specific haplotypes of that gene may be associated with childhood leukemia. These findings raise important questions about the role of mutations in others genes of the MRN complex in childhood leukemia. The aim of this study was to answer the question whether MRE11 and RAD50 alterations may be associated with childhood ALL or AML. We estimated the frequency of constitutional mutations and polymorphisms in selected regions of MRE11, RAD50, and NBN in the group of 220 children diagnosed with childhood leukemias and controls (n=504/2200). The analysis was performed by specific amplification of region of interest by PCR and followed by multi-temperature single-strand conformation polymorphism (PCR-MSSCP) technique. We performed two molecular tests to examine any potential function of the detected the c.551+19G>A SNP in RAD50 gene. To our knowledge, this is the first analysis of the MRE11, RAD50 and NBN genes in childhood leukemia. The frequency of either the AA genotype or A allele of RAD50-rs17166050 were significantly different in controls compared to leukemia group (ALL+AML) (p<0.0019 and p<0.0019, respectively). The cDNA analysis of AA or GA genotypes carriers has not revealed evidence of splicing abnormality of RAD50 pre-mRNA. We measured the allelic-specific expression of G and A alleles at c.551+19G>A and the statistically significant overexpression of the G allele has been observed. Additionally we confirmed the higher incidence of the p.I171V mutation in the leukemia group (7/220) than among controls (12/2400) (p<0.0001). The formerly reported sequence variants in the RAD50 and MRE11 gene may not constitute a

Lingering single-strand breaks trigger Rad51-independent homology-directed repair of collapsed replication forks in the polynucleotide kinase/phosphatase mutant of fission yeast.

Directory of Open Access Journals (Sweden)

Arancha Sanchez

2017-09-01

Full Text Available The DNA repair enzyme polynucleotide kinase/phosphatase (PNKP protects genome integrity by restoring ligatable 5'-phosphate and 3'-hydroxyl termini at single-strand breaks (SSBs. In humans, PNKP mutations underlie the neurological disease known as MCSZ, but these individuals are not predisposed for cancer, implying effective alternative repair pathways in dividing cells. Homology-directed repair (HDR of collapsed replication forks was proposed to repair SSBs in PNKP-deficient cells, but the critical HDR protein Rad51 is not required in PNKP-null (pnk1Δ cells of Schizosaccharomyces pombe. Here, we report that pnk1Δ cells have enhanced requirements for Rad3 (ATR/Mec1 and Chk1 checkpoint kinases, and the multi-BRCT domain protein Brc1 that binds phospho-histone H2A (γH2A at damaged replication forks. The viability of pnk1Δ cells depends on Mre11 and Ctp1 (CtIP/Sae2 double-strand break (DSB resection proteins, Rad52 DNA strand annealing protein, Mus81-Eme1 Holliday junction resolvase, and Rqh1 (BLM/WRN/Sgs1 DNA helicase. Coupled with increased sister chromatid recombination and Rad52 repair foci in pnk1Δ cells, these findings indicate that lingering SSBs in pnk1Δ cells trigger Rad51-independent homology-directed repair of collapsed replication forks. From these data, we propose models for HDR-mediated tolerance of persistent SSBs with 3' phosphate in pnk1Δ cells.

The eIF4E-binding proteins are modifiers of cytoplasmic eIF4E relocalization during the heat shock response.

Science.gov (United States)

Sukarieh, R; Sonenberg, N; Pelletier, J

2009-05-01

Stress granules (SGs) arise as a consequence of cellular stress, contain stalled translation preinitiation complexes, and are associated with cell survival during environmental insults. SGs are dynamic entities with proteins relocating into and out of them during stress. Among the repertoire of proteins present in SGs is eukaryotic initiation factor 4E (eIF4E), a translation factor required for cap-dependent translation and that regulates a rate-limiting step for protein synthesis. Herein, we demonstrate that localization of eIF4E to SGs is dependent on the presence of a family of repressor proteins, eIF4E-binding proteins (4E-BPs). Our results demonstrate that 4E-BPs regulate the SG localization of eIF4E.

Bone Morphogenetic Protein (BMP-4 and BMP-7 regulate differentially Transforming Growth Factor (TGF-β1 in normal human lung fibroblasts (NHLF

Directory of Open Access Journals (Sweden)

Lloyd Clare M

2010-06-01

Full Text Available Abstract Background Airway remodelling is thought to be under the control of a complex group of molecules belonging to the Transforming Growth Factor (TGF-superfamily. The Bone Morphogenetic Proteins (BMPs belong to this family and have been shown to regulate fibrosis in kidney and liver diseases. However, the role of BMPs in lung remodelling remains unclear. BMPs may regulate tissue remodelling in asthma by controlling TGF-β-induced profibrotic functions in lung fibroblasts. Methods Cell cultures were exposed to TGF-β1 alone or in the presence of BMP-4 or BMP-7; control cultures were exposed to medium only. Cell proliferation was assessed by quantification of the incorporation of [3H]-thymidine. The expression of the mRNA encoding collagen type I and IV, tenascin C and fibronectin in normal human lung fibroblasts (NHLF was determined by real-time quantitative PCR and the main results were confirmed by ELISA. Cell differentiation was determined by the analysis of the expression of α-smooth muscle actin (α-SMA by western blot and immunohistochemistry. The effect on matrix metalloproteinase (MMP activity was assessed by zymography. Results We have demonstrated TGF-β1 induced upregulation of mRNAs encoding the extracellular matrix proteins, tenascin C, fibronectin and collagen type I and IV when compared to unstimulated NHLF, and confirmed these results at the protein level. BMP-4, but not BMP-7, reduced TGF-β1-induced extracellular matrix protein production. TGF-β1 induced an increase in the activity of the pro-form of MMP-2 which was inhibited by BMP-7 but not BMP-4. Both BMP-4 and BMP-7 downregulated TGF-β1-induced MMP-13 release compared to untreated and TGF-β1-treated cells. TGF-β1 also induced a myofibroblast-like transformation which was partially inhibited by BMP-7 but not BMP-4. Conclusions Our study suggests that some regulatory properties of BMP-7 may be tissue or cell type specific and unveil a potential regulatory role for

Regulation of cardiac C-protein phosphorylation

International Nuclear Information System (INIS)

Titus, F.L.

1985-01-01

Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased [ 32 P]phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and [ 32 P]phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 μM Iso and 17% in hearts exposed to Iso plus 1 μM methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed

The DNA translocase RAD5A acts independently of the other main DNA repair pathways, and requires both its ATPase and RING domain for activity in Arabidopsis thaliana.

Science.gov (United States)

Klemm, Tobias; Mannuß, Anja; Kobbe, Daniela; Knoll, Alexander; Trapp, Oliver; Dorn, Annika; Puchta, Holger

2017-08-01

Multiple pathways exist to repair DNA damage induced by methylating and crosslinking agents in Arabidopsis thaliana. The SWI2/SNF2 translocase RAD5A, the functional homolog of budding yeast Rad5 that is required for the error-free branch of post-replicative repair, plays a surprisingly prominent role in the repair of both kinds of lesions in Arabidopsis. Here we show that both the ATPase domain and the ubiquitination function of the RING domain of the Arabidopsis protein are essential for the cellular response to different forms of DNA damage. To define the exact role of RAD5A within the complex network of DNA repair pathways, we crossed the rad5a mutant line with mutants of different known repair factors of Arabidopsis. We had previously shown that RAD5A acts independently of two main pathways of replication-associated DNA repair defined by the helicase RECQ4A and the endonuclease MUS81. The enhanced sensitivity of all double mutants tested in this study indicates that the repair of damaged DNA by RAD5A also occurs independently of nucleotide excision repair (AtRAD1), single-strand break repair (AtPARP1), as well as microhomology-mediated double-strand break repair (AtTEB). Moreover, RAD5A can partially complement for a deficient AtATM-mediated DNA damage response in plants, as the double mutant shows phenotypic growth defects. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Fatty acid‐binding protein 4 regulates fatty infiltration after rotator cuff tear by hypoxia‐inducible factor 1 in mice

Science.gov (United States)

Lee, Yong‐Soo; Kim, Ja‐Yeon; Oh, Kyung‐Soo

2017-01-01

Abstract Background Fatty infiltration in skeletal muscle is directly linked to loss of muscle strength and is associated with various adverse physical outcomes such as muscle atrophy, inflammation, insulin resistance, mobility impairments, and even mortality in the elderly. Aging, mechanical unloading, muscle injury, and hormonal imbalance are main causes of muscle fat accumulation, and the fat cells are derived from muscle stem cells via adipogenic differentiation. However, the pathogenesis and molecular mechanisms of fatty infiltration in muscles are still not fully defined. Fatty acid‐binding protein 4 (FABP4) is a carrier protein for fatty acids and is involved in fatty acid uptake, transport, and lipid metabolism. Rotator cuff tear (RCT) usually occurs in the elderly and is closely related with fatty infiltration in injured muscle. To investigate potential mechanisms for fatty infiltration other than adipogenic differentiation of muscle stem cells, we examined the role of FABP4 in muscle fatty infiltration in an RCT mouse model. Methods In the RCT model, we evaluated the expression of FABP4 by qRT‐PCR, western blotting, and immunohistochemical analyses. Histological changes such as inflammation and fat accumulation in the injured muscles were examined immunohistochemically. To evaluate whether hypoxia induces FABP4 expression, the levels of FABP4 mRNA and protein in C3H10T1/2 cells after hypoxia were examined. Using a transient transfection assay in 293T cells, we assessed the promoter activity of FABP4 by hypoxia‐inducible factors (HIFs). Additionally, we evaluated the reduction in FABP4 expression and fat accumulation using specific inhibitors for HIF1 and FABP4, respectively. Results FABP4 expression was significantly increased after RCT in mice, and its expression was localized in the intramuscular fatty region. Rotator cuff tear‐induced FABP4 expression was up‐regulated by hypoxia. HIF1α, which is activated by hypoxia, augmented the promoter

Systems Analyses Reveal Shared and Diverse Attributes of Oct4 Regulation in Pluripotent Cells

DEFF Research Database (Denmark)

Ding, Li; Paszkowski-Rogacz, Maciej; Winzi, Maria

2015-01-01

We combine a genome-scale RNAi screen in mouse epiblast stem cells (EpiSCs) with genetic interaction, protein localization, and "protein-level dependency" studies-a systematic technique that uncovers post-transcriptional regulation-to delineate the network of factors that control the expression...... of Oct4, a key regulator of pluripotency. Our data signify that there are similarities, but also fundamental differences in Oct4 regulation in EpiSCs versus embryonic stem cells (ESCs). Through multiparametric data analyses, we predict that Tox4 is associating with the Paf1C complex, which maintains cell...... identity in both cell types, and validate that this protein-protein interaction exists in ESCs and EpiSCs. We also identify numerous knockdowns that increase Oct4 expression in EpiSCs, indicating that, in stark contrast to ESCs, Oct4 is under active repressive control in EpiSCs. These studies provide...

Regulation of AMPA Receptor Trafficking by Protein Ubiquitination

Directory of Open Access Journals (Sweden)

Jocelyn Widagdo

2017-10-01

Full Text Available The molecular mechanisms underlying plastic changes in the strength and connectivity of excitatory synapses have been studied extensively for the past few decades and remain the most attractive cellular models of learning and memory. One of the major mechanisms that regulate synaptic plasticity is the dynamic adjustment of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA-type glutamate receptor content on the neuronal plasma membrane. The expression of surface AMPA receptors (AMPARs is controlled by the delicate balance between the biosynthesis, dendritic transport, exocytosis, endocytosis, recycling and degradation of the receptors. These processes are dynamically regulated by AMPAR interacting proteins as well as by various post-translational modifications that occur on their cytoplasmic domains. In the last few years, protein ubiquitination has emerged as a major regulator of AMPAR intracellular trafficking. Dysregulation of AMPAR ubiquitination has also been implicated in the pathophysiology of Alzheimer’s disease. Here we review recent advances in the field and provide insights into the role of protein ubiquitination in regulating AMPAR membrane trafficking and function. We also discuss how aberrant ubiquitination of AMPARs contributes to the pathogenesis of various neurological disorders, including Alzheimer’s disease, chronic stress and epilepsy.

Tomosynthesis in the Diagnostic Setting: Changing Rates of BI-RADS Final Assessment over Time.

Science.gov (United States)

Raghu, Madhavi; Durand, Melissa A; Andrejeva, Liva; Goehler, Alexander; Michalski, Mark H; Geisel, Jaime L; Hooley, Regina J; Horvath, Laura J; Butler, Reni; Forman, Howard P; Philpotts, Liane E

2016-10-01

Purpose To evaluate the effect of tomosynthesis in diagnostic mammography on the Breast Imaging Reporting and Data System (BI-RADS) final assessment categories over time. Materials and Methods This retrospective study was approved by the institutional review board. The authors reviewed all diagnostic mammograms obtained during a 12-month interval before (two-dimensional [2D] mammography [June 2, 2010, to June 1, 2011]) and for 3 consecutive years after (tomosynthesis year 1 [2012], tomosynthesis year 2 [2013], and tomosynthesis year 3 [2014]) the implementation of tomosynthesis. The requirement to obtain informed consent was waived. The rates of BI-RADS final assessment categories 1-5 were compared between the 2D and tomosynthesis groups. The positive predictive values after biopsy (PPV3) for BI-RADS category 4 and 5 cases were compared. The mammographic features (masses, architectural distortions, calcifications, focal asymmetries) of lesions categorized as probably benign (BI-RADS category 3) and those for which biopsy was recommended (BI-RADS category 4 or 5) were reviewed. The χ(2) test was used to compare the rates of BI-RADS final assessment categories 1-5 between the two groups, and multivariate logistic regression analysis was performed to compare all diagnostic studies categorized as BI-RADS 3-5. Results There was an increase in the percentage of cases reported as negative or benign (BI-RADS category 1 or 2) with tomosynthesis (58.7% with 2D mammography vs 75.8% with tomosynthesis at year 3, P tomosynthesis at year 3, P tomosynthesis (8.0% with 2D mammography vs 7.8% with tomosynthesis at year 3, P = .2), there was a significant increase in the PPV3 (29.6% vs 50%, respectively; P tomosynthesis use. Conclusion Tomosynthesis in the diagnostic setting resulted in progressive shifts in the BI-RADS final assessment categories over time, with a significant increase in the proportion of studies classified as normal, a continued decrease in the rate of studies

RadWorks Project

Data.gov (United States)

National Aeronautics and Space Administration — The RadWorks project's overarching objective is the maturation and demonstration of affordable, enabling solutions to the radiation-related challenges presented to...

PI-RADS v2 and ADC values: is there room for improvement?

Science.gov (United States)

Jordan, Eric J; Fiske, Charles; Zagoria, Ronald; Westphalen, Antonio C

2018-03-17

To determine the diagnostic accuracy of ADC values in combination with PI-RADS v2 for the diagnosis of clinically significant prostate cancer (CS-PCa) compared to PI-RADS v2 alone. This retrospective study included 155 men whom underwent 3-Tesla prostate MRI and subsequent MR/US fusion biopsies at a single non-academic center from 11/2014 to 3/2016. All scans were performed with a surface coil and included T2, diffusion-weighted, and dynamic contrast-enhanced sequences. Suspicious findings were classified using Prostate Imaging Reporting and Data System (PI-RADS) v2 and targeted using MR/US fusion biopsies. Mixed-effect logistic regression analyses were used to determine the ability of PIRADS v2 alone and combined with ADC values to predict CS-PCa. As ADC categories are more practical in clinical situations than numeric values, an additional model with ADC categories of ≤ 800 and > 800 was performed. A total of 243 suspicious lesions were included, 69 of which were CS-PCa, 34 were Gleason score 3+3 PCa, and 140 were negative. The overall PIRADS v2 score, ADC values, and ADC categories are independent statistically significant predictors of CS-PCa (p values or categories is better discrimination of PI-RADS v2 4 lesions. ADC values and categories help to diagnose CS-PCa when lesions are assigned a PI-RADS v2 score of 4.

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs.

Science.gov (United States)

Rong, Liwei; Livingstone, Mark; Sukarieh, Rami; Petroulakis, Emmanuel; Gingras, Anne-Claude; Crosby, Katherine; Smith, Bradley; Polakiewicz, Roberto D; Pelletier, Jerry; Ferraiuolo, Maria A; Sonenberg, Nahum

2008-07-01

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.

Three additional genes involved in pyrimidine dimer removal in Saccharomyces cerevisiae: RAD7, RAD14, and MMS19

Energy Technology Data Exchange (ETDEWEB)

Prakash, L; Prakash, S

1979-01-01

The ability to remove ultraviolet (uv)-induced pyrimidine dimers from the nuclear DNA of yeast was examined in two radiation-sensitive (rad) mutants and one methyl methanesulfonate-sensitive (mms) mutant of the yeast Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by an endonuclease activity prepared from crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad7, rad14, and mms19 mutants were found to be defective in their ability to remove uv-induced dimers from nuclear DNA. All three mutants belong to the same episatic group as the other mutants involved in excision-repair. All three mutants show enhanced uv-induced mutations. The rad 14 mutant also shows epistatic interactions with genes in the other two uv repair pathways.

Regulations and instructions; Propisi i uputstva

Energy Technology Data Exchange (ETDEWEB)

NONE

1960-07-01

Regulations and instructions for operating the RA reactor consist of the following chapters: general regulations with the fundamental RA reactor characteristics, operating regulations and instructions for the personnel on duty, regulations for accidental conditions, training program for the staff of the Laboratory for reactor operation. Propisi i uputstva za rad reaktora RA sadrze sledeca poglavlja: opsti deo sa kratkim opisom karakteristika reaktora RA, propisi za rad dezurnog osoblja, propisi za slucaj udesa, program obuke osoblja Laboratorije za eksploataciju reaktora RA.

Protein Kinase C-{delta} mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction

Energy Technology Data Exchange (ETDEWEB)

Gao, Feng-Hou [NO.3 People' s Hospital affiliated to Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 201900 (China); The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Wu, Ying-Li [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Meng [Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China); Liu, Chuan-Xu; Wang, Li-Shun [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Guo-Qiang, E-mail: chengq@shsmu.edu.cn [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China)

2009-11-15

We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta ({Delta}PKC-{delta}). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the {Delta}PKC-{delta}, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that {Delta}PKC-{delta} mediated the down-regulation of hnRNP K protein during apoptosis: PKC-{delta} inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-{delta}-deficient apoptotic KG1a cells; conditional induction of {Delta}PKC-{delta} in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of {Delta}PKC-{delta}. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-{delta} down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

HiRadMat: materials under scrutiny

CERN Multimedia

Anaïs Schaeffer

2011-01-01

CERN's new facility, HiRadMat (High Radiation to Materials), which is designed to test materials for the world's future particle accelerators, should be operational and welcoming its first experiments by the end of the year.   The HiRadMat facility, located in the TNC tunnel. The materials used in the LHC and its experiments are exposed to very high-energy particles. The LHC machine experts obviously didn't wait for the first collisions in the world's most powerful accelerator to put the materials through their paces - the equipment was validated following a series of stringent tests. And these tests will get even tougher now, with the arrival of HiRadMat. The tunnel that formerly housed the West Area Neutrino Facility (WANF) has been completely revamped to make way for CERN's latest facility, HiRadMat. Supported by the Radioprotection service, a team from the Engineering (EN) Department handled the dismantling operations from October 2009 to December 2010. "We could only work on disman...

Stereotactic vacuum core biopsy of clustered microcalcifications classified as BI-RADS{sup TM} type 3; Stereotaktische Vakuumstanzbiopsie zur Abklaerung von gruppiertem Mikrokalk der Kategorie BI-RADS{sup TM} 3

Energy Technology Data Exchange (ETDEWEB)

Obenauer, S.; Fischer, U.; Baum, F.; Dammert, S.; Grabbe, E. [Goettingen Univ. (Germany). Abt. fuer Roentgendiagnostik; Fuezesi, L. [Goettingen Univ. (Germany). Zentrum Pathologie

2001-08-01

Purpose: Evaluation of stereotactic vacuum core biopsy of clustered microcalcifications categorized as BI-RADS{sup TM} 3. Material and methods: 86 patients with microcalcification BI-RADS{sup TM} 3 (probably benign, <3% malignant) underwent a stereotactic vacuum core biopsy (Mammotome, Fa. Ethicon Endo-Surgery Breast Care) using a digital stereotactic unit (Mammotest, Fa. Fischer Imaging). The removal of the calcifications was judged by two radiologists in consensus and classified as complete (100%), major (55-99%) or incomplete (<50%). Results: 4/86 patients could not be evaluated by vacuum core biopsy due to the localization of the microcalcifications close to the skin or lack of detection. In 40/82 cases a complete, in 38/82 a major, and in 4/82 an incomplete removal was achieved. Histology revealed 67 cases of fibrocystic changes, 4 papillomas, 4 fibroadenomas, 4 cases of atypical ductal hyperplasia (ADH), and 3 ductal carcinomas in situ (DCIS), one of these with a minimal-invasive tumor component. Patients with ADH were advised to undergo surgical biopsy. Histology revealed complete removal. 7 patients had complications or side-effects. Conclusions: Percutaneous vacuum core biopsy is a reliable minimal-invasive diagnostic method to come to the final diagnosis in patients with clustered microcalcifications categorized BI-RADS{sup TM} 3. However, if malignancy is proven (about 4% of our cases) an open biopsy is necessary. (orig.) [German] Zielsetzung: Evaluation der stereotaktischen Vakuumstanzbiopsie bei gruppierten Mikrokalzifikationen vom Typ BI-RADS{sup TM}3. Material und Methodik: Gruppiert angeordneter Mikrokalk der Kategorie BI-RADS{sup TM} 3 (wahrscheinlich benigne, <3% maligne) wurde bei 86 Patientinnen mit einer stereotaktischen Vakuumstanzbiopsie (Mammotome{sup circledR}, Fa. Ethicon Endo-Surgery Breast Care) an einem digitalen Stereotaxietisch (Mammotest, Fa. Fischer Imaging) abgeklaert. Das Ausmass der bioptisch entfernten Kalizifkationen wurde von

Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress.

Science.gov (United States)

Chastain, Megan; Zhou, Qing; Shiva, Olga; Fadri-Moskwik, Maria; Whitmore, Leanne; Jia, Pingping; Dai, Xueyu; Huang, Chenhui; Ye, Ping; Chai, Weihang

2016-08-02

The telomeric CTC1/STN1/TEN1 (CST) complex has been implicated in promoting replication recovery under replication stress at genomic regions, yet its precise role is unclear. Here, we report that STN1 is enriched at GC-rich repetitive sequences genome-wide in response to hydroxyurea (HU)-induced replication stress. STN1 deficiency exacerbates the fragility of these sequences under replication stress, resulting in chromosome fragmentation. We find that upon fork stalling, CST proteins form distinct nuclear foci that colocalize with RAD51. Furthermore, replication stress induces physical association of CST with RAD51 in an ATR-dependent manner. Strikingly, CST deficiency diminishes HU-induced RAD51 foci formation and reduces RAD51 recruitment to telomeres and non-telomeric GC-rich fragile sequences. Collectively, our findings establish that CST promotes RAD51 recruitment to GC-rich repetitive sequences in response to replication stress to facilitate replication restart, thereby providing insights into the mechanism underlying genome stability maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Yes-associated protein/TEA domain family member and hepatocyte nuclear factor 4-alpha (HNF4α) repress reciprocally to regulate hepatocarcinogenesis in rats and mice.

Science.gov (United States)

Cai, Wang-Yu; Lin, Ling-Yun; Hao, Han; Zhang, Sai-Man; Ma, Fei; Hong, Xin-Xin; Zhang, Hui; Liu, Qing-Feng; Ye, Guo-Dong; Sun, Guang-Bin; Liu, Yun-Jia; Li, Sheng-Nan; Xie, Yuan-Yuan; Cai, Jian-Chun; Li, Bo-An

2017-04-01

Great progress has been achieved in the study of Hippo signaling in regulating tumorigenesis; however, the downstream molecular events that mediate this process have not been completely defined. Moreover, regulation of Hippo signaling during tumorigenesis in hepatocellular carcinoma (HCC) remains largely unknown. In the present study, we systematically investigated the relationship between Yes-associated protein/TEA domain family member (YAP-TEAD) and hepatocyte nuclear factor 4-alpha (HNF4α) in the hepatocarcinogenesis of HCC cells. Our results indicated that HNF4α expression was negatively regulated by YAP1 in HCC cells by a ubiquitin proteasome pathway. By contrast, HNF4α was found to directly associate with TEAD4 to compete with YAP1 for binding to TEAD4, thus inhibiting the transcriptional activity of YAP-TEAD and expression of their target genes. Moreover, overexpression of HNF4α was found to significantly compromise YAP-TEAD-induced HCC cell proliferation and stem cell expansion. Finally, we documented the regulatory mechanism between YAP-TEAD and HNF4α in rat and mouse tumor models, which confirmed our in vitro results. There is a double-negative feedback mechanism that controls TEAD-YAP and HNF4α expression in vitro and in vivo, thereby regulating cellular proliferation and differentiation. Given that YAP acts as a dominant oncogene in HCC and plays a crucial role in stem cell homeostasis and tissue regeneration, manipulating the interaction between YAP, TEADs, and HNF4α may provide a new approach for HCC treatment and regenerative medicine. (Hepatology 2017;65:1206-1221). © 2016 by the American Association for the Study of Liver Diseases.

Usefulness of combined use of contrast-enhanced ultrasound and TI-RADS classification for the differentiation of benign from malignant lesions of thyroid nodules.

Science.gov (United States)

Zhang, Yan; Zhou, Ping; Tian, Shuang-Ming; Zhao, Yong-Feng; Li, Jia-Le; Li, Lan

2017-04-01

To study the thyroid image reporting and data system (TI-RADS) classification and the contrast-enhanced ultrasound (CEUS) enhancement pattern of thyroid nodules, and to determine whether combined use of both methods is helpful in the diagnosis of thyroid nodules. A total of 319 thyroid nodules in 246 patients were assessed with TI-RADS, CEUS and a combination of both methods. The diagnostic performance of TI-RADS, CEUS and a combination of both methods was compared. The accuracy in the diagnosis of thyroid nodules was 90.3 % for TI-RADS, 90.0 % for CEUS and 96.0 % for a combination of both methods respectively. A statistically significant difference was not observed in the diagnostic accuracy of CEUS and TI-RADS (P > 0.05). However, a significant difference was observed between a combination of both methods and either alone (P TI-RADS classifications of 4a and 4b thyroid nodules compared with TI-RADS alone (P  0.05). The improved TI-RADS, when combined with CEUS, could significantly improve the diagnostic accuracy for thyroid nodules, especially for TI-RADS class-4 thyroid nodules. • TI-RADS can be used as the primary diagnostic standard for thyroid nodules • CEUS can be used as an important complement to TI-RADS • The improved TI-RADS can significantly improve the qualitative diagnostic accuracy.

222Rn Determination In Drinking Waters - RAD7 And LSC Technique Comparison

International Nuclear Information System (INIS)

Todorovic, N.; Stojkovic, I.; Nikolov, J.; Tenjovic, B.

2015-01-01

A procedure for the determination of 222Rn in environmental water samples using liquid scintillation counting (LSC) was applied and optimized. For radon determination in drinking water from groundwater and surface water sources by LSC, the EPA Method 913.0 was used. A minimum detectable activity of 0.029 Bq L-1 in a 20 mL glass vial (10 mL water sample mixed with 10 mL of liquid scintillation cocktail) has been achieved during 300 minutes of measurement time. The procedure was compared with RAD7 radon detector measurements. Factors that affect the measurement accuracy and precision of RAD7 radon detector are the sampling technique, sample concentration, sample size, counting time, temperature, relative humidity and background effects. The minimal detectable activity (MDA) for RAD7 technique was found to be 0.1 Bq/L. From obtained results of 222Rn measurements in 15 water samples with different 222Rn activities, correlation between the two techniques applied for measurements of 222Rn in water samples (A less than 400 Bq/L) was determined. There is reasonable agreement (within statistical uncertainties) between the various techniques in most cases, while disagreements most likely come from systematic uncertainties associated with sampling procedures. Discrepancy in determined activities between the two techniques becomes more evident with increased 222Rn activities in water. LSC technique gives in general higher activity concentrations for about 30 percent than RAD7 spectrometer. The interpretation of shown results could be that RAD7 is not properly calibrated for higher activities, since USA reference level of 222Rn concentrations in water is only 11.1 Bq/L (US EPA, Proposed Radon in Drinking Water Regulation). (author).

The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.

Science.gov (United States)

Mittal, Nitish; Guimaraes, Joao C; Gross, Thomas; Schmidt, Alexander; Vina-Vilaseca, Arnau; Nedialkova, Danny D; Aeschimann, Florian; Leidel, Sebastian A; Spang, Anne; Zavolan, Mihaela

2017-09-06

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.

Selective up-regulation of protein kinase C eta in phorbol ester-sensitive versus -resistant EL4 mouse thymoma cells.

Science.gov (United States)

Resnick, M S; Luo, X; Vinton, E G; Sando, J J

1997-06-01

Stimulation of sensitive EL4 mouse thymoma cells (s-EL4) with phorbol esters results in production of interleukin 2 (IL-2), adherence to a plastic substrate, and growth inhibition, whereas a phorbol ester-resistant variant (r-EL4) fails to respond. Previous studies revealed substantially decreased expression of protein kinase C (PKC) epsilon in the r-EL4 versus s-EL4 cells. This work has been extended to examine the more recently described PKC isozymes. Western and Northern analyses revealed a marked decrease in PKC eta and theta in r-EL4 as compared to s-EL4 cells. Treatment of these lines with phorbol ester for 24 h resulted in down-regulation of all PKC isozymes examined except PKC eta, which was up-regulated in the s-EL4 cells at the time of maximal IL-2 production. Two newly isolated EL4 clones, resistant to phorbol ester-induced growth inhibition but still exhibiting the phorbol ester-induced adherence and IL-2 production, both expressed PKC eta and theta. Collectively, these observations suggest a dissociation of growth inhibition from adherence and IL-2 production pathways and a potential role for PKC eta in the latter.

RadNet Air Quality (Fixed Station) Data

Data.gov (United States)

U.S. Environmental Protection Agency — RadNet is a national network of monitoring stations that regularly collect air for analysis of radioactivity. The RadNet network, which has stations in each State,...

NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

International Nuclear Information System (INIS)

Volakakis, Nikolaos; Joodmardi, Eliza; Perlmann, Thomas

2009-01-01

The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPARβ/δ signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPARβ/δ and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.

The small molecule '1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate' and its derivatives regulate global protein synthesis by inactivating eukaryotic translation initiation factor 2-alpha.

Science.gov (United States)

Hong, Mi-Na; Nam, Ky-Youb; Kim, Kyung Kon; Kim, So-Young; Kim, InKi

2016-05-01

By environmental stresses, cells can initiate a signaling pathway in which eukaryotic translation initiation factor 2-alpha (eIF2-α) is involved to regulate the response. Phosphorylation of eIF2-α results in the reduction of overall protein neogenesis, which allows cells to conserve resources and to reprogram energy usage for effective stress control. To investigate the role of eIF2-α in cell stress responses, we conducted a viability-based compound screen under endoplasmic reticulum (ER) stress condition, and identified 1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate (AMC-01) and its derivatives as eIF2-α-inactivating chemical. Molecular characterization of this signaling pathway revealed that AMC-01 induced inactivation of eIF2-α by phosphorylating serine residue 51 in a dose- and time-dependent manner, while the negative control compounds did not affect eIF2-α phosphorylation. In contrast with ER stress induction by thapsigargin, phosphorylation of eIF2-α persisted for the duration of incubation with AMC-01. By pathway analysis, AMC-01 clearly induced the activation of protein kinase RNA-activated (PKR) kinase and nuclear factor-κB (NF-κB), whereas it did not modulate the activity of PERK or heme-regulated inhibitor (HRI). Finally, we could detect a lower protein translation rate in cells incubated with AMC-01, establishing AMC-01 as a potent chemical probe that can regulate eIF2-α activity. We suggest from these data that AMC-01 and its derivative compounds can be used as chemical probes in future studies of the role of eIF2-α in protein synthesis-related cell physiology.

Stratification of mammographic computerized analysis by BI-RADS categories

International Nuclear Information System (INIS)

Lederman, Richard; Leichter, Isaac; Buchbinder, Shalom; Novak, Boris; Bamberger, Philippe; Fields, Scott

2003-01-01

The Breast Imaging Reporting and Data System (BI-RADS) was implemented to standardize characterization of mammographic findings. The purpose of the present study was to evaluate in which BI-RADS categories the changes recommended by computerized mammographic analysis are most beneficial. Archival cases including, 170 masses (101 malignant, 69 benign) and 63 clusters of microcalcifications (MCs; 36 malignant, 27 benign), were evaluated retrospectively, using the BI-RADS categories, by several radiologists, blinded to the pathology results. A computerized system then automatically extracted from the digitized mammogram features characterizing mammographic lesions, which were used to classify the lesions. The results of the computerized classification scheme were compared, by receiver operating characteristics (ROC) analysis, to the conventional interpretation. In the ''low probability of malignancy group'' (excluding BI-RADS categories 4 and 5), computerized analysis improved the A z of the ROC curve significantly, from 0.57 to 0.89. In the ''high probability of malignancy group'' (mostly category 5) the computerized analysis yielded an ROC curve with an A z of 0.99. In the ''intermediate probability of malignancy group'' computerized analysis improved the A z significantly, from 0.66 for to 0.83. Pair-wise analysis showed that in the latter group the modifications resulting from computerized analysis were correct in 83% of cases. Computerized analysis has the ability to improve the performance of the radiologists exactly in the BI-RADS categories with the greatest difficulties in arriving at a correct diagnosis. It increased the performance significantly in the problematic group of ''intermediate probability of malignancy'' and pinpointed all the cases with missed cancers in the ''low probability'' group. (orig.)

The E4 protein; structure, function and patterns of expression

Energy Technology Data Exchange (ETDEWEB)

Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

2013-10-15

The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup �

Simulation of the GCR spectrum in the Mars curiosity rover's RAD detector using MCNP6.

Science.gov (United States)

Ratliff, Hunter N; Smith, Michael B R; Heilbronn, Lawrence

2017-08-01

The paper presents results from MCNP6 simulations of galactic cosmic ray (GCR) propagation down through the Martian atmosphere to the surface and comparison with RAD measurements made there. This effort is part of a collaborative modeling workshop for space radiation hosted by Southwest Research Institute (SwRI). All modeling teams were tasked with simulating the galactic cosmic ray (GCR) spectrum through the Martian atmosphere and the Radiation Assessment Detector (RAD) on-board the Curiosity rover. The detector had two separate particle acceptance angles, 4π and 30 ° off zenith. All ions with Z = 1 through Z = 28 were tracked in both scenarios while some additional secondary particles were only tracked in the 4π cases. The MCNP6 4π absorbed dose rate was 307.3 ± 1.3 µGy/day while RAD measured 233 µGy/day. Using the ICRP-60 dose equivalent conversion factors built into MCNP6, the simulated 4π dose equivalent rate was found to be 473.1 ± 2.4 µSv/day while RAD reported 710 µSv/day. Copyright © 2017 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.

Mouse Rad9b is essential for embryonic development and promotes resistance to DNA damage

Science.gov (United States)

Leloup, Corinne; Hopkins, Kevin M.; Wang, Xiangyuan; Zhu, Aiping; Wolgemuth, Debra J.; Lieberman, Howard B.

2010-01-01

RAD9 participates in promoting resistance to DNA damage, cell cycle checkpoint control, DNA repair, apoptosis, embryogenesis, and regulation of transcription. A paralogue of RAD9 (named RAD9B) has been identified. To define the function of mouse Rad9b (Mrad9b), embryonic stem (ES) cells with a targeted gene deletion were constructed and used to generate Mrad9b mutant mice. Mrad9b−/− embryos are resorbed after E7.5 while some of the heterozygotes die between E12.5 and a few days after birth. Mrad9b is expressed in embryonic brain and Mrad9b+/− embryos exhibit abnormal neural tube closure. Mrad9b−/− mouse embryonic fibroblasts are not viable. Mrad9b−/− ES cells are more sensitive to gamma rays and mitomycin C than Mrad9b+/+ controls, but show normal gamma-ray-induced G2/M checkpoint control. There is no evidence of spontaneous genomic instability in Mrad9b−/− cells. Our findings thus indicate that Mrad9b is essential for embryonic development and mediates resistance to certain DNA damaging agents. PMID:20842695

RadNet Air Data From Sacramento, CA

Science.gov (United States)

This page presents radiation air monitoring and air filter analysis data for Sacramento, CA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

RadNet Air Data From Honolulu, HI

Science.gov (United States)

This page presents radiation air monitoring and air filter analysis data for Honolulu, HI from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

RadNet Air Data From Houston, TX

Science.gov (United States)

This page presents radiation air monitoring and air filter analysis data for Houston, TX from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

RadNet Air Data From Austin, TX

Science.gov (United States)

This page presents radiation air monitoring and air filter analysis data for Austin, TX from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

RadNet Air Data From Orlando, FL

Science.gov (United States)

This page presents radiation air monitoring and air filter analysis data for Orlando, FL from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

Effects of the rad52 gene on recombination in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Prakash, S.; Prakash, L.; Burke, W.; Montelone, B.A.

1980-01-01

Effects of the rad 52 mutation in Saccharomyces cerevisiae on meiotic, γ-ray-induced, uv-induced and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Both intra and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the his1-1/his1-315 and trp-5-2/trp5-48 heteroalleles. Gene-centromere recombination also was not observed in rad/52/rad52 diploids. No γ-ray- or uv-induced intragenic mitotic recombination is seen in rad52/rad52 diploids. The rate of spontaneous mitotic recombination is lowered five-fold at the his1-1/his1-315 and leu1-c/leu1-12 heteroalleles. Spontaneous reversion rates of both his1-1 and his1-315 were elevated 10 to 20 fold in rad52/rad52 diploids. The RAD52 gene function is required for spontaneous mitotic recombination, uv- and γ-ray-induced mitotic recombination and mitotic recombination

Performance of the RAD-57 pulse CO-oximeter compared with standard laboratory carboxyhemoglobin measurement.

Science.gov (United States)

Touger, Michael; Birnbaum, Adrienne; Wang, Jessica; Chou, Katherine; Pearson, Darion; Bijur, Polly

2010-10-01

We assess agreement between carboxyhemoglobin levels measured by the Rad-57 signal extraction pulse CO-oximeter (RAD), a Food and Drug Administration-approved device for noninvasive bedside measurement, and standard laboratory arterial or venous measurement in a sample of emergency department (ED) patients with suspected carbon monoxide poisoning. The study was a cross-sectional cohort design using a convenience sample of adult and pediatric ED patients in a Level I trauma, burn, and hyperbaric oxygen referral center. Measurement of RAD carboxyhemoglobin was performed simultaneously with blood sampling for laboratory determination of carboxyhemoglobin level. The difference between the measures for each patient was calculated as laboratory carboxyhemoglobin minus carboxyhemoglobin from the carbon monoxide oximeter. The limits of agreement from a Bland-Altman analysis are calculated as the mean of the differences between methods ±1.96 SDs above and below the mean. Median laboratory percentage carboxyhemoglobin level was 2.3% (interquartile range 1 to 8.5; range 0% to 38%). The mean difference between laboratory carboxyhemoglobin values and RAD values was 1.4% carboxyhemoglobin (95% confidence interval [CI] 0.2% to 2.6%). The limits of agreement of differences of measurement made with the 2 devices were -11.6% and 14.4% carboxyhemoglobin. This range exceeded the value of ±5% carboxyhemoglobin defined a priori as clinically acceptable. RAD correctly identified 11 of 23 patients with laboratory values greater than 15% carboxyhemoglobin (sensitivity 48%; 95% CI 27% to 69%). There was one case of a laboratory carboxyhemoglobin level less than 15%, in which the RAD device gave a result greater than 15% (specificity of RAD 96/97=99%; 95% CI 94% to 100%). In the range of carboxyhemoglobin values measured in this sample, the level of agreement observed suggests RAD measurement may not be used interchangeably with standard laboratory measurement. Copyright © 2010 American

The leukodystrophy protein FAM126A (hyccin) regulates PtdIns(4)P synthesis at the plasma membrane.

Science.gov (United States)

Baskin, Jeremy M; Wu, Xudong; Christiano, Romain; Oh, Michael S; Schauder, Curtis M; Gazzerro, Elisabetta; Messa, Mirko; Baldassari, Simona; Assereto, Stefania; Biancheri, Roberta; Zara, Federico; Minetti, Carlo; Raimondi, Andrea; Simons, Mikael; Walther, Tobias C; Reinisch, Karin M; De Camilli, Pietro

2016-01-01

Genetic defects in myelin formation and maintenance cause leukodystrophies, a group of white matter diseases whose mechanistic underpinnings are poorly understood. Hypomyelination and congenital cataract (HCC), one of these disorders, is caused by mutations in FAM126A, a gene of unknown function. We show that FAM126A, also known as hyccin, regulates the synthesis of phosphatidylinositol 4-phosphate (PtdIns(4)P), a determinant of plasma membrane identity. HCC patient fibroblasts exhibit reduced PtdIns(4)P levels. FAM126A is an intrinsic component of the plasma membrane phosphatidylinositol 4-kinase complex that comprises PI4KIIIα and its adaptors TTC7 and EFR3 (refs 5,7). A FAM126A-TTC7 co-crystal structure reveals an all-α-helical heterodimer with a large protein-protein interface and a conserved surface that may mediate binding to PI4KIIIα. Absence of FAM126A, the predominant FAM126 isoform in oligodendrocytes, destabilizes the PI4KIIIα complex in mouse brain and patient fibroblasts. We propose that HCC pathogenesis involves defects in PtdIns(4)P production in oligodendrocytes, whose specialized function requires massive plasma membrane expansion and thus generation of PtdIns(4)P and downstream phosphoinositides. Our results point to a role for FAM126A in supporting myelination, an important process in development and also following acute exacerbations in multiple sclerosis.

RAD51B in Familial Breast Cancer

OpenAIRE

Pelttari, L.M.; Khan, S.; et al.,

2016-01-01

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737\\ud and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast\\ud cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer\\ud predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the\\ud coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for\\ud identifi...

RAD51B in familial breast cancer

OpenAIRE

Pelttari, LM; Khan, S; Vuorela, M; Kiiski, JI; Vilske, S; Nevanlinna, V; Ranta, S; Schleutker, J; Winqvist, R; Kallioniemi, A; Dörk, T; Bogdanova, NV; Figueroa, J; Pharoah, PDP; Schmidt, MK

2016-01-01

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possi...

Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

Science.gov (United States)

Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

2012-01-01

The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells.

Science.gov (United States)

Schaletzki, Yvonne; Kromrey, Marie-Luise; Bröderdorf, Susanne; Hammer, Elke; Grube, Markus; Hagen, Paul; Sucic, Sonja; Freissmuth, Michael; Völker, Uwe; Greinacher, Andreas; Rauch, Bernhard H; Kroemer, Heyo K; Jedlitschky, Gabriele

2017-01-05

The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit β3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.

Stratification of mammographic computerized analysis by BI-RADS categories

Energy Technology Data Exchange (ETDEWEB)

Lederman, Richard [Department of Radiology, Hadassah University Hospital, Ein Kerem, Jerusalem (Israel); Leichter, Isaac [Department of Electro-Optics, Jerusalem College of Technology, P.O.B. 16031, Jerusalem (Israel); Buchbinder, Shalom [Department of Radiology of The Montefiore Medical Center, The University Hospital for the Albert Einstein College of Medicine, Bronx, New York (United States); Novak, Boris [Department of Applied Mathematics, Jerusalem College of Technology, P.O.B. 16031, Jerusalem 91160 (Israel); Bamberger, Philippe [Department of Electronics, Jerusalem College of Technology, POB 16031, Jerusalem (Israel); Fields, Scott [Department of Radiology, Hadassah University Hospital, Mt. Scopus, Jerusalem (Israel)

2003-02-01

The Breast Imaging Reporting and Data System (BI-RADS) was implemented to standardize characterization of mammographic findings. The purpose of the present study was to evaluate in which BI-RADS categories the changes recommended by computerized mammographic analysis are most beneficial. Archival cases including, 170 masses (101 malignant, 69 benign) and 63 clusters of microcalcifications (MCs; 36 malignant, 27 benign), were evaluated retrospectively, using the BI-RADS categories, by several radiologists, blinded to the pathology results. A computerized system then automatically extracted from the digitized mammogram features characterizing mammographic lesions, which were used to classify the lesions. The results of the computerized classification scheme were compared, by receiver operating characteristics (ROC) analysis, to the conventional interpretation. In the ''low probability of malignancy group'' (excluding BI-RADS categories 4 and 5), computerized analysis improved the A{sub z}of the ROC curve significantly, from 0.57 to 0.89. In the ''high probability of malignancy group'' (mostly category 5) the computerized analysis yielded an ROC curve with an A {sub z}of 0.99. In the ''intermediate probability of malignancy group'' computerized analysis improved the A {sub z}significantly, from 0.66 for to 0.83. Pair-wise analysis showed that in the latter group the modifications resulting from computerized analysis were correct in 83% of cases. Computerized analysis has the ability to improve the performance of the radiologists exactly in the BI-RADS categories with the greatest difficulties in arriving at a correct diagnosis. It increased the performance significantly in the problematic group of ''intermediate probability of malignancy'' and pinpointed all the cases with missed cancers in the ''low probability'' group. (orig.)

Comparison of clastogen-induced gene expression profiles in wild-type and DNA repair-deficient Rad54/Rad54B cells

Directory of Open Access Journals (Sweden)

van Benthem Jan

2010-01-01

Full Text Available Abstract Background Previously we found that Rad54/Rad54B cells are more sensitive towards mitomycin C (MMC as compared to wild-type (WT cells. This difference in sensitivity was absent upon exposure to other clastogens like bleomycin (BLM and γ-radiation. In order to get further insight into possible underlying mechanisms, gene expression changes in WT and Rad54/Rad54B MEFs (mouse embryonic fibroblasts after exposure to the clastogens MMC and BLM were investigated. Exposures of these cells to mutagens (N-ac-AAF and ENU and vehicle were taken as controls. Results Most exposures resulted in an induction of DNA damage signaling and apoptosis genes and a reduced expression of cell division genes in cells of both genotypes. As expected, responses to N-ac-AAF were very similar in both genotypes. ENU exposure did not lead to significant gene expression changes in cells of both genotypes, presumably due to its short half-life. Gene expression responses to clastogens, however, showed a genotype-dependent effect for BLM and MMC. MMC treated Rad54/Rad54B MEFs showed no induction of p53-signaling, DNA damage response and apoptosis as seen for all the other treatments. Conclusion These data support our finding that different types of clastogens exist and that responses to these types depend on the DNA repair status of the cells.

STAG2 and Rad21 mammalian mitotic cohesins are implicated in meiosis

OpenAIRE

Prieto, Ignacio; Pezzi, Nieves; Buesa, José M.; Kremer, Leonor; Barthelemy, Isabel; Carreiro, Candelas; Roncal, Fernando; Martínez, Alicia; Gómez, Lucio; Fernández, Raúl; Martínez-A, Carlos; Barbero, José L.

2002-01-01

STAG/SA proteins are specific cohesin complex subunits that maintain sister chromatid cohesion in mitosis and meiosis. Two members of this family, STAG1/SA1 and STAG2/SA2,‡ are classified as mitotic cohesins, as they are found in human somatic cells and in Xenopus laevis as components of the cohesinSA1 and cohesinSA2 complexes, in which the shared subunits are Rad21/SCC1, SMC1 and SMC3 proteins. A recently reported third family member, STAG3, is germinal cell-specific and is a subunit of the ...

NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

Energy Technology Data Exchange (ETDEWEB)

Volakakis, Nikolaos; Joodmardi, Eliza [Ludwig Institute for Cancer Research Ltd., Box 240, S-17177 Stockholm (Sweden); Perlmann, Thomas, E-mail: thomas.perlmann@licr.ki.se [Ludwig Institute for Cancer Research Ltd., Box 240, S-17177 Stockholm (Sweden); The Department of Cell and Molecular Biology, Karolinska Institute, S-17177 Stockholm (Sweden)

2009-12-25

The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPAR{beta}/{delta} signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPAR{beta}/{delta} and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.

The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination

DEFF Research Database (Denmark)

Burgess, Rebecca C; Sebesta, Marek; Sisakova, Alexandra

2013-01-01

Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54...... and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage...

Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish.

Science.gov (United States)

Botthof, Jan Gregor; Bielczyk-Maczyńska, Ewa; Ferreira, Lauren; Cvejic, Ana

2017-05-30

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

Effects of the rad52 gene on recombination in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Prakash, S.; Prakash, L.; Burke, W.; Montelone, B.A.

1979-01-01

Effects of the rad52 mutation in Saccharomyces cerevisiae on meiotic, γ-ray-induced, uv-induced, and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Intra- and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the hisl-1/hisl-315 and trp5-2/trp5-48 heteroalleles. Gene-centromere recombination was also not observed in rad52/rad52 diploids. No γ-ray-induced intragenic mitotic recombination is seen in rad52/rad52 diploids and uv-induced intragenic recombination is greatly reduced. However, spontaneous mitotic recombination is not similarly affected. The RAD52 gene thus functions in recombination in meiosis and in γ-ray and uv-induced mitotic recombination but not in spontaneous mitotic recombination

Thioredoxin 1 regulation of protein S-desulfhydration

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Youngjun Ju

2016-03-01

Full Text Available The importance of H2S in biology and medicine has been widely recognized in recent years, and protein S-sulfhydration is proposed to mediate the direct actions of H2S bioactivity in the body. Thioredoxin 1 (Trx1 is an important reducing enzyme that cleaves disulfides in proteins and acts as an S-denitrosylase. The regulation of Trx1 on protein S-sulfhydration is unclear. Here we showed that Trx1 facilitates protein S-desulfhydration. Overexpression of Trx1 attenuated the basal level and H2S-induced protein S-sulfhydration by direct interaction with S-sulfhydrated proteins, i.e., glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase. In contrast, knockdown of Trx1 mRNA expression by short interfering RNA or blockage of Trx1 redox activity with PX12 or 2,4-dinitrochlorobenzene enhanced protein S-sulfhydration. Mutation of cysteine-32 but not cysteine-35 in the Trp–Cys32–Gly–Pro–Cys35 motif eliminated the binding of Trx1 with S-sulfhydrated proteins and abolished the S-desulfhydrating effect of Trx1. All these data suggest that Trx1 acts as an S-desulfhydrase.

Iron-regulated proteins (IRPS of leptospira biflexa serovar Patoc strain Patoc I

Directory of Open Access Journals (Sweden)

Sritharan M

2004-01-01

Full Text Available BACKGROUND: Iron deficiency has been shown to induce the expression of siderophores and their receptors, the iron-regulated membrane proteins in a number of bacterial systems. In this study, the response of Leptospira biflexa serovar Patoc strain Patoc I to conditions of iron deprivation was assessed and the expression of siderophores and iron-regulated proteins is reported. MATERIALS AND METHODS: Two methods were used for establishing conditions of iron deprivation. One method consisted of addition of the iron chelators ethylenediamine-N, N′-diacetic acid (EDDA and ethylenediamine di-o-hydroxyphenylacetic acid (EDDHPA and the second method involved the addition of iron at 0.02 µg Fe/mL. Alternatively, iron sufficient conditions were achieved by omitting the chelators in the former method and adding 4 µg Fe/mL of the medium in the latter protocol. Triton X-114 extraction of the cells was done to isolate the proteins in the outer membrane (detergent phase, periplasmic space (aqueous phase and the protoplasmic cylinder (cell pellet. The proteins were subjected to SDS-PAGE for analysis. RESULTS: In the presence of the iron-chelators, four iron-regulated proteins (IRPs of apparent molecular masses of 82, 64, 60 and 33 kDa were expressed. The 82-kDa protein was seen only in the aqueous phase, while the other three proteins were seen in both the aqueous and detergent fractions. These proteins were not identified in organisms grown in the absence of the iron chelators. The 64, 60 and the 33 kDa proteins were also demonstrated in organisms grown in media with 0.02 µg Fe/mL. In addition, a 24 kDa protein was found to be down-regulated at this concentration of iron as compared to the high level of expression in organisms grown with 4 µg Fe/mL. The blue CAS agar plates with top agar containing 0.02µg Fe/mL showed a colour change to orange-red. CONCLUSION: The expression of siderophores and iron-regulated proteins under conditions of iron deprivation

CXC chemokine receptor 4 expression and stromal cell-derived factor-1alpha-induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin-4 and interleukin-10

DEFF Research Database (Denmark)

Jinquan, T; Quan, S; Jacobi, H H

2000-01-01

-10. IL-4 and IL-10 up- or down-regulated CXCR4 mRNA expression in CD4+ T lymphocytes, respectively, as detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Scatchard analysis revealed a type of CXCR4 with affinity (Kd approximately 6.3 nM), and approximately 70....... The regulation of CXCR4 expression in CD4+ T lymphocytes by IL-4 and IL-10 could be blocked by a selective inhibitor of protein kinase (staurosporine) or by a selective inhibitor of cAMP- and cGMP-dependent protein kinase (H-8), indicating that these cytokines regulate CXCR4 on CD4+ T lymphocytes via both c......AMP and cGMP signalling pathways. The fact that cyclosporin A or ionomycin were able to independently change the CXCR4 expression and block the effects of IL-4 and IL-10 on CXCR4 expression implied that the capacity of IL-4 and IL-10 to regulate CXCR4 on CD4+ T lymphocytes is not linked to calcium...

Wrecked regulation of intrinsically disordered proteins in diseases: Pathogenicity of deregulated regulators

Directory of Open Access Journals (Sweden)

Vladimir N. Uversky

2014-07-01

Full Text Available Biologically active proteins without stable tertiary structure are common in all known proteomes. Functions of these intrinsically disordered proteins (IDPs are typically related to regulation, signaling and control. Cellular levels of these important regulators are tightly regulated by a variety mechanisms ranging from firmly controlled expression to precisely targeted degradation. Functions of IDPs are controlled by binding to specific partners, alternative splicing, and posttranslational modifications among other means. In the norm, right amounts of precisely activated IDPs have to be present in right time at right places. Wrecked regulation brings havoc to the ordered world of disordered proteins, leading to protein misfolding, misidentification, and missignaling that give rise to numerous human diseases, such as cancer, cardiovascular disease, neurodegenerative diseases, and diabetes. Among factors inducing pathogenic transformations of IDPs are various cellular mechanisms, such as chromosomal translocations, damaged splicing, altered expression, frustrated posttranslational modifications, aberrant proteolytic degradation, and defective trafficking. This review presents some of the aspects of deregulated regulation of IDPs leading to human diseases.

First ultrasound diagnosis of BI-RADS 3 lesions in young patients: Can 6-months follow-up be sufficient to assess stability?

Energy Technology Data Exchange (ETDEWEB)

Marcon, Magda, E-mail: magda.marcon@usz.ch [Institute of Diagnostic and Interventional Radiology, University Hospital Zurich (Switzerland); Frauenfelder, Thomas, E-mail: thomas.frauenfelder@usz.ch [Institute of Diagnostic and Interventional Radiology, University Hospital Zurich (Switzerland); Becker, Anton S., E-mail: anton.becker@usz.ch [Institute of Diagnostic and Interventional Radiology, University Hospital Zurich (Switzerland); Dedes, Konstantin J., E-mail: Konstantin.dedes@usz.ch [Department of Gynecology, University Hospital Zurich (Switzerland); Boss, Andreas, E-mail: andreas.boss@usz.ch [Institute of Diagnostic and Interventional Radiology, University Hospital Zurich (Switzerland)

2017-04-15

Objectives: To evaluate the outcome of repeated short-term follow-up with ultrasound in no high-risk young patients with a BI-RADS3 lesion at first examination. Methods: In this IRB-approved study 492 women, aged 18–34 years (mean ± standard deviation, 28 ± 4.5 years) with first breast ultrasound examination in 2012–2014 were retrospectively evaluated. Inclusion criteria were: at least one BI-RADS3 lesion and (a) biopsy/surgical excision or (b) follow-up of at least 18 months (including a 6-month follow-up). BI-RADS category assigned during follow-up and pathologic findings in cases undergoing biopsy/surgical excision were collected. At the 6- and 18-month follow-up the recommended biopsy rates (RBR) and the corresponding positive predictive value (PPV) were calculated. Results: In 97 patients, 151 BI-RADS3 lesions were identified. Biopsy/surgical excision was initially performed in 25/151 (16.5%) lesions. After 6-month, category was downgraded to BI-RADS1/2 in 23/126 (15.3%) and upgraded to BI-RADS4 in 9/126 lesions (7.1%). Pathological diagnosis of these lesions was fibroadenoma in 5 and benign phyllodes tumor in 4 cases (RBR 7%, PPV{sub bio} 44.4%). After 18-month one lesion was classified BI-RADS4 and pathological diagnosis was fibroadenoma (RBR 1.1%, PPV{sub bio} 0%). Conclusions: Our preliminary data show that follow-up imaging performed after 18 months from a first BI-RADS3 diagnosis does not affect clinical treatment and 6-month follow-up may be sufficient to assess the stability of probably benign lesions.

First ultrasound diagnosis of BI-RADS 3 lesions in young patients: Can 6-months follow-up be sufficient to assess stability?

International Nuclear Information System (INIS)

Marcon, Magda; Frauenfelder, Thomas; Becker, Anton S.; Dedes, Konstantin J.; Boss, Andreas

2017-01-01

Objectives: To evaluate the outcome of repeated short-term follow-up with ultrasound in no high-risk young patients with a BI-RADS3 lesion at first examination. Methods: In this IRB-approved study 492 women, aged 18–34 years (mean ± standard deviation, 28 ± 4.5 years) with first breast ultrasound examination in 2012–2014 were retrospectively evaluated. Inclusion criteria were: at least one BI-RADS3 lesion and (a) biopsy/surgical excision or (b) follow-up of at least 18 months (including a 6-month follow-up). BI-RADS category assigned during follow-up and pathologic findings in cases undergoing biopsy/surgical excision were collected. At the 6- and 18-month follow-up the recommended biopsy rates (RBR) and the corresponding positive predictive value (PPV) were calculated. Results: In 97 patients, 151 BI-RADS3 lesions were identified. Biopsy/surgical excision was initially performed in 25/151 (16.5%) lesions. After 6-month, category was downgraded to BI-RADS1/2 in 23/126 (15.3%) and upgraded to BI-RADS4 in 9/126 lesions (7.1%). Pathological diagnosis of these lesions was fibroadenoma in 5 and benign phyllodes tumor in 4 cases (RBR 7%, PPV bio 44.4%). After 18-month one lesion was classified BI-RADS4 and pathological diagnosis was fibroadenoma (RBR 1.1%, PPV bio 0%). Conclusions: Our preliminary data show that follow-up imaging performed after 18 months from a first BI-RADS3 diagnosis does not affect clinical treatment and 6-month follow-up may be sufficient to assess the stability of probably benign lesions.

Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes

International Nuclear Information System (INIS)

Cole, G.M.; Mortimer, R.K.

1989-01-01

The Saccharomyces cerevisiae RAD54 gene is transcriptionally regulated by a broad spectrum of DNA-damaging agents. Induction of RAD54 by DNA-damaging agents is under positive control. Sequences responsible for DNA damage induction (the DRS element) lie within a 29-base-pair region from -99 to -70 from the most proximal transcription start site. This inducible promoter element is functionally separable from a poly(dA-dT) region immediately downstream which is required for constitutive expression. Deletions which eliminate induction of RAD54 transcription by DNA damage but do not affect constitutive expression have no effect on growth or survival of noninducible strains relative to wild-type strains in the presence of DNA-damaging agents. The DRS element is also not required for homothallic mating type switching, transcriptional induction of RAD54 during meiosis, meiotic recombination, or spontaneous or X-ray-induced mitotic recombination. We find no phenotype for a lack of induction of RAD54 message via the damage-inducible DRS, which raises significant questions about the physiology of DNA damage induction in S. cerevisiae

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

Science.gov (United States)

Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

2016-07-29

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Physical mapping and cloning of RAD56

DEFF Research Database (Denmark)

Mathiasen, David P; Gallina, Irene; Germann, Susanne Manuela

2013-01-01

Here we report the physical mapping of the rad56-1 mutation to the NAT3 gene, which encodes the catalytic subunit of the NatB N-terminal acetyltransferase in Saccharomyces cerevisiae. Mutation of RAD56 causes sensitivity to X-rays, methyl methanesulfonate, zeocin, camptothecin and hydroxyurea...

PI-RADS classification. Structured reporting for MRI of the prostate

International Nuclear Information System (INIS)

Roethke, Matthias; Schlemmer, H.P.; Blondin, D.; Franiel, T.

2013-01-01

Purpose: To flesh out the ESUR guidelines for the standardized interpretation of multiparametric magnetic resonance imaging (mMRI) for the detection of prostate cancer and to present a graphic reporting scheme for improved communication of findings to urologists. Materials and Methods: The ESUR has recently published a structured reporting system for mMRI of the prostate (PI-RADS). This system involves the use of 5-point Likert scales for grading the findings obtained with different MRI techniques. The mMRI includes T2-weighted MRI, diffusion-weighted imaging, dynamic contrast-enhanced MRI, and MR spectroscopy. In a first step, the fundamentals of technical implementation were determined by consensus, taking into account in particular the German-speaking community. Then, representative images were selected by consensus on the basis of examinations of the three institutions. In addition, scoring intervals for an aggregated PI-RADS score were determined in consensus. Results: The multiparametric methods were discussed critically with regard to implementation and the current status. Criteria used for grading mMRI findings with the PI-RADS classification were concretized by succinct examples. Using the consensus table for aggregated scoring in a clinical setting, a diagnosis of suspected prostate cancer should be made if the PI-RADS score is 4 or higher (≥ 10 points if 3 techniques are used or ≥ 13 points if 4 techniques are used). Finally, a graphic scheme was developed for communicating mMRI prostate findings. Conclusion: Structured reporting according to the ESUR guidelines contributes to quality assurance by standardizing prostate mMRI, and it facilities the communication of findings to urologists. (orig.)

RAD51B in Familial Breast Cancer

DEFF Research Database (Denmark)

Pelttari, Liisa M; Khan, Sofia; Vuorela, Mikko

2016-01-01

Common variation on 14q24.1, close to RAD51B, has been associated with breast cancer: rs999737 and rs2588809 with the risk of female breast cancer and rs1314913 with the risk of male breast cancer. The aim of this study was to investigate the role of RAD51B variants in breast cancer predisposition......, particularly in the context of familial breast cancer in Finland. We sequenced the coding region of RAD51B in 168 Finnish breast cancer patients from the Helsinki region for identification of possible recurrent founder mutations. In addition, we studied the known rs999737, rs2588809, and rs1314913 SNPs and RAD......51B haplotypes in 44,791 breast cancer cases and 43,583 controls from 40 studies participating in the Breast Cancer Association Consortium (BCAC) that were genotyped on a custom chip (iCOGS). We identified one putatively pathogenic missense mutation c.541C>T among the Finnish cancer patients...

Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break.

Directory of Open Access Journals (Sweden)

M Scott Brown

2015-12-01

Full Text Available The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs. Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt Rad51 filaments and also by one or more short Dmc1 filaments.

Challenges in biotechnology at LLNL: from genes to proteins; TOPICAL

International Nuclear Information System (INIS)

Albala, J S

1999-01-01

This effort has undertaken the task of developing a link between the genomics, DNA repair and structural biology efforts within the Biology and Biotechnology Research Program at LLNL. Through the advent of the I.M.A.G.E. (Integrated Molecular Analysis of Genomes and their Expression) Consortium, a world-wide effort to catalog the largest public collection of genes, accepted and maintained within BBRP, it is now possible to systematically express the protein complement of these to further elucidate novel gene function and structure. The work has ensued in four phases, outlined as follows: (1) Gene and System selection; (2) Protein expression and purification; (3) Structural analysis; and (4) biological integration. Proteins to be expressed have been those of high programmatic interest. This includes, in particular, proteins involved in the maintenance of genome integrity, particularly those involved in the repair of DNA damage, including ERCC1, ERCC4, XRCC2, XRCC3, XRCC9, HEX1, APN1, p53, RAD51B, RAD51C, and RAD51. Full-length cDNA cognates of selected genes were isolated, and cloned into baculovirus-based expression vectors. The baculoviral expression system for protein over-expression is now well-established in the Albala laboratory. Procedures have been successfully optimized for full-length cDNA clining into expression vectors for protein expression from recombinant constructs. This includes the reagents, cell lines, techniques necessary for expression of recombinant baculoviral constructs in Spodoptera frugiperda (Sf9) cells. The laboratory has also generated a high-throughput baculoviral expression paradigm for large scale expression and purification of human recombinant proteins amenable to automation

Effect of complete protein 4.1R deficiency on ion transport properties of murine erythrocytes

International Nuclear Information System (INIS)

Rivera, Alicia; De Franceschi, Lucia; Peters, Luanne L.; Gascard, Philippe; Mohandas, Narla; Brugnara, Carlo

2006-01-01

Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1-/-; Shi TS et al., J. Clin. Invest. 103:331,1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1-/- mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1-/- erythrocytes was markedly activated by exposure to hypertonic conditions (18.2+- 3.2 in 4.1 -/- vs.9.8 +- 1.3 mmol/1013 cell x h in control mice), with an abnormal dependence on osmolarity, (K0.5=417 +- 42 in 4.1 -/- vs. 460 +- 35 mOsmin control mice) suggestive of an up-regulated functional state. While the affinity for internal protons was not altered (K0.5= 489.7 +- 0.7 vs.537.0 +- 0.56 nM in control mice), the Vmax of the H-induced Na/H exchange activity was markedly elevated in 4.1-/- erythrocytes Vmax 91.47 Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1-/-; Shi TSet al., J. Clin. Invest. 103:331,1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1-/- mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1-/- erythrocytes was markedly activated by exposure to hypertonic conditions (18.2 +- 3.2 in 4.1 -/- vs. 9.8 +- 1.3mmol/1013 cell x h in

O-GlcNAc transferase regulates transcriptional activity of human Oct4.

Science.gov (United States)

Constable, Sandii; Lim, Jae-Min; Vaidyanathan, Krithika; Wells, Lance

2017-10-01

O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

Science.gov (United States)

Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen-Ling; Bishop, Douglas K.; Haber, James E.

2015-01-01

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

Mongoose: Creation of a Rad-Hard MIPS R3000

Science.gov (United States)

Lincoln, Dan; Smith, Brian

1993-01-01

This paper describes the development of a 32 Bit, full MIPS R3000 code-compatible Rad-Hard CPU, code named Mongoose. Mongoose progressed from contract award, through the design cycle, to operational silicon in 12 months to meet a space mission for NASA. The goal was the creation of a fully static device capable of operation to the maximum Mil-883 derated speed, worst-case post-rad exposure with full operational integrity. This included consideration of features for functional enhancements relating to mission compatibility and removal of commercial practices not supported by Rad-Hard technology. 'Mongoose' developed from an evolution of LSI Logic's MIPS-I embedded processor, LR33000, code named Cobra, to its Rad-Hard 'equivalent', Mongoose. The term 'equivalent' is used to infer that the core of the processor is functionally identical, allowing the same use and optimizations of the MIPS-I Instruction Set software tool suite for compilation, software program trace, etc. This activity was started in September of 1991 under a contract from NASA-Goddard Space Flight Center (GSFC)-Flight Data Systems. The approach affected a teaming of NASA-GSFC for program development, LSI Logic for system and ASIC design coupled with the Rad-Hard process technology, and Harris (GASD) for Rad-Hard microprocessor design expertise. The program culminated with the generation of Rad-Hard Mongoose prototypes one year later.

Annex VII - Diagrams: 1. Reactor operation (1960-1977); 2. Mean daily reactor power density in 1977; 3. Monthly reactor power for 1977; 4. percent of utilization of experimental space in 1977; Prilog VII - Dijagrami: 1. Rad reaktora (MWh) po godinama (1960-1977); 2. Srednja dnevna snaga reaktora u 1977. godini; 3. Rad reaktora (MWh) po mesecima za 1977. godinu i 4. Procenat iskoriscenja eksperimentalnog prostora u 1977. godini

Energy Technology Data Exchange (ETDEWEB)

NONE

1977-12-15

This Annex includes the following diagrams: 1. Annual Reactor RA power production (MWh) for the period from 1960-1977; 2. Mean daily reactor power density MW in 1977; 3. Monthly reactor power production (MWh) for 1977; 4. percent of utilization of experimental space in 1977. [Serbo-Croat] Ovaj prilog sadrzi dijagrame: 1. Rad reaktora (MWh) po godinama (1960-1977); 2. Srednja dnevna snaga reaktora u 1977. godini; 3. Rad reaktora (MWh) po mesecima za 1977. godinu i 4. Procenat iskoriscenja eksperimentalnog prostora u 1977. godini.

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

Science.gov (United States)

Dohn, Michael R; Mundell, Nathan A; Sawyer, Leah M; Dunlap, Julie A; Jessen, Jason R

2013-11-01

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular

Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d.

Science.gov (United States)

Kero, Darko; Vukojevic, Katarina; Stazic, Petra; Sundov, Danijela; Mardesic Brakus, Snjezana; Saraga-Babic, Mirna

2017-10-02

Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19 INK4d . p19 INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19 INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19 INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19 INK4d throughout the investigated period indicates that p19 INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19 INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.

Controlling parasympathetic regulation of heart rate: a gatekeeperrole for RGS proteins in the sinoatrial node

Directory of Open Access Journals (Sweden)

Alexandra eMighiu

2012-06-01

Full Text Available Neurotransmitters released from sympathetic and parasympathetic nerve terminals in the SAN exert their effects via G-protein-coupled receptors. Integration of these different G-protein signals within pacemaker cells of the sinoatrial node (SAN is critical for proper regulation of heart rate and function. For example, excessive parasympathetic signaling can be associated with sinus node dysfunction and supraventricular arrhythmias. Our previous work has shown that one member of the regulator of G-protein signaling (RGS protein family, RGS4, is highly and selectively expressed in pacemaker cells of the SAN. Consistent with its role as an inhibitor of parasympathetic signaling, RGS4-knockout mice have reduced basal heart rates and enhanced negative chronotropic responses to parasympathetic agonists. Moreover, RGS4 appears to be an important part of SA nodal myocyte signaling pathways that mediate G protein-coupled inwardly-rectifying potassium channel (GIRK channel activation/deactivation and desensitization. Since RGS4 acts immediately downstream of M2 muscarinic receptors, it is tempting to speculate that RGS4 functions as a master regulator of parasympathetic signaling upstream of GIRKs, HCNs and L-type Ca2+ channels in the SAN. Thus, loss of RGS4 function may lead to increased susceptibility to conditions associated with increased parasympathetic signaling, including bradyarrhythmia, sinus node dysfunction, and atrial fibrillation.

RadSTraM: Radiological Source Tracking and Monitoring, Phase II Final Report

Energy Technology Data Exchange (ETDEWEB)

Warren, Tracy A [ORNL; Walker, Randy M [ORNL; Hill, David E [ORNL; Gross, Ian G [ORNL; Smith, Cyrus M [ORNL; Abercrombie, Robert K [ORNL

2008-12-01

This report focuses on the technical information gained from the Radiological Source Tracking and Monitoring (RadSTraM) Phase II investigation and its implications. The intent of the RadSTraM project was to determine the feasibility of tracking radioactive materials in commerce, particularly International Atomic Energy Agency (IAEA) Category 3 and 4 materials. Specifically, Phase II of the project addressed tracking radiological medical isotopes in commerce. These categories of materials are susceptible to loss or theft but the problem is not being addressed by other agencies.

RadSTraM: Radiological Source Tracking and Monitoring, Phase II Final Report

International Nuclear Information System (INIS)

Warren, Tracy A.; Walker, Randy M.; Hill, David E.; Gross, Ian G.; Smith, Cyrus M.; Abercrombie, Robert K.

2008-01-01

This report focuses on the technical information gained from the Radiological Source Tracking and Monitoring (RadSTraM) Phase II investigation and its implications. The intent of the RadSTraM project was to determine the feasibility of tracking radioactive materials in commerce, particularly International Atomic Energy Agency (IAEA) Category 3 and 4 materials. Specifically, Phase II of the project addressed tracking radiological medical isotopes in commerce. These categories of materials are susceptible to loss or theft but the problem is not being addressed by other agencies

Regulated eukaryotic DNA replication origin firing with purified proteins.

Science.gov (United States)

Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

2015-03-26

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

Regulation of human protein S gene (PROS1) transcription

NARCIS (Netherlands)

Wolf, Cornelia de

2006-01-01

This thesis describes the investigation of the transcriptional regulation of the gene for anticoagulant plasma Protein S, PROS1. Protein S is a cofactor for Protein C in the Protein C anticoagulant pathway. The coagulation cascade is negatively regulated by this pathway through inactivation of

Ankyrin repeat and SOCS box containing protein 4 (Asb-4 colocalizes with insulin receptor substrate 4 (IRS4 in the hypothalamic neurons and mediates IRS4 degradation

Directory of Open Access Journals (Sweden)

Xia Zefeng

2011-09-01

Full Text Available Abstract Background The arcuate nucleus of the hypothalamus regulates food intake. Ankyrin repeat and SOCS box containing protein 4 (Asb-4 is expressed in neuropeptide Y and proopiomelanocortin (POMC neurons in the arcuate nucleus, target neurons in the regulation of food intake and metabolism by insulin and leptin. However, the target protein(s of Asb-4 in these neurons remains unknown. Insulin receptor substrate 4 (IRS4 is an adaptor molecule involved in the signal transduction by both insulin and leptin. In the present study we examined the colocalization and interaction of Asb-4 with IRS4 and the involvement of Asb-4 in insulin signaling. Results In situ hybridization showed that the expression pattern of Asb-4 was consistent with that of IRS4 in the rat brain. Double in situ hybridization showed that IRS4 colocalized with Asb-4, and both Asb-4 and IRS4 mRNA were expressed in proopiomelanocortin (POMC and neuropeptide Y (NPY neurons within the arcuate nucleus of the hypothalamus. In HEK293 cells co-transfected with Myc-tagged Asb-4 and Flag-tagged IRS4, Asb-4 co-immunoprecipitated with IRS4; In these cells endogenous IRS4 also co-immunoprecipitated with transfected Myc-Asb-4; Furthermore, Asb-4 co-immunoprecipitated with IRS4 in rat hypothalamic extracts. In HEK293 cells over expression of Asb-4 decreased IRS4 protein levels and deletion of the SOCS box abolished this effect. Asb-4 increased the ubiquitination of IRS4; Deletion of SOCS box abolished this effect. Expression of Asb-4 decreased both basal and insulin-stimulated phosphorylation of AKT at Thr308. Conclusions These data demonstrated that Asb-4 co-localizes and interacts with IRS4 in hypothalamic neurons. The interaction of Asb-4 with IRS4 in cell lines mediates the degradation of IRS4 and decreases insulin signaling.

Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues.

Science.gov (United States)

Schulz, Timothy A; Choi, Mal-Gi; Raychaudhuri, Sumana; Mears, Jason A; Ghirlando, Rodolfo; Hinshaw, Jenny E; Prinz, William A

2009-12-14

Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein-related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.

The Saccharomyces cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA damage inducible and functions in a novel error-free postreplication repair mechanism

Energy Technology Data Exchange (ETDEWEB)

McDonald, J. P. [NIH, Bethesda, MD. (United States); Levine, A. S.; Woodgate, R.

1997-12-15

Damage-inducible mutagenesis in prokaryotes is largely dependent upon the activity of the UmuD'C-like proteins. Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized Saccharomyces cerevisiae DNA repair gene related to the Escherichia coli dinB, umuC and S. cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis. Similar to its prokaryotic homologues, RAD30 was found to be damage inducible. Like many S. cerevisiae genes involved in error-prone DNA repair, epistasis analysis clearly places RAD30 in the RAD6 group and rad30 mutants display moderate UV sensitivity reminiscent of rev mutants. However, unlike rev mutants, no defect in UV-induced reversion was seen in rad30 strains. While rad6 and rad18 are both epistatic to rad30, no epistasis was observed with rev1, rev3, rev7 or rad5, all of which are members of the RAD6 epistasis group. These findings suggest that RD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5. (author)

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert

2014-01-01

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean

2014-07-10

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Survival and growth of yeast without telomere capping by Cdc13 in the absence of Sgs1, Exo1, and Rad9.

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Hien-Ping Ngo

2010-08-01

Full Text Available Maintenance of telomere capping is absolutely essential to the survival of eukaryotic cells. Telomere capping proteins, such as Cdc13 and POT1, are essential for the viability of budding yeast and mammalian cells, respectively. Here we identify, for the first time, three genetic modifications that allow budding yeast cells to survive without telomere capping by Cdc13. We found that simultaneous inactivation of Sgs1, Exo1, and Rad9, three DNA damage response (DDR proteins, is sufficient to allow cell division in the absence of Cdc13. Quantitative amplification of ssDNA (QAOS was used to show that the RecQ helicase Sgs1 plays an important role in the resection of uncapped telomeres, especially in the absence of checkpoint protein Rad9. Strikingly, simultaneous deletion of SGS1 and the nuclease EXO1, further reduces resection at uncapped telomeres and together with deletion of RAD9 permits cell survival without CDC13. Pulsed-field gel electrophoresis studies show that cdc13-1 rad9Delta sgs1Delta exo1Delta strains can maintain linear chromosomes despite the absence of telomere capping by Cdc13. However, with continued passage, the telomeres of such strains eventually become short and are maintained by recombination-based mechanisms. Remarkably, cdc13Delta rad9Delta sgs1Delta exo1Delta strains, lacking any Cdc13 gene product, are viable and can grow indefinitely. Our work has uncovered a critical role for RecQ helicases in limiting the division of cells with uncapped telomeres, and this may provide one explanation for increased tumorigenesis in human diseases associated with mutations of RecQ helicases. Our results reveal the plasticity of the telomere cap and indicate that the essential role of telomere capping is to counteract specific aspects of the DDR.

RadNet Air Data From Fort Smith, AR

Science.gov (United States)

This page presents radiation air monitoring and air filter analysis data for Fort Smith, AR from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

RadNet Air Data From Little Rock, AR

Science.gov (United States)

This page presents radiation air monitoring and air filter analysis data for Little Rock, AR from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

RadNet Air Data From Mason City, IA

Science.gov (United States)

This page presents radiation air monitoring and air filter analysis data for Mason City, IA from EPA's RadNet system. RadNet is a nationwide network of monitoring stations that measure radiation in air, drinking water and precipitation.

Reach Address Database (RAD)

Data.gov (United States)

U.S. Environmental Protection Agency — The Reach Address Database (RAD) stores the reach address of each Water Program feature that has been linked to the underlying surface water features (streams,...

Stealing the spotlight: CUL4-DDB1 ubiquitin ligase docks WD40-repeat proteins to destroy

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Zhang Hui

2007-02-01

Full Text Available Abstract Recent investigation of Cullin 4 (CUL4 has ushered this class of multiprotein ubiquitin E3 ligases to center stage as critical regulators of diverse processes including cell cycle regulation, developmental patterning, DNA replication, DNA damage and repair, and epigenetic control of gene expression. CUL4 associates with DNA Damage Binding protein 1 (DDB1 to assemble an ubiquitin E3 ligase that targets protein substrates for ubiquitin-dependent proteolysis. CUL4 ligase activity is also regulated by the covalent attachment of the ubiquitin-like protein NEDD8 to CUL4, or neddylation, and the COP9 signalosome complex (CSN that removes this important modification. Recently, multiple WD40-repeat proteins (WDR were found to interact with DDB1 and serve as the substrate-recognition subunits of the CUL4-DDB1 ubiquitin ligase. As more than 150–300 WDR proteins exist in the human genome, these findings impact a wide array of biological processes through CUL4 ligase-mediated proteolysis. Here, we review the recent progress in understanding the mechanism of CUL4 ubiquitin E3 ligase and discuss the architecture of CUL4-assembled E3 ubiquitin ligase complexes by comparison to CUL1-based E3s (SCF. Then, we will review several examples to highlight the critical roles of CUL4 ubiquitin ligase in genome stability, cell cycle regulation, and histone lysine methylation. Together, these studies provide insights into the mechanism of this novel ubiquitin ligase in the regulation of important biological processes.

Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism

International Nuclear Information System (INIS)

Saxena, Sunil K.; Horiuchi, Hisanori; Fukuda, Mitsunori

2006-01-01

Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays with two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins

Inhibition of HCV replication by oxysterol-binding protein-related protein 4 (ORP4 through interaction with HCV NS5B and alteration of lipid droplet formation.

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In-Woo Park

Full Text Available Hepatitis C virus (HCV RNA replication involves complex interactions among the 3'x RNA element within the HCV 3' untranslated region, viral and host proteins. However, many of the host proteins remain unknown. In this study, we devised an RNA affinity chromatography /2D/MASS proteomics strategy and identified nine putative 3' X-associated host proteins; among them is oxysterol-binding protein-related protein 4 (ORP4, a cytoplasmic receptor for oxysterols. We determined the relationship between ORP4 expression and HCV replication. A very low level of constitutive ORP4 expression was detected in hepatocytes. Ectopically expressed ORP4 was detected in the endoplasmic reticulum and inhibited luciferase reporter gene expression in HCV subgenomic replicon cells and HCV core expression in JFH-1-infected cells. Expression of ORP4S, an ORP4 variant that lacked the N-terminal pleckstrin-homology domain but contained the C-terminal oxysterol-binding domain also inhibited HCV replication, pointing to an important role of the oxysterol-binding domain in ORP4-mediated inhibition of HCV replication. ORP4 was found to associate with HCV NS5B and its expression led to inhibition of the NS5B activity. ORP4 expression had little effect on intracellular lipid synthesis and secretion, but it induced lipid droplet formation in the context of HCV replication. Taken together, these results demonstrate that ORP4 is a negative regulator of HCV replication, likely via interaction with HCV NS5B in the replication complex and regulation of intracellular lipid homeostasis. This work supports the important role of lipids and their metabolism in HCV replication and pathogenesis.

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

International Nuclear Information System (INIS)

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

2005-01-01

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian

Tousled-like kinase-dependent phosphorylation of Rad9 plays a role in cell cycle progression and G2/M checkpoint exit.

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Ryan Kelly

Full Text Available Genomic integrity is preserved by checkpoints, which act to delay cell cycle progression in the presence of DNA damage or replication stress. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1 complex is a PCNA-like clamp that is loaded onto DNA at structures resulting from damage and is important for initiating and maintaining the checkpoint response. Rad9 possesses a C-terminal tail that is phosphorylated constitutively and in response to cell cycle position and DNA damage. Previous studies have identified tousled-like kinase 1 (TLK1 as a kinase that may modify Rad9. Here we show that Rad9 is phosphorylated in a TLK-dependent manner in vitro and in vivo, and that T355 within the C-terminal tail is the primary targeted residue. Phosphorylation of Rad9 at T355 is quickly reduced upon exposure to ionizing radiation before returning to baseline later in the damage response. We also show that TLK1 and Rad9 interact constitutively, and that this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, we demonstrate via siRNA-mediated depletion that TLK1 is required for progression through S-phase in normally cycling cells, and that cells lacking TLK1 display a prolonged G2/M arrest upon exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 has previously been shown to be transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, we propose that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn serves to regulate the DNA damage response.

BET Bromodomain Proteins Brd2, Brd3 and Brd4 Selectively Regulate Metabolic Pathways in the Pancreatic β-Cell.

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Jude T Deeney

Full Text Available Displacement of Bromodomain and Extra-Terminal (BET proteins from chromatin has promise for cancer and inflammatory disease treatments, but roles of BET proteins in metabolic disease remain unexplored. Small molecule BET inhibitors, such as JQ1, block BET protein binding to acetylated lysines, but lack selectivity within the BET family (Brd2, Brd3, Brd4, Brdt, making it difficult to disentangle contributions of each family member to transcriptional and cellular outcomes. Here, we demonstrate multiple improvements in pancreatic β-cells upon BET inhibition with JQ1 or BET-specific siRNAs. JQ1 (50-400 nM increases insulin secretion from INS-1 cells in a concentration dependent manner. JQ1 increases insulin content in INS-1 cells, accounting for increased secretion, in both rat and human islets. Higher concentrations of JQ1 decrease intracellular triglyceride stores in INS-1 cells, a result of increased fatty acid oxidation. Specific inhibition of both Brd2 and Brd4 enhances insulin transcription, leading to increased insulin content. Inhibition of Brd2 alone increases fatty acid oxidation. Overlapping yet discrete roles for individual BET proteins in metabolic regulation suggest new isoform-selective BET inhibitors may be useful to treat insulin resistant/diabetic patients. Results imply that cancer and diseases of chronic inflammation or disordered metabolism are related through shared chromatin regulatory mechanisms.

BET Bromodomain Proteins Brd2, Brd3 and Brd4 Selectively Regulate Metabolic Pathways in the Pancreatic β-Cell.