Disparate requirements for the Walker A and B ATPase motifs ofhuman RAD51D in homologous recombination

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Wiese, Claudia; Hinz, John M.; Tebbs, Robert S.; Nham, Peter B.; Urbin, Salustra S.; Collins, David W.; Thompson, Larry H.; Schild, David

2006-04-21

In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks. Ectopic expression of wild type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs.

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

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

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

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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.

Enhancement of the RAD51 Recombinase Activity by the Tumor Suppressor PALB2

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Dray, Eloise; Etchin, Julia; Wiese, Claudia; Saro, Dorina; Williams, Gareth J.; Hammel, Michal; Yu, Xiong; Galkin, Vitold E.; Liu, Dongqing; Tsai, Miaw-Sheue; Sy, Shirley M-H.; Egelman, Edward; Chen, Junjie; Sung, Patrick; Schild, D.

2010-08-24

Homologous recombination mediated by the RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-stranded breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2 in the enhancement of RAD51's ability to form the D-loop. We show that PALB2 binds DNA and physically interacts with RAD51. Importantly, while PALB2 alone stimulates D-loop formation, a cooperative effect is seen with RAD51AP1, an enhancer of RAD51. This stimulation stems from PALB2's ability to function with RAD51 and RAD51AP1 to assemble the synaptic complex. Our results help unveil a multi-faceted role of PALB2 in chromosome damage repair. Since PALB2 mutations can cause breast and other tumors or lead to Fanconi anemia, our findings are important for understanding the mechanism of tumor suppression in humans.

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

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)

OsRAD51C Is Essential for Double Strand Break Repair in Rice Meiosis

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Ding eTang

2014-05-01

Full Text Available RAD51C is one of the RAD51 paralogs that plays an important role in DNA double-strand break repair by homologous recombination. Here, we identified and characterized OsRAD51C, the rice homolog of human RAD51C. The Osrad51c mutant plant is normal in vegetative growth but exhibits complete male and female sterility. Cytological investigation revealed that homologous pairing and synapsis were severely disrupted. Massive chromosome fragmentation occurred during metaphase I in Osrad51c meiocytes, and was fully suppressed by the CRC1 mutation. Immunofluorescence analysis showed that OsRAD51C localized onto the chromosomes from leptotene to early pachytene during prophase I, and that normal loading of OsRAD51C was dependent on OsREC8, PAIR2, and PAIR3. Additionally, ZEP1 did not localize properly in Osrad51c, indicating that OsRAD51C is required for synaptonemal complex assembly. Our study also provided evidence in support of a functional divergence in RAD51C among organisms.

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

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.

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

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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

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.

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.

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

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

Science.gov (United States)

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.

RAD51 interconnects between DNA replication, DNA repair and immunity.

Science.gov (United States)

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.

Congenital Mirror Movements Due to RAD51: Cosegregation with a Nonsense Mutation in a Norwegian Pedigree and Review of the Literature

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Oriane Trouillard

2016-11-01

Full Text Available Background: Autosomal dominant congenital mirror movements (CMM is a neurodevelopmental disorder characterized by early onset involuntary movements of one side of the body that mirror intentional movements on the contralateral side; these persist throughout life in the absence of other neurological symptoms. The main culprit genes responsible for this condition are RAD51 and DCC. This condition has only been reported in a few families, and the molecular mechanisms linking RAD51 mutations and mirror movements (MM are poorly understood. Methods: We collected demographic, clinical, and genetic data of a new family with CMM due to a truncating mutation of RAD51. We reviewed the literature to identify all reported patients with CMM due to RAD51 mutations. Results: We identified a heterozygous nonsense mutation c.760C>T (p.Arg254∗ in eight subjects: four with obvious and disabling MM, and four with a mild phenotype. Including our new family, we identified 32 patients from 6 families with CMM linked to RAD51 variants. Discussion: Our findings further support the involvement of RAD51 in CMM pathogenesis. Possible molecular mechanisms involved in CMM pathogenesis are discussed.

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...

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

Science.gov (United States)

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

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.

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.

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.

Regulation of homologous recombination repair protein Rad51 by Ku70

International Nuclear Information System (INIS)

Du Liqing; Liu Qiang; Wang Yan; Xu Chang; Cao Jia; Fu Yue; Chen Fenghua; Fan Feiyue

2013-01-01

Objective: To explore the regulative effect of non-homologous end joining (NHEJ)protein Ku70 on homologous recombination repair protein Rad51, and to investigate the synergistic mechanism of homologous recombination repair in combination with NHEJ. Methods: Observed Rad51 protein expression after transfect Ku70 small interfering RNA or Ku70 plasmid DNA into tumor cells using Western blot. Results: Expression of Rad51 was obviously reduced after pretreated with Ku70 small interfering RNA. And with the increasing expression of Ku70 protein after transfection of Ku70 plasmid DNA PGCsi3.0-hKu70 into tumor cell lines, the Rad51 protein expression was increased. Conclusion: Ku70 protein has regulating effect on gene expression of Rad51, and it might participate in the collaboration between homologous recombination repair and NHEJ. (authors)

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

DEFF Research Database (Denmark)

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....

Three new genetic loci (R1210C in CFH, variants in COL8A1 and RAD51B are independently related to progression to advanced macular degeneration.

Directory of Open Access Journals (Sweden)

Johanna M Seddon

Full Text Available To assess the independent impact of new genetic variants on conversion to advanced stages of AMD, controlling for established risk factors, and to determine the contribution of genes in predictive models.In this prospective longitudinal study of 2765 individuals, 777 subjects progressed to neovascular disease (NV or geographic atrophy (GA in either eye over 12 years. Recently reported genetic loci were assessed for their independent effects on incident advanced AMD after controlling for 6 established loci in 5 genes, and demographic, behavioral, and macular characteristics. New variants which remained significantly related to progression were then added to a final multivariate model to assess their independent effects. The contribution of genes to risk models was assessed using reclassification tables by determining risk within cross-classified quintiles for alternative models.THREE NEW GENETIC VARIANTS WERE SIGNIFICANTLY RELATED TO PROGRESSION: rare variant R1210C in CFH (hazard ratio (HR 2.5, 95% confidence interval [CI] 1.2-5.3, P = 0.01, and common variants in genes COL8A1 (HR 2.0, 95% CI 1.1-3.5, P = 0.02 and RAD51B (HR 0.8, 95% CI 0.60-0.97, P = 0.03. The area under the curve statistic (AUC was significantly higher for the 9 gene model (.884 vs the 0 gene model (.873, P = .01. AUC's for the 9 vs 6 gene models were not significantly different, but reclassification analyses indicated significant added information for more genes, with adjusted odds ratios (OR for progression within 5 years per one quintile increase in risk score of 2.7, P<0.001 for the 9 vs 6 loci model, and OR 3.5, P<0.001 for the 9 vs. 0 gene model. Similar results were seen for NV and GA.Rare variant CFH R1210C and common variants in COL8A1 and RAD51B plus six genes in previous models contribute additional predictive information for advanced AMD beyond macular and behavioral phenotypes.

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.

Location of RAD51-like protein during meiotic prophase in Eimeria tenella.

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Del Cacho, Emilio; Gallego, Margarita; Pagés, Marc; Barbero, José Luís; Monteagudo, Luís; Sánchez-Acedo, Caridad

2011-05-31

This study focuses on reporting events in Eimeria tenella oocysts from early to late prophase I in terms of RAD51 protein in association with the synaptonemal complex formed between homologous chromosomes. The aim of the study was the sequential localization of RAD51 protein, which is involved in the repair of double-strand breaks (DSBs) on the eimerian chromosomes as they synapse and desynapse. Structural Maintenance of Chromosome protein SMC3, which plays a role in synaptonemal complex formation, was labeled to identify initiation and progress of chromosome synapsis and desynapsis in parallel with the appearance and disappearance of RAD51 foci. Antibodies directed against RAD51 and cohesin subunit SMC3 proteins were labeled with either fluorescence or colloidal gold to visualize RAD51 protein foci and synaptonemal complexes. RAD51 protein localization during prophase I was studied on meiotic chromosomes spreads obtained from oocysts at different points in time after the start of sporulation. The present findings showed that foci detected with the antibody directed against RAD51 protein first appeared at the pre-leptotene stage before homologous chromosomes began pairing. Subsequently, the foci were detected in association with the lateral elements at the precise sites where synapsis were in progress. These findings lead us to suggest that in E. tenella, homologous chromosome pairing was a DSB-dependent mechanism and reinforced the participation of RAD51 protein in meiotic homology search, alignment and pairing of chromosomes. Copyright © 2010 Elsevier B.V. All rights reserved.

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

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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.

Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

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Callender, Tracy L; Laureau, Raphaelle; Wan, Lihong; Chen, Xiangyu; Sandhu, Rima; Laljee, Saif; Zhou, Sai; Suhandynata, Ray T; Prugar, Evelyn; Gaines, William A; Kwon, YoungHo; Börner, G Valentin; Nicolas, Alain; Neiman, Aaron M; Hollingsworth, Nancy M

2016-08-01

During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.

Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

Directory of Open Access Journals (Sweden)

Tracy L Callender

2016-08-01

Full Text Available During meiosis, programmed double strand breaks (DSBs are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i phosphorylation of Rad54 by Mek1 and (ii binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.

Resolving RAD51C function in late stages of homologous recombination

Directory of Open Access Journals (Sweden)

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.

Effect of Rad 51 overexpression on chromosomal stability and radiation sensitivity in tumour cells

International Nuclear Information System (INIS)

Jend, C.; Stuerzbecher, H.W.; Dikomey, E.; Borgmann, K.

2004-01-01

The present study was dedicated to examining the effects of Rad51 overexpression on genomic instability, expressed in terms of chromosomal aberrations in G1 and G2 phases following X-ray irradiation. For this purpose an osteosarcoma cell line (Ui-OS) which shows inducing Rad51 overexpression (UiRad5-2) after stable transfection was compared with an isogenetic line (UiLacZ) which overexpresses beta-galactosidase instead of Rad51 [de

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...

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

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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.

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

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Henson, Sarah E. [Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Tsai, Shih-Chang [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Malone, Cindy Sue [Department of Biology, California State University Northridge, Northridge, CA 91330 (United States); Soghomonian, Shahe V. [Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Ouyang, Yan [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Wall, Randolph [Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Molecular Biology Institute and Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Marahrens, York [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States) and Molecular Biology Institute and Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States)]. E-mail: YMarahrens@mednet.ucla.edu; Teitell, Michael A. [Molecular Biology Institute and Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States) and Department of Pathology and Laboratory Medicine, California NanoSystems Institute, and Institute for Stem Cell Biology and Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States)]. E-mail: mteitell@ucla.edu

2006-10-10

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.

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

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)

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

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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.

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

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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.

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

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

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

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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.

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...

The role of Rad 51 protein in radioresistance of spheroid model of Du 145 prostate carcinoma cell line

International Nuclear Information System (INIS)

Taghizadeh, M.; Khoei, S.; Nikoofar, A. R.; Ghamsari, L.; Goliaei, B.

2009-01-01

Rad 51 is a protein with critical role in double strand break repair. Down-regulation of this protein has a significant effect in radiosensitivity of some cell lines like prostate carcinoma. Compared to monolayer cell culture model, the spheroids are more resistant to radiation. The aim of the current study was to determine the Rad 51 protein level in Du 145 spheroids, and monolayer cells before and after exposure to gamma irradiation. Materials and Methods: In the present study, western blot was used to determine the level of Rad 51 protein in Du 145 cell line grown as monolayer and spheroid. Results: Western blot analysis showed that in the spheroid cells, Rad 51 had an elevated level before and after radiation in comparison with monolayer cells. Higher doses of radiation induced elevated expression of Rad 51 protein in both culture models.The level of at protein after exposure to gamma rays had been time-dependent. Conclusion: Rad 51 might act as a mediator of radiation resistance in tumor cells. Repression of Rad 51 activity could be a prominent strategy to overcome radiation resistance of tumors.

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

DEFF Research Database (Denmark)

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 of an E. coli RecA and yeast RAD51 homolog, radA, an allele of the uvsC in Aspergillus nidulans and its mutator effects.

Science.gov (United States)

Seong, K Y; Chae, S K; Kang, H S

1997-04-30

An E. coli RecA and yeast RAD51 homolog from Aspergillus nidulans, radA, has been cloned by screening genomic and cDNA libraries with a PCR-amplified probe. This probe was generated using primers carrying the conserved sequences of eukaryotic RecA homologs. The deduced amino acid sequence revealed two conserved Walker-A and -B type nucleotide-binding domains and exhibited 88%, 60%, and 53% identity with Mei-3 of Neurospora crassa, rhp51+ of Schizosaccharomyces pombe, and Rad51 of Saccharomyces cerevisiae, respectively. radA null mutants constructed by replacing the whole coding region with a selection marker showed high methyl methanesulfonate (MMS) sensitivity. Heterozygous diploids of radA disruptant with the uvsC114 mutant failed to complement with respect to MMS-sensitivity, indicating that radA is an allele of uvsC. In selecting spontaneous forward selenate resistant mutations, mutator effects were observed in radA null mutants similarly to those shown in uvsC114 mutant strains.

Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

DEFF Research Database (Denmark)

Gao, Min; Wei, Wei; Li, Ming Hua

2014-01-01

resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs...

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

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

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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)

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

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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.

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

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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.

RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA.

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Wolf, Christine; Rapp, Alexander; Berndt, Nicole; Staroske, Wolfgang; Schuster, Max; Dobrick-Mattheuer, Manuela; Kretschmer, Stefanie; König, Nadja; Kurth, Thomas; Wieczorek, Dagmar; Kast, Karin; Cardoso, M Cristina; Günther, Claudia; Lee-Kirsch, Min Ae

2016-05-27

Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA.

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)

Evidence that a recombinationless strain, rad 51, of Saccharomyces cerevisiae lacks the budding cell resistance to γ-rays

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Hama-Inaba, Hiroko; Saeki, Tetsuya

1975-01-01

The radiosensitivities of a wild-type and x-ray sensitive mutant, rad 51 (defective in genetic recombination) of Saccharomyces cerevisiae to γ-rays were compared, using non-synchronized and partially synchronized cultures. The rad 51 cells, either haploid or diploid, showed only very small changes in radiosensitivity during cell growth, whereas the wild-type cells, especially haploid, showed the well-known budding resistance. The heterozygous (wild/rad 51) diploid cells showed in a survival curve a remarkable budding resistance and sigmoidal inactivation kinetics similar to those of wild-type homozygous diploid cells. (author)

Three new genetic loci (R1210C in CFH, variants in COL8A1 and RAD51B) are independently related to progression to advanced macular degeneration.

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Seddon, Johanna M; Reynolds, Robyn; Yu, Yi; Rosner, Bernard

2014-01-01

To assess the independent impact of new genetic variants on conversion to advanced stages of AMD, controlling for established risk factors, and to determine the contribution of genes in predictive models. In this prospective longitudinal study of 2765 individuals, 777 subjects progressed to neovascular disease (NV) or geographic atrophy (GA) in either eye over 12 years. Recently reported genetic loci were assessed for their independent effects on incident advanced AMD after controlling for 6 established loci in 5 genes, and demographic, behavioral, and macular characteristics. New variants which remained significantly related to progression were then added to a final multivariate model to assess their independent effects. The contribution of genes to risk models was assessed using reclassification tables by determining risk within cross-classified quintiles for alternative models. THREE NEW GENETIC VARIANTS WERE SIGNIFICANTLY RELATED TO PROGRESSION: rare variant R1210C in CFH (hazard ratio (HR) 2.5, 95% confidence interval [CI] 1.2-5.3, P = 0.01), and common variants in genes COL8A1 (HR 2.0, 95% CI 1.1-3.5, P = 0.02) and RAD51B (HR 0.8, 95% CI 0.60-0.97, P = 0.03). The area under the curve statistic (AUC) was significantly higher for the 9 gene model (.884) vs the 0 gene model (.873), P = .01. AUC's for the 9 vs 6 gene models were not significantly different, but reclassification analyses indicated significant added information for more genes, with adjusted odds ratios (OR) for progression within 5 years per one quintile increase in risk score of 2.7, Padvanced AMD beyond macular and behavioral phenotypes.

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

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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 ...

Homologous Recombination Repair Signaling in Chemical Carcinogenesis: Prolonged Particulate Hexavalent Chromium Exposure Suppresses the Rad51 Response in Human Lung Cells

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Qin, Qin; Xie, Hong; Wise, Sandra S.; Browning, Cynthia L.; Thompson, Kelsey N.; Holmes, Amie L.; Wise, John Pierce

2014-01-01

The aim of this study was to focus on hexavalent chromium, [Cr(VI)], a chemical carcinogen and major public health concern, and consider its ability to impact DNA double strand break repair. We further focused on particulate Cr(VI), because it is the more potent carcinogenic form of Cr(VI). DNA double strand break repair serves to protect cells against the detrimental effects of DNA double strand breaks. For particulate Cr(VI), data show DNA double strand break repair must be overcome for neoplastic transformation to occur. Acute Cr(VI) exposures reveal a robust DNA double strand break repair response, however, longer exposures have not been considered. Using the comet assay, we found longer exposures to particulate zinc chromate induced concentration-dependent increases in DNA double strand breaks indicating breaks were occurring throughout the exposure time. Acute (24 h) exposure induced DNA double strand break repair signaling by inducing Mre11 foci formation, ATM phosphorylation and phosphorylated ATM foci formation, Rad51 protein levels and Rad51 foci formation. However, longer exposures reduced the Rad51 response. These data indicate a major chemical carcinogen can simultaneously induce DNA double strand breaks and alter their repair and describe a new and important aspect of the carcinogenic mechanism for Cr(VI). PMID:25173789

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.

RAD51 potentiates synergistic effects of chemotherapy with PCI-24781 and cis-diamminedichloroplatinum on gastric cancer

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He, Wei-Ling; Li, Yu-Huang; Hou, Wei-Jian; Ke, Zun-Fu; Chen, Xin-Lin; Lu, Li-Ya; Cai, Shi-Rong; Song, Wu; Zhang, Chang-Hua; He, Yu-Long

2014-01-01

AIM: To explore the efficacy of PCI-24781, a broad-spectrum, hydroxamic acid-derived histone deacetylase inhibitor, in the treatment of gastric cancer (GC). METHODS: With or without treatment of PCI-24781 and/or cis-diamminedichloroplatinum (CDDP), GC cell lines were subjected to functional analysis, including cell growth, apoptosis and clonogenic assays. Chromatin immunoprecipitation and luciferase reporter assays were used to determine the interacting molecules and the activity of the enzyme. An in vivo study was carried out in GC xenograft mice. Cell culture-based assays were represented as mean ± SD. ANOVA tests were used to assess differences across groups. All pairwise comparisons between tumor weights among treatment groups were made using the Tukey-Kramer method for multiple comparison adjustment to control experimental-wise type I error rates. Significance was set at P PCI-24781 significantly reduced the growth of the GC cells, enhanced cell apoptosis and suppressed clonogenicity, and these effects synergized with the effects of CDDP. PCI-24781 modulated the cell cycle and significantly reduced the expression of RAD51, which is related to homologous recombination. Depletion of RAD51 augmented the biological functions of PCI-24781, CDDP and the combination treatment, whereas overexpressing RAD51 had the opposite effects. Increased binding of the transcription suppressor E2F4 on the RAD51 promoter appeared to play a major role in these processes. Furthermore, significant suppression of tumor growth and weight in vivo was obtained following PCI-24781 treatment, which synergized with the anticancer effect of CDDP. CONCLUSION: These data suggest that RAD51 potentiates the synergistic effects of chemotherapy with PCI-24781 and CDDP on GC. PMID:25110436

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

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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.

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.

Radioresistance of chordoma cells is associated with the ATM/ATR pathway, in which RAD51 serves as an important downstream effector.

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Zhang, Chao; Wang, Bing; Li, Lei; Li, Yawei; Li, Pengzhi; Lv, Guohua

2017-09-01

Surgery followed by radiotherapy is the standard treatment for chordomas, which are a rare but low-grade type of bone cancer arising from remnants of the embryonic notochord. However, disease recurrence following radiotherapy is common, most likely due to endogenous DNA repair mechanisms that promote cell survival upon radiation strikes. The ataxia telangiectasia mutated/ataxia telangiectasia mutated and Rad3 related (ATM/ATR)-mediated pathway has a critical role in DNA repair mechanisms; however, it has rarely been investigated in chordomas. In the present study, the expression of signal molecules related to the ATM/ATR pathway in chordoma tissues and adjacent normal tissues were initially examined using immunohistochemistry and western blot analysis. Chordoma U-CH1 and U-CH2 cells were subsequently used to investigate cell responses to ionizing radiation and the potential protective actions mediated by the ATM/ATR pathway. Phosphorylated (p)-ATM, p-ATR, γ-H2A histone family, member X (H2AX) and RAD51 were significantly upregulated in chordoma tissues relative to adjacent normal tissues (PATM, γ-H2AX and RAD51 expression in U-CH1 cells (PATM, p-ATR and RAD51 levels in U-CH2 cells (PATM/ATR pathway, in which RAD51 serves as an important downstream effector. Thus, RAD51 presents a promising therapeutic target for improving the outcome of radiotherapy treatment in chordomas.

Icotinib hydrochloride enhances chemo- and radiosensitivity by inhibiting EGFR signaling and attenuating RAD51 expression and function in Hela S3 cells

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Wang X

2018-03-01

Full Text Available Xuanxuan Wang, Yanjun Gu, Hai Liu, Liming Shi, Xiaonan Sun Department of Radiation Oncology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Background: Radiotherapy and cisplatin-based chemotherapy are currently considered as standard treatments employed for advanced cervical cancer (CC. However, patients with local recurrence or distant metastasis continue to have poor outcomes. EGFR overexpression correlated with chemo/radioresistance, and disease failure has been well proved in the previous studies. Hence, the aim of this study was to explore the therapeutic efficacy and underlying mechanism of the sensitization to radiation or cisplatin of icotinib hydrochloride (IH, a high-selective EGFR tyrosine kinase inhibitor (TKI, in the Hela S3 human CC cell line.Methods: Cell proliferation was measured with cell counting kit-8 (CCK-8 assay. Flow cytometry analysis was performed to examine cell cycle distribution and apoptosis. The phosphorylation of EGFR and its downstream signaling molecules were measured by Western blot analysis. γ-H2AX foci and RAD51 foci in the cellular nucleus were visualized using immunofluoresence staining. Expression levels of RAD51 in the whole cells and subceullar fractions were detected to demonstrate the impact of IH on DNA repair. Results: IH can significantly inhibit cell proliferation, redistribute cell cycle, enhance apoptosis and impair DNA damage response of Hela S3 cells following radiation or cisplatin treatment through suppressing the activation of the EGFR signaling pathway and attenuating the expression and function of homologous recombination (HR protein RAD51.Conclusion: This study suggests that IH is a potential sensitizer in radiotherapy and cisplatin-based chemotherapy for CC and RAD51 may serve as a prognosis biomarker for this combination treatment. Keywords: icotinib hydrochloride, cervical cancer, EGFR, radiotherapy, chemotherapy

Associations of common variants at 1p11.2 and 14q24.1 (RAD51L1) with breast cancer risk and heterogeneity by tumor subtype

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Figueroa, Jonine D; Garcia-Closas, Montserrat; Humphreys, Manjeet

2011-01-01

A genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) at 1p11.2 and 14q24.1 (RAD51L1) as breast cancer susceptibility loci. The initial GWAS suggested stronger effects for both loci for estrogen receptor (ER)-positive tumors. Using data from the Breast Cancer As...

Simultaneous ATM/BRCA1/RAD51 expression variations associated with prognostic factors in Iranian sporadic breast cancer patients.

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Hallajian, Zeinab; Mahjoubi, Frouzandeh; Nafissi, Nahid

2017-07-01

DNA double-strand breaks (DSBs) as a serious lesion are repaired by non-homologous end-joining and homologous recombination pathways. ATM, BRCA1, RAD51 genes are involved in HR pathways. While some studies have revealed individual expression changes of these genes in different types of cancer, there are limited studies attempting to evaluate correlation of expression variations of these genes in breast cancer pathogenesis. This study aimed to determine RAD51, ATM and BRCA1 gene expression level and its association with clinicopathological factors in fresh breast cancer tissues. Moreover, this study evaluates potential correlations among expression levels of these genes. 50 breast cancer tissues were collected and examined for BRCA1, RAD51 and ATM gene expression by Real Time PCR. Expression changes were analyzed with REST software version 2009. mRNA expression was reduced in all these three genes when compared with β-Actin as a control gene (P value  ATM, BRCA1 and RAD51 gene down expression (P value  ATM with stage (P value  < 0.05), necrosis (P value  < 0.05), perineural invasion (P value  < 0.05), vascular invasion (P value  < 0.01), malignancy (P value  ≤ 0.001), PR (P value  < 0.05) and ER status (P value  < 0.01). In addition, there was a significant association between down expression of BRCA1 with Ki67 (P value  ≤ 0.001). Moreover, there was a significant association between down expression of RAD51 with lymph node involvement (P value  < 0.01), auxiliary lymph node metastasis (P value  = 0.01), age (P = 0.001), grade (P value  < 0.05) and PR status (P value  < 0.05). This study suggests association between expression changes in several DSB repair genes in a common functional pathway in breast cancer and the significant association between abnormal expression of these genes and important clinical prognostic factors.

Associations of common variants at 1p11.2 and 14q24.1 (RAD51L1) with breast cancer risk and heterogeneity by tumor subtype

DEFF Research Database (Denmark)

Figueroa, Jonine D; Garcia-Closas, Montserrat; Humphreys, Manjeet

2011-01-01

for tumors of lower grade (case-only P= 6.7 × 10(-3)) and lobular histology (case-only P= 0.01). SNPs at 14q24.1 were associated with risk for most tumor subtypes evaluated, including triple-negative breast cancers, which has not been described previously. Our results underscore the need for large pooling......A genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) at 1p11.2 and 14q24.1 (RAD51L1) as breast cancer susceptibility loci. The initial GWAS suggested stronger effects for both loci for estrogen receptor (ER)-positive tumors. Using data from the Breast Cancer......10483813 (r(2)= 0.98) at 14q24.1 (RAD51L1), for up to 46 036 invasive breast cancer cases and 46 930 controls from 39 studies. Analyses by tumor characteristics focused on subjects reporting to be white women of European ancestry and were based on 25 458 cases, of which 87% had ER data. The SNP at 1p11...

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

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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.

Specific inhibition of Wee1 kinase and Rad51 recombinase: A strategy to enhance the sensitivity of leukemic T-cells to ionizing radiation-induced DNA double-strand breaks

International Nuclear Information System (INIS)

Havelek, Radim; Cmielova, Jana; Kralovec, Karel; Bruckova, Lenka; Bilkova, Zuzana; Fousova, Ivana; Sinkorova, Zuzana; Vavrova, Jirina; Rezacova, Martina

2014-01-01

Highlights: • Pre-treatment with the inhibitors increased the sensitivity of Jurkat cells to irradiation. • Combining both inhibitors together resulted in a G2 cell cycle arrest abrogation in Jurkat. • Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24 h upon irradiation. • Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction in MOLT-4 cells. • When dosed together, the combination decreased MOLT-4 cell survival. - Abstract: Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a part of their anticancer treatment. The objectives of the current study were to investigate the effects of the small molecule inhibitors of Wee1 kinase II (681641) and Rad51 (RI-1) on cell cycle progression, DNA double-strand breaks repair and apoptosis following ionizing radiation exposure in human leukemic T-cells Jurkat and MOLT-4. Pre-treatment with the Wee1 681641 or Rad51 RI-1 inhibitor alone increased the sensitivity of Jurkat cells to irradiation, however combining both inhibitors together resulted in a further enhancement of apoptosis. Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24 h upon irradiation. MOLT-4 cells were less affected by inhibitors application prior to ionizing radiation exposure. Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction; however Wee1 681641 increased ionizing radiation-induced cell death in MOLT-4 cells

Immunochip analysis identifies association of the RAD50/IL13 region with human longevity.

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Flachsbart, Friederike; Ellinghaus, David; Gentschew, Liljana; Heinsen, Femke-Anouska; Caliebe, Amke; Christiansen, Lene; Nygaard, Marianne; Christensen, Kaare; Blanché, Hélène; Deleuze, Jean-François; Derbois, Céline; Galan, Pilar; Büning, Carsten; Brand, Stephan; Peters, Anette; Strauch, Konstantin; Müller-Nurasyid, Martina; Hoffmann, Per; Nöthen, Markus M; Lieb, Wolfgang; Franke, Andre; Schreiber, Stefan; Nebel, Almut

2016-06-01

Human longevity is characterized by a remarkable lack of confirmed genetic associations. Here, we report on the identification of a novel locus for longevity in the RAD50/IL13 region on chromosome 5q31.1 using a combined European sample of 3208 long-lived individuals (LLI) and 8919 younger controls. First, we performed a large-scale association study on 1458 German LLI (mean age 99.0 years) and 6368 controls (mean age 57.2 years) by targeting known immune-associated loci covered by the Immunochip. The analysis of 142 136 autosomal single nucleotide polymorphisms (SNPs) revealed an Immunochip-wide significant signal (PI mmunochip  = 7.01 × 10(-9) ) for the SNP rs2075650 in the TOMM40/APOE region, which has been previously described in the context of human longevity. To identify novel susceptibility loci, we selected 15 markers with PI mmunochip  association at SNP rs2706372 replicated in the French study collection and showed a similar trend in the Danish participants and was also significant in a meta-analysis of the combined French and Danish data after adjusting for multiple testing. In a meta-analysis of all three samples, rs2706372 reached a P-value of PI mmunochip+Repl  = 5.42 × 10(-7) (OR = 1.20; 95% CI = 1.12-1.28). SNP rs2706372 is located in the extended RAD50/IL13 region. RAD50 seems a plausible longevity candidate due to its involvement in DNA repair and inflammation. Further studies are needed to identify the functional variant(s) that predispose(s) to a long and healthy life. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

ATM/ATR-mediated phosphorylation of PALB2 promotes RAD51 function

DEFF Research Database (Denmark)

Ahlskog, Johanna K; Larsen, Brian D; Achanta, Kavya

2016-01-01

DNA damage activates the ATM and ATR kinases that coordinate checkpoint and DNA repair pathways. An essential step in homology-directed repair (HDR) of DNA breaks is the formation of RAD51 nucleofilaments mediated by PALB2-BRCA2; however, roles of ATM and ATR in this critical step of HDR are poor...... function, as the PALB2-dependent checkpoint response is normal in cells expressing the phospho-deficient PALB2 mutant. Collectively, our findings highlight a critical importance of PALB2 phosphorylation as a novel regulatory step in genome maintenance after genotoxic stress....

Inducibility of nuclear Rad51 foci after DNA damage distinguishes all Fanconi anemia complementation groups from D1/BRCA2

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Godthelp, Barbara C. [Department of Toxicogenetics, Leiden University Medical Center, Wassenaarseweg 72, NL-2333 AL Leiden (Netherlands); Wiegant, Wouter W. [Department of Toxicogenetics, Leiden University Medical Center, Wassenaarseweg 72, NL-2333 AL Leiden (Netherlands); Waisfisz, Quinten [Department of Clinical Genetics and Human Genetics, Free University Medical Center, Van der Boechorststraat 7, NL-1081 BT Amsterdam (Netherlands); Medhurst, Annette L. [Department of Clinical Genetics and Human Genetics, Free University Medical Center, Van der Boechorststraat 7, NL-1081 BT Amsterdam (Netherlands); Arwert, Fre [Department of Clinical Genetics and Human Genetics, Free University Medical Center, Van der Boechorststraat 7, NL-1081 BT Amsterdam (Netherlands); Joenje, Hans [Department of Clinical Genetics and Human Genetics, Free University Medical Center, Van der Boechorststraat 7, NL-1081 BT Amsterdam (Netherlands); Zdzienicka, Malgorzata Z. [Department of Toxicogenetics, Leiden University Medical Center, Wassenaarseweg 72, NL-2333 AL Leiden (Netherlands) and Department of Molecular Cell Genetics, Collegium Medicum, N. Copernicus University, Bydgoszcz (Poland)]. E-mail: m.z.zdzienicka@lumc.nl

2006-02-22

Fanconi anemia (FA) is a cancer susceptibility disorder characterized by chromosomal instability and hypersensitivity to DNA cross-linking agents. So far 11 complementation groups have been identified, from which only FA-D1/BRCA2 and FA-J are defective downstream of the central FANCD2 protein as cells from these groups are capable of monoubiquitinating FANCD2. In this study we show that cells derived from patients from the new complementation groups, FA-I, FA-J and FA-L are all proficient in DNA damage induced Rad51 foci formation, making the cells from FA-D1/BRCA2 patients that are defective in this process the sole exception. Although FA-B patient HSC230 was previously reported to also have biallelic BRCA2 mutations, we found normal Rad51 foci formation in cells from this patient, consistent with the recent identification of an X-linked gene being mutated in four unrelated FA-B patients. Thus, our data show that none of the FA proteins, except BRCA2, are required to sequester Rad51 into nuclear foci. Since cells from the FA-D1 and FA-J patient groups are both able to monoubiquitinate FANCD2, the 'Rad51 foci phenotype' provides a convenient assay to distinguish between these two groups. Our results suggest that FANCJ and FANCD1/BRCA2 are part of the integrated FANC/BRCA DNA damage response pathway or, alternatively, that they represent sub-pathways in which only FANCD1/BRCA2 is directly connected to the process of homologous recombination.

Inducibility of nuclear Rad51 foci after DNA damage distinguishes all Fanconi anemia complementation groups from D1/BRCA2

International Nuclear Information System (INIS)

Godthelp, Barbara C.; Wiegant, Wouter W.; Waisfisz, Quinten; Medhurst, Annette L.; Arwert, Fre; Joenje, Hans; Zdzienicka, Malgorzata Z.

2006-01-01

Fanconi anemia (FA) is a cancer susceptibility disorder characterized by chromosomal instability and hypersensitivity to DNA cross-linking agents. So far 11 complementation groups have been identified, from which only FA-D1/BRCA2 and FA-J are defective downstream of the central FANCD2 protein as cells from these groups are capable of monoubiquitinating FANCD2. In this study we show that cells derived from patients from the new complementation groups, FA-I, FA-J and FA-L are all proficient in DNA damage induced Rad51 foci formation, making the cells from FA-D1/BRCA2 patients that are defective in this process the sole exception. Although FA-B patient HSC230 was previously reported to also have biallelic BRCA2 mutations, we found normal Rad51 foci formation in cells from this patient, consistent with the recent identification of an X-linked gene being mutated in four unrelated FA-B patients. Thus, our data show that none of the FA proteins, except BRCA2, are required to sequester Rad51 into nuclear foci. Since cells from the FA-D1 and FA-J patient groups are both able to monoubiquitinate FANCD2, the 'Rad51 foci phenotype' provides a convenient assay to distinguish between these two groups. Our results suggest that FANCJ and FANCD1/BRCA2 are part of the integrated FANC/BRCA DNA damage response pathway or, alternatively, that they represent sub-pathways in which only FANCD1/BRCA2 is directly connected to the process of homologous recombination

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.

The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma

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Guo Yajun

2006-01-01

Full Text Available Abstract Background Previously we have generated the monoclonal antibody SM5-1 by using a subtractive immunization protocol of human melanoma. This antibody exhibits a high sensitivity for primary melanomas of 99% (248/250 tested and for metastatic melanoma of 96% (146/151 tested in paraffin embedded sections. This reactivity is superior to the one obtained by HMB-45, anti-MelanA or anti-Tyrosinase and is comparable to anti-S100. However, as compared to anti-S100, the antibody SM5-1 is highly specific for melanocytic lesions since 40 different neoplasms were found to be negative for SM5-1 by immunohistochemistry. The antigen recognized by SM5-1 is unknown. Methods In order to characterize the antigen recognized by mAb SM5-1, a cDNA library was constructed from the metastatic human melanoma cell line SMMUpos in the Uni-ZAP lambda phage and screened by mAb SM5-1. The cDNA clones identified by this approach were then sequenced and subsequently analyzed. Results Sequence analysis of nine independent overlapping clones (length 3100–5600 bp represent fibronectin cDNA including the ED-A, but not the ED-B region which are produced by alternative splicing. The 89aa splicing variant of the IIICS region was found in 8/9 clones and the 120aa splicing variant in 1/9 clones, both of which are included in the CS1 region of fibronectin being involved in melanoma cell adhesion and spreading. Conclusion The molecule recognized by SM5-1 is a melanoma associated FN variant expressed by virtually all primary and metastatic melanomas and may play an important role in melanoma formation and progression. This antibody is therefore not only of value in immunohistochemistry, but potentially also for diagnostic imaging and immunotherapy.

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

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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.

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.

An integrated in silico approach to analyze the involvement of single amino acid polymorphisms in FANCD1/BRCA2-PALB2 and FANCD1/BRCA2-RAD51 complex.

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Doss, C George Priya; Nagasundaram, N

2014-11-01

Fanconi anemia (FA) is an autosomal recessive human disease characterized by genomic instability and a marked increase in cancer risk. The importance of FANCD1 gene is manifested by the fact that deleterious amino acid substitutions were found to confer susceptibility to hereditary breast and ovarian cancers. Attaining experimental knowledge about the possible disease-associated substitutions is laborious and time consuming. The recent introduction of genome variation analyzing in silico tools have the capability to identify the deleterious variants in an efficient manner. In this study, we conducted in silico variation analysis of deleterious non-synonymous SNPs at both functional and structural level in the breast cancer and FA susceptibility gene BRCA2/FANCD1. To identify and characterize deleterious mutations in this study, five in silico tools based on two different prediction methods namely pathogenicity prediction (SIFT, PolyPhen, and PANTHER), and protein stability prediction (I-Mutant 2.0 and MuStab) were analyzed. Based on the deleterious scores that overlap in these in silico approaches, and the availability of three-dimensional structures, structure analysis was carried out with the major mutations that occurred in the native protein coded by FANCD1/BRCA2 gene. In this work, we report the results of the first molecular dynamics (MD) simulation study performed to analyze the structural level changes in time scale level with respect to the native and mutated protein complexes (G25R, W31C, W31R in FANCD1/BRCA2-PALB2, and F1524V, V1532F in FANCD1/BRCA2-RAD51). Analysis of the MD trajectories indicated that predicted deleterious variants alter the structural behavior of BRCA2-PALB2 and BRCA2-RAD51 protein complexes. In addition, statistical analysis was employed to test the significance of these in silico tool predictions. Based on these predictions, we conclude that the identification of disease-related SNPs by in silico methods, in combination with MD

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.

Development and application of the Chinese adult female computational phantom Rad-HUMAN

International Nuclear Information System (INIS)

Wu, Yican; Cheng, Mengyun; Wang, Wen; Fan, Yanchang; Zhao, Kai; He, Tao; Pei, Xi; Shang, Leiming; Chen, Chaobin; Long, Pengcheng; Cao, Ruifen; Wang, Guozhong; Zhou, Shaoheng; Yu, Shengpeng; Hu, Liqin; Zeng, Q.

2013-01-01

Rad-HUMAN is a whole-body numerical phantom of a Chinese adult woman which contains 46 organs and tissues and was created by MCAM6 software using the color photographs of the Chinese Visible Human dataset. This dataset was obtained from a 22-year old Chinese female cadaver judged to represent normal human anatomy as much as possible. The density and elemental composition recommended in the ICRP Publication 89 and in the ICRU report 44 were assigned to the organ and tissue in Rad-HUMAN for radiation protection purpose. The last step was to implement the anatomical data into a Monte Carlo code. Rad-HUMAN contains more than 28.8 billion tiny volume units, which produces an accurately whole-body numerical phantom of a Chinese adult female

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...

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

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.

HELQ promotes RAD51 paralogue-dependent repair to avert germ cell loss and tumorigenesis

DEFF Research Database (Denmark)

Adelman, Carrie A.; Lolo, Rafal L.; Birkbak, Nicolai Juul

2013-01-01

Repair of interstrand crosslinks (ICLs) requires the coordinated action of the intra-S-phase checkpoint and the Fanconi anaemia pathway, which promote ICL incision, translesion synthesis and homologous recombination (reviewed in refs 1, 2). Previous studies have implicated the 3'-5' superfamily 2......, phenotype than the null, indicative of haploinsufficiency. We establish that HELQ interacts directly with the RAD51 paralogue complex BCDX2 and functions in parallel to the Fanconi anaemia pathway to promote efficient homologous recombination at damaged replication forks. Thus, our results reveal a critical...

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.

Identification of the meiotic toolkit in diatoms and exploration of meiosis-specific SPO11 and RAD51 homologs in the sexual species Pseudo-nitzschia multistriata and Seminavis robusta.

Science.gov (United States)

Patil, Shrikant; Moeys, Sara; von Dassow, Peter; Huysman, Marie J J; Mapleson, Daniel; De Veylder, Lieven; Sanges, Remo; Vyverman, Wim; Montresor, Marina; Ferrante, Maria Immacolata

2015-11-14

Sexual reproduction is an obligate phase in the life cycle of most eukaryotes. Meiosis varies among organisms, which is reflected by the variability of the gene set associated to the process. Diatoms are unicellular organisms that belong to the stramenopile clade and have unique life cycles that can include a sexual phase. The exploration of five diatom genomes and one diatom transcriptome led to the identification of 42 genes potentially involved in meiosis. While these include the majority of known meiosis-related genes, several meiosis-specific genes, including DMC1, could not be identified. Furthermore, phylogenetic analyses supported gene identification and revealed ancestral loss and recent expansion in the RAD51 family in diatoms. The two sexual species Pseudo-nitzschia multistriata and Seminavis robusta were used to explore the expression of meiosis-related genes: RAD21, SPO11-2, RAD51-A, RAD51-B and RAD51-C were upregulated during meiosis, whereas other paralogs in these families showed no differential expression patterns, suggesting that they may play a role during vegetative divisions. An almost identical toolkit is shared among Pseudo-nitzschia multiseries and Fragilariopsis cylindrus, as well as two species for which sex has not been observed, Phaeodactylum tricornutum and Thalassiosira pseudonana, suggesting that these two may retain a facultative sexual phase. Our results reveal the conserved meiotic toolkit in six diatom species and indicate that Stramenopiles share major modifications of canonical meiosis processes ancestral to eukaryotes, with important divergences in each Kingdom.

Rad9 Has a Functional Role in Human Prostate Carcinogenesis

Science.gov (United States)

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

Germline Mutations in PALB2, BRCA1, and RAD51C, Which Regulate DNA Recombination Repair, in Patients with Gastric Cancer

Science.gov (United States)

Sahasrabudhe, Ruta; Lott, Paul; Bohorquez, Mabel; Toal, Ted; Estrada, Ana P.; Suarez, John J.; Brea-Fernández, Alejandro; Cameselle-Teijeiro, José; Pinto, Carla; Ramos, Irma; Mantilla, Alejandra; Prieto, Rodrigo; Corvalan, Alejandro; Norero, Enrique; Alvarez, Carolina; Tapia, Teresa; Carvallo, Pilar; Gonzalez, Luz M.; Cock-Rada, Alicia; Solano, Angela; Neffa, Florencia; Valle, Adriana Della; Yau, Chris; Soares, Gabriela; Borowsky, Alexander; Hu, Nan; He, Li-Ji; Han, Xiao-You; Taylor, Philip R.; Goldstein, Alisa M.; Torres, Javier; Echeverry, Magdalena; Ruiz-Ponte, Clara; Teixeira, Manuel R.; Carvajal Carmona, Luis G.

2016-01-01

Up to 10% of cases of gastric cancer are familial, but so far, only mutations in CDH1 have been associated with gastric cancer risk. To identify genetic variants that affect risk for gastric cancer, we collected blood samples from 28 patients with hereditary diffuse gastric cancer (HDGC) not associated with mutations in CDH1 and performed whole-exome sequence analysis. We then analyzed sequences of candidate genes in 333 independent HDGC and non-HDGC cases. We identified 11 cases with mutations in PALB2, BRCA1, or RAD51C genes, which regulate homologous DNA recombination. We found these mutations in 2 of 31 patients with HDGC (6.5%) and 9 of 331 patients with sporadic gastric cancer (2.8%). Most of these mutations had been previously associated with other types of tumors and partially co-segregated with gastric cancer in our study. Tumors that developed in patients with these mutations had a mutation signature associated with somatic homologous recombination deficiency. Our findings indicate that defects in homologous recombination increase risk for gastric cancer. PMID:28024868

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.

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

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

Energy Technology Data Exchange (ETDEWEB)

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.

Comprehensive Pathway-Based Association Study of DNA Repair Gene Variants and the Risk of Nasopharyngeal Carcinoma

Science.gov (United States)

Qin, Hai-De; Shugart, Yin Yao; Bei, Jin-Xin; Pan, Qing-Hua; Chen, Lina; Feng, Qi-Sheng; Chen, Li-Zhen; Huang, Wei; Liu, Jian Jun; Jorgensen, Timothy J.; Zeng, Yi-Xin; Jia, Wei-Hua

2011-01-01

DNA repair plays a central role in protecting against environmental carcinogenesis, and genetic variants of DNA repair genes have been reported to be associated with several human malignancies. To assess whether DNA gene variants were associated with nasopharyngeal carcinoma (NPC) risk, a candidate gene association study was conducted among the Cantonese population within the Guangdong Province, China --the ethnic group with the highest risk for NPC. A two-stage study design was utilized. In the discovery stage, 676 tagging SNPs covering 88 DNA repair genes were genotyped in a matched case-control study (cases/controls = 755/755). Eleven SNPs with Ptrend Cantonese population (cases/controls = 1,568/1,297). Two of the SNPs (rs927220 and rs11158728) – both in RAD51L1 – remained strongly associated with NPC. The SNP rs927220 had a significant Pcombined of 5.55 × 10−5, with OR = 1.20 (95%CI = 1.10 to 1.30), Bonferroni corrected P = 0.0381. The other SNP (rs11158728), which is in strong LD with rs927220 (r2 = 0.7), had a significant Pcombined of 2.0 × 10−4, Bonferroni corrected P = 0.1372. Gene-environment interaction analysis suggested that the exposures of salted-fish consumption and cigarette smoking had potential interactions with DNA repair gene variations, but need to be further investigated. Our findings support the notion that DNA repair genes, in particular RAD51L1, play a role in NPC etiology and development. PMID:21368091

In vitro radiation and chemotherapy sensitivity of established cell lines of human small cell lung cancer and its large cell morphological variants

International Nuclear Information System (INIS)

Carney, D.N.; Mitchell, J.B.; Kinsella, T.J.

1983-01-01

The in vitro response to radiation and chemotherapeutic drugs of cell lines established from 7 patients with small cell (SC) lung cancer were tested using a soft agarose clonogenic assay. Five cell lines retained the typical morphological and biochemical amine precursor uptake decarboxylation characteristics of SC, while two cell lines had undergone ''transformation'' to large cell (LC) morphological variants with loss of amine precursor uptake decarboxylation cell characteristics of SC. The radiation survival curves for the SC lines were characterized by D0 values ranging from 51 to 140 rads and extrapolation values (n) ranging from 1.0 to 3.3. While the D0 values of the radiation survival curves of the LC variants were similar (91 and 80 rads), the extrapolation values were 5.6 and 11.1 In vitro chemosensitivity testing of the cell lines revealed an excellent correlation between prior treatment status of the patient and in vitro sensitivity or resistance. No correlation was observed between in vitro chemosensitivity and radiation response. These data suggest that transformation of SC to LC with loss of amine precursor uptake and decarboxylation characteristics is associated with a marked increase in radiation resistance (n) in vitro. The observation of a 2- to 5-fold increase in survival of the LC compared to the SC lines following 200 rads suggests that the use of larger daily radiation fractions and/or radiation-sensitizing drugs might lead to a significantly greater clinical response in patients with LC morphology. This clinical approach may have a major impact on patient response and survival

Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation

Science.gov (United States)

Zelensky, Alex N.; Sanchez, Humberto; Ristic, Dejan; Vidic, Iztok; van Rossum-Fikkert, Sari E.; Essers, Jeroen; Wyman, Claire; Kanaar, Roland

2013-01-01

Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair. PMID:23666627

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

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

Conceptos de ataque frente a variantes defensivas 6:0 y 5:1 [Concepts of attacks against the variants defensive 6:0 and 5:1

Directory of Open Access Journals (Sweden)

Juan de Dios Román Seco

2009-12-01

Full Text Available Resumen: La filosofía del juego de ataque contra diferentes Sistemas Defensivos tiene una raíz basada en los Principios Tácticos Colectivos del Ataque que se orienta y apoya a nivel práctico en los medios tácticos colectivos. Las diferentes estructuras colectivas en defensa exigen cumplir aquellos y saber elegir los medios tácticos adecuados. En síntesis, no existen "recetas" para resolver problemas sino la utilización de los medios tácticos adecuados. La estrategia tiene su fundamento y lleva al éxito siempre que se defina a partir de buenas bases tácticas. Palabras clave: Ataque, Defensa 6:0, Defensa 5.1 Abstract: The philosophy of the attack game against different Defensive Systems has a root based on the Principles Tactical Communities of the Attack that it is guided and it supports at practical level in the collective tactical means. The different collective structures in defense demand to complete those and to know how to choose the appropriate tactical means. In synthesis, recipes don't exist to solve problems but the use of the appropriate tactical means. The strategy has its foundation and it takes to the success whenever he/she is defined starting from good tactical bases. Key words: Attacks, variant defensive 6:0, variant defensive 5:1.

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)

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...

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

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

CRISPR Technology Reveals RAD(51)-ical Mechanisms of Repair in Roundworms: An Educational Primer for Use with "Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans".

Science.gov (United States)

Turcotte, Carolyn A; Andrews, Nicolas P; Sloat, Solomon A; Checchi, Paula M

2016-11-01

The mechanisms cells use to maintain genetic fidelity via DNA repair and the accuracy of these processes have garnered interest from scientists engaged in basic research to clinicians seeking improved treatment for cancer patients. Despite the continued advances, many details of DNA repair are still incompletely understood. In addition, the inherent complexity of DNA repair processes, even at the most fundamental level, makes it a challenging topic. This primer is meant to assist both educators and students in using a recent paper, "Promotion of homologous recombination by SWS-1 in complex with RAD-51 paralogs in Caenorhabditis elegans," to understand mechanisms of DNA repair. The goals of this primer are to highlight and clarify several key techniques utilized, with special emphasis on the clustered, regularly interspaced, short palindromic repeats technique and the ways in which it has revolutionized genetics research, as well as to provide questions for deeper in-class discussion. Copyright © 2016 by the Genetics Society of America.

Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators.

Directory of Open Access Journals (Sweden)

Yumiko Kurokawa

2008-04-01

Full Text Available In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog-mediated homologous recombination (HR and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA, which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1-mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA.

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

Specific absorbed fractions of electrons and photons for Rad-HUMAN phantom using Monte Carlo method

International Nuclear Information System (INIS)

Wang Wen; Hu Liqin; Cheng Mengyun; Long Pengcheng

2015-01-01

The specific absorbed fractions (SAF) for self- and cross-irradiation are effective tools for the internal dose estimation of inhalation and ingestion intakes of radionuclides. A set of SAFs of photons and electrons were calculated using the Rad-HUMAN phantom, which is a computational voxel phantom of a Chinese adult female that was created using the color photographic image of the Chinese Visible Human (CVH) data set by the FDS Team. The model can represent most Chinese adult female anatomical characteristics and can be taken as an individual phantom to investigate the difference of internal dose with Caucasians. In this study, the emission of mono-energetic photons and electrons of 10 keV to 4 MeV energy were calculated using the Monte Carlo particle transport calculation code MCNP. Results were compared with the values from ICRP reference and ORNL models. The results showed that SAF from the Rad-HUMAN have similar trends but are larger than those from the other two models. The differences were due to the racial and anatomical differences in organ mass and inter-organ distance. The SAFs based on the Rad-HUMAN phantom provide an accurate and reliable data for internal radiation dose calculations for Chinese females. (authors)

Prevalence of deleterious germline variants in risk genes including BRCA1/2 in consecutive ovarian cancer patients (AGO-TR-1.

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Philipp Harter

Full Text Available Identification of families at risk for ovarian cancer offers the opportunity to consider prophylactic surgery thus reducing ovarian cancer mortality. So far, identification of potentially affected families in Germany was solely performed via family history and numbers of affected family members with breast or ovarian cancer. However, neither the prevalence of deleterious variants in BRCA1/2 in ovarian cancer in Germany nor the reliability of family history as trigger for genetic counselling has ever been evaluated.Prospective counseling and germline testing of consecutive patients with primary diagnosis or with platinum-sensitive relapse of an invasive epithelial ovarian cancer. Testing included 25 candidate and established risk genes. Among these 25 genes, 16 genes (ATM, BRCA1, BRCA2, CDH1, CHEK2, MLH1, MSH2, MSH6, NBN, PMS2, PTEN, PALB2, RAD51C, RAD51D, STK11, TP53 were defined as established cancer risk genes. A positive family history was defined as at least one relative with breast cancer or ovarian cancer or breast cancer in personal history.In total, we analyzed 523 patients: 281 patients with primary diagnosis of ovarian cancer and 242 patients with relapsed disease. Median age at primary diagnosis was 58 years (range 16-93 and 406 patients (77.6% had a high-grade serous ovarian cancer. In total, 27.9% of the patients showed at least one deleterious variant in all 25 investigated genes and 26.4% in the defined 16 risk genes. Deleterious variants were most prevalent in the BRCA1 (15.5%, BRCA2 (5.5%, RAD51C (2.5% and PALB2 (1.1% genes. The prevalence of deleterious variants did not differ significantly between patients at primary diagnosis and relapse. The prevalence of deleterious variants in BRCA1/2 (and in all 16 risk genes in patients <60 years was 30.2% (33.2% versus 10.6% (18.9% in patients ≥60 years. Family history was positive in 43% of all patients. Patients with a positive family history had a prevalence of deleterious variants

The Molecular Basis of Double-Strand DNA Break Repair: The Critical Structure of the RAD52/RPA Complex

National Research Council Canada - National Science Library

Jackson, Dobra

2001-01-01

.... RAD52 has specific interactions with RAD51, RPA and DNA (1,2,3). The binding of RAD52 to ends of double-strand breaks has been found to be a key initiation step to DNA repair by homologous recombination...

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...

Altered endoribonuclease activity of apurinic/apyrimidinic endonuclease 1 variants identified in the human population.

Directory of Open Access Journals (Sweden)

Wan Cheol Kim

Full Text Available Apurinic/apyrimidinic endonuclease 1 (APE1 is the major mammalian enzyme in the DNA base excision repair pathway and cleaves the DNA phosphodiester backbone immediately 5' to abasic sites. APE1 also has 3'-5' DNA exonuclease and 3' DNA phosphodiesterase activities, and regulates transcription factor DNA binding through its redox regulatory function. The human APE1 has recently been shown to endonucleolytically cleave single-stranded regions of RNA. Towards understanding the biological significance of the endoribonuclease activity of APE1, we examined eight different amino acid substitution variants of APE1 previously identified in the human population. Our study shows that six APE1 variants, D148E, Q51H, I64V, G241R, R237A, and G306A, exhibit a 76-85% reduction in endoribonuclease activity against a specific coding region of the c-myc RNA, yet fully retain the ability to cleave apurinic/apyrimidinic DNA. We found that two APE1 variants, L104R and E126D, exhibit a unique RNase inhibitor-resistant endoribonuclease activity, where the proteins cleave c-myc RNA 3' of specific single-stranded guanosine residues. Expression of L104R and E126D APE1 variants in bacterial Origami cells leads to a 60-80% reduction in colony formation and a 1.5-fold increase in cell doubling time, whereas the other variants, which exhibit diminished endoribonuclease activity, had no effect. These data indicate that two human APE1 variants exhibit a unique endoribonuclease activity, which correlates with their ability to induce cytotoxicity or slow down growth in bacterial cells and supports the notion of their biological functionality.

Human papillomavirus type-16 variants in Quechua aboriginals from Argentina.

Science.gov (United States)

Picconi, María Alejandra; Alonio, Lidia Virginia; Sichero, Laura; Mbayed, Viviana; Villa, Luisa Lina; Gronda, Jorge; Campos, Rodolfo; Teyssié, Angélica

2003-04-01

Cervical carcinoma is the leading cause of cancer death in Quechua indians from Jujuy (northwestern Argentina). To determine the prevalence of HPV-16 variants, 106 HPV-16 positive cervical samples were studied, including 33 low-grade squamous intraepithelial lesions (LSIL), 28 high-grade squamous intraepithelial lesions (HSIL), 9 invasive cervical cancer (ICC), and 36 samples from women with normal colposcopy and cytology. HPV genome variability was examined in the L1 and E6 genes by PCR-hybridization. In a subset of 20 samples, a LCR fragment was also analyzed by PCR-sequencing. Most variants belonged to the European branch with subtle differences that depended on the viral gene fragment studied. Only about 10% of the specimens had non-European variants, including eight Asian-American, two Asian, and one North-American-1. E6 gene analysis revealed that 43% of the samples were identical to HPV-16 prototype, while 57% corresponded to variants. Interestingly, the majority (87%) of normal smears had HPV-16 prototype, whereas variants were detected mainly in SIL and ICC. LCR sequencing yielded 80% of variants, including 69% of European, 19% Asian-American, and 12% Asian. We identified a new variant, the Argentine Quechua-51 (AQ-51), similar to B-14 plus two additional changes: G7842-->A and A7837-->C; phylogenetic inference allocated it in the Asian-American branch. The high proportion of European variants may reflect Spanish colonial influence on these native Inca descendants. The predominance of HPV-16 variants in pathologic samples when compared to normal controls could have implications for the natural history of cervical lesions. Copyright 2003 Wiley-Liss, Inc.

Evaluation of Selected CYP51A1 Polymorphisms in View of Interactions with Substrate and Redox Partner

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Tadeja Režen

2017-06-01

Full Text Available Cholesterol is essential for development, growth, and maintenance of organisms. Mutations in cholesterol biosynthetic genes are embryonic lethal and few polymorphisms have been so far associated with pathologies in humans. Previous analyses show that lanosterol 14α-demethylase (CYP51A1 from the late part of cholesterol biosynthesis has only a few missense mutations with low minor allele frequencies and low association with pathologies in humans. The aim of this study is to evaluate the role of amino acid changes in the natural missense mutations of the hCYP51A1 protein. We searched SNP databases for existing polymorphisms of CYP51A1 and evaluated their effect on protein function. We found rare variants causing detrimental missense mutations of CYP51A1. Some missense variants were also associated with a phenotype in humans. Two missense variants have been prepared for testing enzymatic activity in vitro but failed to produce a P450 spectrum. We performed molecular modeling of three selected missense variants to evaluate the effect of the amino acid substitution on potential interaction with its substrate and the obligatory redox partner POR. We show that two of the variants, R277L and especially D152G, have possibly lower binding potential toward obligatory redox partner POR. D152G and R431H have also potentially lower affinity toward the substrate lanosterol. We evaluated the potential effect of damaging variants also using data from other in vitro CYP51A1 mutants. In conclusion, we propose to include damaging CYP51A1 variants into personalized diagnostics to improve genetic counseling for certain rare disease phenotypes.

Prolonged Particulate Hexavalent Chromium Exposure Suppresses Homologous Recombination Repair in Human Lung Cells.

Science.gov (United States)

Browning, Cynthia L; Qin, Qin; Kelly, Deborah F; Prakash, Rohit; Vanoli, Fabio; Jasin, Maria; Wise, John Pierce

2016-09-01

Genomic instability is one of the primary models of carcinogenesis and a feature of almost all cancers. Homologous recombination (HR) repair protects against genomic instability by maintaining high genomic fidelity during the repair of DNA double strand breaks. The defining step of HR repair is the formation of the Rad51 nucleofilament, which facilitates the search for a homologous sequence and invasion of the template DNA strand. Particulate hexavalent chromium (Cr(VI)), a human lung carcinogen, induces DNA double strand breaks and chromosome instability. Since the loss of HR repair increases Cr(VI)-induced chromosome instability, we investigated the effect of extended Cr(VI) exposure on HR repair. We show acute (24 h) Cr(VI) exposure induces a normal HR repair response. In contrast, prolonged (120 h) exposure to particulate Cr(VI) inhibited HR repair and Rad51 nucleofilament formation. Prolonged Cr(VI) exposure had a profound effect on Rad51, evidenced by reduced protein levels and Rad51 mislocalization to the cytoplasm. The response of proteins involved in Rad51 nuclear import and nucleofilament formation displayed varying responses to prolonged Cr(VI) exposure. BRCA2 formed nuclear foci after prolonged Cr(VI) exposure, while Rad51C foci formation was suppressed. These results suggest that particulate Cr(VI), a major chemical carcinogen, inhibits HR repair by targeting Rad51, causing DNA double strand breaks to be repaired by a low fidelity, Rad51-independent repair pathway. These results further enhance our understanding of the underlying mechanism of Cr(VI)-induced chromosome instability and thus, carcinogenesis. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Human papillomavirus type 16 molecular variants in Guarani Indian women from Misiones, Argentina.

Science.gov (United States)

Tonon, Sergio Andrés; Basiletti, Jorge; Badano, Ines; Alonio, Lidia Virginia; Villa, Luisa Lina; Teyssie, Angélica Rita; Picconi, María Alejandra

2007-01-01

To identify human papillomavirus type 16 (HPV16) E6 and L1 molecular variants infecting Guarani Indian women settled in Misiones, Argentina, a region with a high prevalence of cervical cancer. Some intratypic molecular variants of HPV16 have been associated with greater oncogenic risk, but their implication in the etiology of cervical cancer is still uncertain. Seventy HPV16 positive cervical samples from Guarani Indian women settled in two different areas of Misiones, Argentina, (34 from the northern area and 36 from the central area), were analyzed. Thirty-seven had normal cytology, 18 had a low-grade squamous intraepithelial lesion (LGSIL), and 15 a high-grade squamous intraepithelial lesion (HGSIL). HPV16 E6 and L1 molecular variants were identified by PCR, followed by dot blot hybridization with 23 and 12 biotinylated oligonucleotide probes, respectively. The frequency of HPV16 variants over the Guarani population was 51% EP (European prototype), 32% E-350G, 9% Af1-a (African 1), 4% E-6862C, 3% Af2-a, and 1% AA-a (Asian-American). The distribution of variants was not homogeneous in the two areas under analysis, with the northern area being more diverse showing 74% of European variants, while the central area presented exclusively E variants. No statistically significant association was found between any particular variant and grade of cervical lesion. This study reports for the first time HPV16 E6 and L1 molecular variants infecting women from an aboriginal community inhabiting a rainforest region of South America. The presence of E class variants could be attributed primarily to contacts with the Spanish conquerors, and Af variants from African slaves introduced later in the South American continent.

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...

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

Directory of Open Access Journals (Sweden)

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.

Preferential binding of yeast Rad4-Rad23 complex to damaged DNA

International Nuclear Information System (INIS)

Jansen, L.E.T.; Verhage, R.A.; Brouwer, J.

1998-01-01

The yeast Rad4 and Rad23 proteins form a complex that is involved in nucleotide excision repair (NER). Their function in this process is not known yet, but genetic data suggest that they act in an early step in NER. We have purified an epitope-tagged Rad4.Rad23 (tRad4. Rad23) complex from yeast cells, using a clone overproducing Rad4 with a hemagglutinin-tag at its C terminus. tRad4.Rad23 complex purified by both conventional and immuno-affinity chromatography complements the in vitro repair defect of rad4 and rad23 mutant extracts, demonstrating that these proteins are functional in NER. Using electrophoretic mobility shift assays, we show preferential binding of the tRad4.Rad23 complex to damaged DNA in vitro. UV-irradiated, as well as N-acetoxy-2-(acetylamino)fluorene-treated DNA, is efficiently bound by the protein complex. These data suggest that Rad4.Rad23 interacts with DNA damage during NER and may play a role in recognition of the damage

Single Cell Analysis of Human RAD18-Dependent DNA Post-Replication Repair by Alkaline Bromodeoxyuridine Comet Assay

Science.gov (United States)

Mórocz, Mónika; Gali, Himabindu; Raskó, István; Downes, C. Stephen; Haracska, Lajos

2013-01-01

Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population. PMID:23936422

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

Repair of pyrimidine dimers in radiation-sensitive mutants rad3, rad4, rad6, and rad9 of Saccharomyces cerevisiae. [nicking

Energy Technology Data Exchange (ETDEWEB)

Prakash, L [Rochester Univ., N.Y. (USA). Dept. of Radiation Biology and Biophysics; Rochester Univ., N.Y. (USA). School of Medicine and Dentistry)

1977-10-01

The ability to remove ultraviolet-induced pyrimidine dimers was examined in four radiation-sensitive mutants of Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by either the T4 uv-endonuclease or an endonuclease activity found in crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad3 and rad4 mutants are shown to be defective in dimer excision whereas the rad6 and rad9 mutants are proficient in dimer excision.

Prevalence of pathogenic germline variants detected by multigene sequencing in unselected Japanese patients with ovarian cancer.

Science.gov (United States)

Hirasawa, Akira; Imoto, Issei; Naruto, Takuya; Akahane, Tomoko; Yamagami, Wataru; Nomura, Hiroyuki; Masuda, Kiyoshi; Susumu, Nobuyuki; Tsuda, Hitoshi; Aoki, Daisuke

2017-12-22

Pathogenic germline BRCA1 , BRCA2 ( BRCA1/2 ), and several other gene variants predispose women to primary ovarian, fallopian tube, and peritoneal carcinoma (OC), although variant frequency and relevance information is scarce in Japanese women with OC. Using targeted panel sequencing, we screened 230 unselected Japanese women with OC from our hospital-based cohort for pathogenic germline variants in 75 or 79 OC-associated genes. Pathogenic variants of 11 genes were identified in 41 (17.8%) women: 19 (8.3%; BRCA1 ), 8 (3.5%; BRCA2 ), 6 (2.6%; mismatch repair genes), 3 (1.3%; RAD51D ), 2 (0.9%; ATM ), 1 (0.4%; MRE11A ), 1 ( FANCC ), and 1 ( GABRA6 ). Carriers of BRCA1/2 or any other tested gene pathogenic variants were more likely to be diagnosed younger, have first or second-degree relatives with OC, and have OC classified as high-grade serous carcinoma (HGSC). After adjustment for these variables, all 3 features were independent predictive factors for pathogenic variants in any tested genes whereas only the latter two remained for variants in BRCA1/2 . Our data indicate similar variant prevalence in Japanese patients with OC and other ethnic groups and suggest that HGSC and OC family history may facilitate genetic predisposition prediction in Japanese patients with OC and referring high-risk patients for genetic counseling and testing.

Identifying pathogenicity of human variants via paralog-based yeast complementation.

Directory of Open Access Journals (Sweden)

Fan Yang

2017-05-01

Full Text Available To better understand the health implications of personal genomes, we now face a largely unmet challenge to identify functional variants within disease-associated genes. Functional variants can be identified by trans-species complementation, e.g., by failure to rescue a yeast strain bearing a mutation in an orthologous human gene. Although orthologous complementation assays are powerful predictors of pathogenic variation, they are available for only a few percent of human disease genes. Here we systematically examine the question of whether complementation assays based on paralogy relationships can expand the number of human disease genes with functional variant detection assays. We tested over 1,000 paralogous human-yeast gene pairs for complementation, yielding 34 complementation relationships, of which 33 (97% were novel. We found that paralog-based assays identified disease variants with success on par with that of orthology-based assays. Combining all homology-based assay results, we found that complementation can often identify pathogenic variants outside the homologous sequence region, presumably because of global effects on protein folding or stability. Within our search space, paralogy-based complementation more than doubled the number of human disease genes with a yeast-based complementation assay for disease variation.

Disruption of mouse RAD54 reduces ionizing radiation resistance and homologous recombination.

NARCIS (Netherlands)

J. Essers (Jeroen); R.W. Hendriks (Rudi); S.M.A. Swagemakers (Sigrid); C. Troelstra (Christine); J. de Wit (Jan); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan); R. Kanaar (Roland)

1997-01-01

textabstractDouble-strand DNA break (DSB) repair by homologous recombination occurs through the RAD52 pathway in Saccharomyces cerevisiae. Its biological importance is underscored by the conservation of many RAD52 pathway genes, including RAD54, from fungi to humans. We have analyzed the phenotype

Studies on emerging radiation leukemia virus variants in C57BL/Ka mice

International Nuclear Information System (INIS)

Rassart, E.; Shang, M.; Boie, Y.; Jolicoeur, P.

1986-01-01

To analyze the emergence of radiation leukemia virus (RadLV) variants in primary X-ray-induced C57BL/Ka thymoma and to identify the virus responsible for the very high leukemogenic potential of passaged Kaplan strain BL/VL3 preparation, we cloned several primary and passaged ecotropic RadLV infectious genomes. By restriction analysis, we found that BL/VL3 cells harbor three related but different ecotropic RadLVs. Their restriction map differs significantly from those of primary RadLVs. Hybridization analysis also indicated that BL/VL3 and primary RadLVs differ in their p15E and long terminal repeat (LTR) regions. The LTR sequence of primary weakly leukemogenic RadLV has only one change, a C-rich sequence, generating a 6-base-pair direct repeat just in front of the promotor. The LTR of the primary nonleukemogenic RadLV only showed few base changes, mainly clustered in R and U5. The LTR from a moderately leukemogenic passaged BL/VL3 RadLV had conserved the C-rich sequence and acquired a 43-base-pair direct repeat in U3 and several other point mutations, small insertions, and deletions scattered in U3, R, and U5. All cloned primary RadLVs were fibrotropic, and some were weakly leukemogenic. All cloned BL/VL3 RadLVs were thymotropic and nonfibrotropic. The block of their replication was found to be after the synthesis of unintegrated linear and supercoiled viral DNA. Most of the BL/VL3 RadLVs were moderately leukemogenic, and one (V-13) was highly leukemogenic, being as virulent as the Moloney strain. We propose a model for the emergence of the RadLV variants and show that the virus responsible for the high leukemogenic potential of BL/VL3 preparation is a nondefective, ecotropic, lymphotropic, nonfibrotropic, unique retrovirus which most likely arose from a parental primary RadLV similar to those studied here

Increased interreader agreement in diagnosis of hepatocellular carcinoma using an adapted LI-RADS algorithm

Energy Technology Data Exchange (ETDEWEB)

Becker, Anton S., E-mail: anton.becker@usz.ch; Barth, Borna K.; Marquez, Paulo H.; Donati, Olivio F.; Ulbrich, Erika J.; Karlo, Christoph; Reiner, Cäcilia S.; Fischer, Michael A.

2017-01-15

Purpose: To evaluate a simplified Liver Imaging Reporting and Data System (LI-RADS) algorithm to improve interreader agreement while maintaining diagnostic performance for HCC. Materials and methods: MRI scans of 84 cirrhotic patients with 104 distinct liver observations were retrospectively selected to equivocally match each of the LI-RADS grades (LR1-5) using histopathology and imaging follow up as standard of reference. Four independent radiologists categorized all observations as benign (LR1-2) or potentially malignant (LR3-5) and determined LI-RADS based imaging features including observation size, arterial phase hyperenhancement, washout, capsule appearance and threshold growth for LR3-5 observations and timed their readouts. LR3-5 observations were categorized according to the LI-RADS v2014 algorithm and according to a modified LI-RADS (mLI-RADS) version. Diagnostic performance and Interreader agreement were determined for LI-RADS and mLI-RADS using receiver operating characteristics (ROC) and Fleiss’ and Cohen’s Kappa analysis respectively. Results: ROC analysis revealed equal diagnostic performance for LI-RADS and mLI-RADS (area under the ROC curve = 0.91). Interreader agreement was higher using mLI-RADS as compared to current LI-RADS showing an improved overall (κ = 0.53 ± 0.04 vs. 0.45 ± 0.04), and pair-wise agreement between most readers (κ range 0.44-0.62 vs. 0.35-0.60) at a reduced median evaluation time (51 vs. 62 s per observation, p < 0.0001). Conclusion: Focusing on observation size and washout criteria using a modified, stepwise LI-RADS decision tree for LR3-5 observations results in higher interobserver reliability and faster categorization while maintaining diagnostic accuracy.

Sensing Conformational Changes in DNA upon Ligand Binding Using QCM-D. Polyamine Condensation and Rad51 Extension of DNA Layers

KAUST Repository

Sun, Lu

2014-10-16

© 2014 American Chemical Society. Biosensors, in which binding of ligands is detected through changes in the optical or electrochemical properties of a DNA layer confined to the sensor surface, are important tools for investigating DNA interactions. Here, we investigate if conformational changes induced in surface-attached DNA molecules upon ligand binding can be monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. DNA duplexes containing 59-184 base pairs were formed on QCM-D crystals by stepwise assembly of synthetic oligonucleotides of designed base sequences. The DNA films were exposed to the cationic polyamines spermidine and spermine, known to condense DNA molecules in bulk experiments, or to the recombination protein Rad51, known to extend the DNA helix. The binding and dissociation of the ligands to the DNA films were monitored in real time by measurements of the shifts in resonance frequency (Δf) and in dissipation (ΔD). The QCM-D data were analyzed using a Voigt-based model for the viscoelastic properties of polymer films in order to evaluate how the ligands affect thickness and shear viscosity of the DNA layer. Binding of spermine shrinks all DNA layers and increases their viscosity in a reversible fashion, and so does spermidine, but to a smaller extent, in agreement with its lower positive charge. SPR was used to measure the amount of bound polyamines, and when combined with QCM-D, the data indicate that the layer condensation leads to a small release of water from the highly hydrated DNA films. The binding of Rad51 increases the effective layer thickness of a 59bp film, more than expected from the know 50% DNA helix extension. The combined results provide guidelines for a QCM-D biosensor based on ligand-induced structural changes in DNA films. The QCM-D approach provides high discrimination between ligands affecting the thickness and the structural properties of the DNA layer differently. The reversibility of the film

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.

Breast imaging reporting and data system (BI-RADS) lexicon for breast MRI: Interobserver variability in the description and assignment of BI-RADS category

International Nuclear Information System (INIS)

El Khoury, Mona; Lalonde, Lucie; David, Julie; Labelle, Maude; Mesurolle, Benoit; Trop, Isabelle

2015-01-01

Highlights: • The use of BI-RADS lexicon in interpreting breast MRI examinations is beneficial. • Our study shows: (a) moderate to substantial agreement between observers and (b) better agreement in interpreting mass than non-mass enhancement (NME). • Careful analysis of the NME should be done to help detect cancer as early as possible. - Abstract: Purpose: To retrospectively evaluate interobserver variability between breast radiologists when describing abnormal enhancement on breast MR examinations and assigning a BI-RADS category using the Breast Imaging Reporting and Data System (BI-RADS) terminology. Materials and methods: Five breast radiologists blinded to patients’ medical history and pathologic results retrospectively and independently reviewed 257 abnormal areas of enhancement on breast MRI performed in 173 women. Each radiologist described the focal enhancement using BI-RADS terminology and assigned a final BI-RADS category. Krippendorff's α coefficient of agreement was used to asses interobserver variability. Results: All radiologists agreed on the morphology of enhancement in 183/257 (71%) lesions, yielding a substantial agreement (Krippendorff's α = 0.71). Moderate agreement was obtained for mass descriptors – shape, margins and internal enhancement – (α = 0.55, 0.51 and 0.45 respectively) and NME (non-mass enhancement) descriptors – distribution and internal enhancement – (α = 0.54 and 0.43). Overall substantial agreement was obtained for BI-RADS category assignment (α = 0.71). It was however only moderate (α = 0.38) for NME compared to mass (α = 0.80). Conclusion: Our study shows good agreement in describing mass and NME on a breast MR examination but a better agreement in predicting malignancy for mass than NME

Breast imaging reporting and data system (BI-RADS) lexicon for breast MRI: Interobserver variability in the description and assignment of BI-RADS category

Energy Technology Data Exchange (ETDEWEB)

El Khoury, Mona, E-mail: monelkhoury@gmail.com [Centre Hospitalier Universitaire de Montréal, Breast Centre, Radiology Department, 3840 Rue Saint Urbain, Montréal, QC H2W1T8 (Canada); Lalonde, Lucie; David, Julie; Labelle, Maude [Centre Hospitalier Universitaire de Montréal, Breast Centre, Radiology Department, 3840 Rue Saint Urbain, Montréal, QC H2W1T8 (Canada); Mesurolle, Benoit [Centre Hospitalier Universitaire de McGill, Cedar Breast Centre, Radiology Department, 687 Pine Avenue West, Montreal, QC H3A1A1 (Canada); Trop, Isabelle [Centre Hospitalier Universitaire de Montréal, Breast Centre, Radiology Department, 3840 Rue Saint Urbain, Montréal, QC H2W1T8 (Canada)

2015-01-15

Highlights: • The use of BI-RADS lexicon in interpreting breast MRI examinations is beneficial. • Our study shows: (a) moderate to substantial agreement between observers and (b) better agreement in interpreting mass than non-mass enhancement (NME). • Careful analysis of the NME should be done to help detect cancer as early as possible. - Abstract: Purpose: To retrospectively evaluate interobserver variability between breast radiologists when describing abnormal enhancement on breast MR examinations and assigning a BI-RADS category using the Breast Imaging Reporting and Data System (BI-RADS) terminology. Materials and methods: Five breast radiologists blinded to patients’ medical history and pathologic results retrospectively and independently reviewed 257 abnormal areas of enhancement on breast MRI performed in 173 women. Each radiologist described the focal enhancement using BI-RADS terminology and assigned a final BI-RADS category. Krippendorff's α coefficient of agreement was used to asses interobserver variability. Results: All radiologists agreed on the morphology of enhancement in 183/257 (71%) lesions, yielding a substantial agreement (Krippendorff's α = 0.71). Moderate agreement was obtained for mass descriptors – shape, margins and internal enhancement – (α = 0.55, 0.51 and 0.45 respectively) and NME (non-mass enhancement) descriptors – distribution and internal enhancement – (α = 0.54 and 0.43). Overall substantial agreement was obtained for BI-RADS category assignment (α = 0.71). It was however only moderate (α = 0.38) for NME compared to mass (α = 0.80). Conclusion: Our study shows good agreement in describing mass and NME on a breast MR examination but a better agreement in predicting malignancy for mass than NME.

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

CDKL5 variants

Science.gov (United States)

Kalscheuer, Vera M.; Hennig, Friederike; Leonard, Helen; Downs, Jenny; Clarke, Angus; Benke, Tim A.; Armstrong, Judith; Pineda, Mercedes; Bailey, Mark E.S.; Cobb, Stuart R.

2017-01-01

Objective: To provide new insights into the interpretation of genetic variants in a rare neurologic disorder, CDKL5 deficiency, in the contexts of population sequencing data and an updated characterization of the CDKL5 gene. Methods: We analyzed all known potentially pathogenic CDKL5 variants by combining data from large-scale population sequencing studies with CDKL5 variants from new and all available clinical cohorts and combined this with computational methods to predict pathogenicity. Results: The study has identified several variants that can be reclassified as benign or likely benign. With the addition of novel CDKL5 variants, we confirm that pathogenic missense variants cluster in the catalytic domain of CDKL5 and reclassify a purported missense variant as having a splicing consequence. We provide further evidence that missense variants in the final 3 exons are likely to be benign and not important to disease pathology. We also describe benign splicing and nonsense variants within these exons, suggesting that isoform hCDKL5_5 is likely to have little or no neurologic significance. We also use the available data to make a preliminary estimate of minimum incidence of CDKL5 deficiency. Conclusions: These findings have implications for genetic diagnosis, providing evidence for the reclassification of specific variants previously thought to result in CDKL5 deficiency. Together, these analyses support the view that the predominant brain isoform in humans (hCDKL5_1) is crucial for normal neurodevelopment and that the catalytic domain is the primary functional domain. PMID:29264392

RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress

DEFF Research Database (Denmark)

Bhowmick, Rahul; Minocherhomji, Sheroy; Hickson, Ian D

2016-01-01

Homologous recombination (HR) is necessary to counteract DNA replication stress. Common fragile site (CFS) loci are particularly sensitive to replication stress and undergo pathological rearrangements in tumors. At these loci, replication stress frequently activates DNA repair synthesis in mitosis...... replication stress at CFS loci during S-phase. In contrast, MiDAS is RAD52 dependent, and RAD52 is required for the timely recruitment of MUS81 and POLD3 to CFSs in early mitosis. Our results provide further mechanistic insight into MiDAS and define a specific function for human RAD52. Furthermore, selective...

Homologous recombination contributes to the repair of DNA double-strand breaks induced by high-energy iron ions

Energy Technology Data Exchange (ETDEWEB)

Zafar, Faria; Seidler, Sara B.; Kronenberg, Amy; Schild, David; Wiese, Claudia

2010-06-29

To test the contribution of homologous recombinational repair (HRR) in repairing DNA damaged sites induced by high-energy iron ions, we used: (1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We show that in response to iron ions, HRR contributes to cell survival in rodent cells, and that HRR-deficiency abrogates RAD51 foci formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 foci formation. For human cells irradiated with iron ions, cell survival is decreased, and, in p53 mutant cells, the levels of mutagenesis are increased when HRR is impaired. Human cells synchronized in S phase exhibit more pronounced resistance to iron ions as compared with cells in G1 phase, and this increase in radioresistance is diminished by RAD51 knockdown. These results implicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged particle irradiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival in response to high-energy high LET radiation.

Homologous recombination contributes to the repair of DNA double-strand breaks induced by high-energy iron ions

International Nuclear Information System (INIS)

Zafar, Faria; Seidler, Sara B.; Kronenberg, Amy; Schild, David; Wiese, Claudia

2010-01-01

To test the contribution of homologous recombinational repair (HRR) in repairing DNA damaged sites induced by high-energy iron ions, we used: (1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We show that in response to iron ions, HRR contributes to cell survival in rodent cells, and that HRR-deficiency abrogates RAD51 foci formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 foci formation. For human cells irradiated with iron ions, cell survival is decreased, and, in p53 mutant cells, the levels of mutagenesis are increased when HRR is impaired. Human cells synchronized in S phase exhibit more pronounced resistance to iron ions as compared with cells in G1 phase, and this increase in radioresistance is diminished by RAD51 knockdown. These results implicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged particle irradiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival in response to high-energy high LET radiation.

A global evolutionary and metabolic analysis of human obesity gene risk variants.

Science.gov (United States)

Castillo, Joseph J; Hazlett, Zachary S; Orlando, Robert A; Garver, William S

2017-09-05

It is generally accepted that the selection of gene variants during human evolution optimized energy metabolism that now interacts with our obesogenic environment to increase the prevalence of obesity. The purpose of this study was to perform a global evolutionary and metabolic analysis of human obesity gene risk variants (110 human obesity genes with 127 nearest gene risk variants) identified using genome-wide association studies (GWAS) to enhance our knowledge of early and late genotypes. As a result of determining the mean frequency of these obesity gene risk variants in 13 available populations from around the world our results provide evidence for the early selection of ancestral risk variants (defined as selection before migration from Africa) and late selection of derived risk variants (defined as selection after migration from Africa). Our results also provide novel information for association of these obesity genes or encoded proteins with diverse metabolic pathways and other human diseases. The overall results indicate a significant differential evolutionary pattern for the selection of obesity gene ancestral and derived risk variants proposed to optimize energy metabolism in varying global environments and complex association with metabolic pathways and other human diseases. These results are consistent with obesity genes that encode proteins possessing a fundamental role in maintaining energy metabolism and survival during the course of human evolution. Copyright © 2017. Published by Elsevier B.V.

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.

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

Science.gov (United States)

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.

Human genomic disease variants: a neutral evolutionary explanation.

Science.gov (United States)

Dudley, Joel T; Kim, Yuseob; Liu, Li; Markov, Glenn J; Gerold, Kristyn; Chen, Rong; Butte, Atul J; Kumar, Sudhir

2012-08-01

Many perspectives on the role of evolution in human health include nonempirical assumptions concerning the adaptive evolutionary origins of human diseases. Evolutionary analyses of the increasing wealth of clinical and population genomic data have begun to challenge these presumptions. In order to systematically evaluate such claims, the time has come to build a common framework for an empirical and intellectual unification of evolution and modern medicine. We review the emerging evidence and provide a supporting conceptual framework that establishes the classical neutral theory of molecular evolution (NTME) as the basis for evaluating disease- associated genomic variations in health and medicine. For over a decade, the NTME has already explained the origins and distribution of variants implicated in diseases and has illuminated the power of evolutionary thinking in genomic medicine. We suggest that a majority of disease variants in modern populations will have neutral evolutionary origins (previously neutral), with a relatively smaller fraction exhibiting adaptive evolutionary origins (previously adaptive). This pattern is expected to hold true for common as well as rare disease variants. Ultimately, a neutral evolutionary perspective will provide medicine with an informative and actionable framework that enables objective clinical assessment beyond convenient tendencies to invoke past adaptive events in human history as a root cause of human disease.

Common genetic variants influence human subcortical brain structures.

Science.gov (United States)

Hibar, Derrek P; Stein, Jason L; Renteria, Miguel E; Arias-Vasquez, Alejandro; Desrivières, Sylvane; Jahanshad, Neda; Toro, Roberto; Wittfeld, Katharina; Abramovic, Lucija; Andersson, Micael; Aribisala, Benjamin S; Armstrong, Nicola J; Bernard, Manon; Bohlken, Marc M; Boks, Marco P; Bralten, Janita; Brown, Andrew A; Chakravarty, M Mallar; Chen, Qiang; Ching, Christopher R K; Cuellar-Partida, Gabriel; den Braber, Anouk; Giddaluru, Sudheer; Goldman, Aaron L; Grimm, Oliver; Guadalupe, Tulio; Hass, Johanna; Woldehawariat, Girma; Holmes, Avram J; Hoogman, Martine; Janowitz, Deborah; Jia, Tianye; Kim, Sungeun; Klein, Marieke; Kraemer, Bernd; Lee, Phil H; Olde Loohuis, Loes M; Luciano, Michelle; Macare, Christine; Mather, Karen A; Mattheisen, Manuel; Milaneschi, Yuri; Nho, Kwangsik; Papmeyer, Martina; Ramasamy, Adaikalavan; Risacher, Shannon L; Roiz-Santiañez, Roberto; Rose, Emma J; Salami, Alireza; Sämann, Philipp G; Schmaal, Lianne; Schork, Andrew J; Shin, Jean; Strike, Lachlan T; Teumer, Alexander; van Donkelaar, Marjolein M J; van Eijk, Kristel R; Walters, Raymond K; Westlye, Lars T; Whelan, Christopher D; Winkler, Anderson M; Zwiers, Marcel P; Alhusaini, Saud; Athanasiu, Lavinia; Ehrlich, Stefan; Hakobjan, Marina M H; Hartberg, Cecilie B; Haukvik, Unn K; Heister, Angelien J G A M; Hoehn, David; Kasperaviciute, Dalia; Liewald, David C M; Lopez, Lorna M; Makkinje, Remco R R; Matarin, Mar; Naber, Marlies A M; McKay, D Reese; Needham, Margaret; Nugent, Allison C; Pütz, Benno; Royle, Natalie A; Shen, Li; Sprooten, Emma; Trabzuni, Daniah; van der Marel, Saskia S L; van Hulzen, Kimm J E; Walton, Esther; Wolf, Christiane; Almasy, Laura; Ames, David; Arepalli, Sampath; Assareh, Amelia A; Bastin, Mark E; Brodaty, Henry; Bulayeva, Kazima B; Carless, Melanie A; Cichon, Sven; Corvin, Aiden; Curran, Joanne E; Czisch, Michael; de Zubicaray, Greig I; Dillman, Allissa; Duggirala, Ravi; Dyer, Thomas D; Erk, Susanne; Fedko, Iryna O; Ferrucci, Luigi; Foroud, Tatiana M; Fox, Peter T; Fukunaga, Masaki; Gibbs, J Raphael; Göring, Harald H H; Green, Robert C; Guelfi, Sebastian; Hansell, Narelle K; Hartman, Catharina A; Hegenscheid, Katrin; Heinz, Andreas; Hernandez, Dena G; Heslenfeld, Dirk J; Hoekstra, Pieter J; Holsboer, Florian; Homuth, Georg; Hottenga, Jouke-Jan; Ikeda, Masashi; Jack, Clifford R; Jenkinson, Mark; Johnson, Robert; Kanai, Ryota; Keil, Maria; Kent, Jack W; Kochunov, Peter; Kwok, John B; Lawrie, Stephen M; Liu, Xinmin; Longo, Dan L; McMahon, Katie L; Meisenzahl, Eva; Melle, Ingrid; Mohnke, Sebastian; Montgomery, Grant W; Mostert, Jeanette C; Mühleisen, Thomas W; Nalls, Michael A; Nichols, Thomas E; Nilsson, Lars G; Nöthen, Markus M; Ohi, Kazutaka; Olvera, Rene L; Perez-Iglesias, Rocio; Pike, G Bruce; Potkin, Steven G; Reinvang, Ivar; Reppermund, Simone; Rietschel, Marcella; Romanczuk-Seiferth, Nina; Rosen, Glenn D; Rujescu, Dan; Schnell, Knut; Schofield, Peter R; Smith, Colin; Steen, Vidar M; Sussmann, Jessika E; Thalamuthu, Anbupalam; Toga, Arthur W; Traynor, Bryan J; Troncoso, Juan; Turner, Jessica A; Valdés Hernández, Maria C; van 't Ent, Dennis; van der Brug, Marcel; van der Wee, Nic J A; van Tol, Marie-Jose; Veltman, Dick J; Wassink, Thomas H; Westman, Eric; Zielke, Ronald H; Zonderman, Alan B; Ashbrook, David G; Hager, Reinmar; Lu, Lu; McMahon, Francis J; Morris, Derek W; Williams, Robert W; Brunner, Han G; Buckner, Randy L; Buitelaar, Jan K; Cahn, Wiepke; Calhoun, Vince D; Cavalleri, Gianpiero L; Crespo-Facorro, Benedicto; Dale, Anders M; Davies, Gareth E; Delanty, Norman; Depondt, Chantal; Djurovic, Srdjan; Drevets, Wayne C; Espeseth, Thomas; Gollub, Randy L; Ho, Beng-Choon; Hoffmann, Wolfgang; Hosten, Norbert; Kahn, René S; Le Hellard, Stephanie; Meyer-Lindenberg, Andreas; Müller-Myhsok, Bertram; Nauck, Matthias; Nyberg, Lars; Pandolfo, Massimo; Penninx, Brenda W J H; Roffman, Joshua L; Sisodiya, Sanjay M; Smoller, Jordan W; van Bokhoven, Hans; van Haren, Neeltje E M; Völzke, Henry; Walter, Henrik; Weiner, Michael W; Wen, Wei; White, Tonya; Agartz, Ingrid; Andreassen, Ole A; Blangero, John; Boomsma, Dorret I; Brouwer, Rachel M; Cannon, Dara M; Cookson, Mark R; de Geus, Eco J C; Deary, Ian J; Donohoe, Gary; Fernández, Guillén; Fisher, Simon E; Francks, Clyde; Glahn, David C; Grabe, Hans J; Gruber, Oliver; Hardy, John; Hashimoto, Ryota; Hulshoff Pol, Hilleke E; Jönsson, Erik G; Kloszewska, Iwona; Lovestone, Simon; Mattay, Venkata S; Mecocci, Patrizia; McDonald, Colm; McIntosh, Andrew M; Ophoff, Roel A; Paus, Tomas; Pausova, Zdenka; Ryten, Mina; Sachdev, Perminder S; Saykin, Andrew J; Simmons, Andy; Singleton, Andrew; Soininen, Hilkka; Wardlaw, Joanna M; Weale, Michael E; Weinberger, Daniel R; Adams, Hieab H H; Launer, Lenore J; Seiler, Stephan; Schmidt, Reinhold; Chauhan, Ganesh; Satizabal, Claudia L; Becker, James T; Yanek, Lisa; van der Lee, Sven J; Ebling, Maritza; Fischl, Bruce; Longstreth, W T; Greve, Douglas; Schmidt, Helena; Nyquist, Paul; Vinke, Louis N; van Duijn, Cornelia M; Xue, Luting; Mazoyer, Bernard; Bis, Joshua C; Gudnason, Vilmundur; Seshadri, Sudha; Ikram, M Arfan; Martin, Nicholas G; Wright, Margaret J; Schumann, Gunter; Franke, Barbara; Thompson, Paul M; Medland, Sarah E

2015-04-09

The highly complex structure of the human brain is strongly shaped by genetic influences. Subcortical brain regions form circuits with cortical areas to coordinate movement, learning, memory and motivation, and altered circuits can lead to abnormal behaviour and disease. To investigate how common genetic variants affect the structure of these brain regions, here we conduct genome-wide association studies of the volumes of seven subcortical regions and the intracranial volume derived from magnetic resonance images of 30,717 individuals from 50 cohorts. We identify five novel genetic variants influencing the volumes of the putamen and caudate nucleus. We also find stronger evidence for three loci with previously established influences on hippocampal volume and intracranial volume. These variants show specific volumetric effects on brain structures rather than global effects across structures. The strongest effects were found for the putamen, where a novel intergenic locus with replicable influence on volume (rs945270; P = 1.08 × 10(-33); 0.52% variance explained) showed evidence of altering the expression of the KTN1 gene in both brain and blood tissue. Variants influencing putamen volume clustered near developmental genes that regulate apoptosis, axon guidance and vesicle transport. Identification of these genetic variants provides insight into the causes of variability in human brain development, and may help to determine mechanisms of neuropsychiatric dysfunction.

Implications of a RAD54L polymorphism (2290C/T) in human meningiomas as a risk factor and/or a genetic marker

International Nuclear Information System (INIS)

Leone, Paola E; Mendiola, Marta; Alonso, Javier; Paz-y-Miño, César; Pestaña, Angel

2003-01-01

RAD54L (OMIM 603615, Locus Link 8438) has been proposed as a candidate oncosupressor in tumours bearing a non-random deletion of 1p32, such as breast or colon carcinomas, lymphomas and meningiomas. In a search for RAD54L mutations in 29 menigiomas with allelic deletions in 1p, the only genetic change observed was a silent C/T transition at nucleotide 2290 in exon 18. In this communication the possible association of the 2290C/T polymorphism with the risk of meningiomas was examined. In addition, the usefulness of this polymorphism as a genetic marker within the meningioma consensus deletion region in 1p32 was also verified. The present study comprises 287 blood control samples and 70 meningiomas from Spain and Ecuador. Matched blood samples were only available from Spanish patients. The frequency of the rare allele-T and heterozygotes for the 2290C/T polymorphism in the blood of Spanish meningioma patients and in the Ecuadorian meningioma tumours was higher than in the control population (P < 0.05). Four other rare variants (2290C/G, 2299C/G, 2313G/A, 2344A/G) were found within 50 bp at the 3' end of RAD54L. Frequent loss of heterozygosity for the 2290C/T SNP in meningiomas allowed to further narrow the 1p32 consensus region of deletion in meningiomas to either 2.08 Mbp – within D1S2713 (44.35 Mbp) and RAD54L (46.43 Mbp) – or to 1.47 Mbp – within RAD54L and D1S2134 (47.90 Mbp) – according to recent gene mapping results. The statistical analysis of genotypes at the 2290C/T polymorphism suggest an association between the rare T allele and the development of meningeal tumours. This polymorphism can be used as a genetic marker inside the consensus deletion region at 1p32 in meningiomas

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

A PYY Q62P variant linked to human obesity

Energy Technology Data Exchange (ETDEWEB)

Ahituv, Nadav; Kavaslar, Nihan; Schackwitz, Wendy; Ustaszewska,Anna; Collier, John Michael; Hebert, Sybil; Doelle, Heather; Dent,Robert; Pennacchio, Len A.; McPherson, Ruth

2005-06-27

Members of the pancreatic polypeptide family and the irreceptors have been implicated in the control of food intake in rodents and humans. To investigate whether nucleotide changes in these candidate genes result in abnormal weight in humans, we sequenced the coding exons and splice sites of seven family members (NPY, PYY, PPY, NPY1R, NPY2R, NPY4R, and NPY5R) in a large cohort of extremely obese (n=379) and lean (n=378) individuals. In total we found eleven rare non-synonymous variants, four of which exhibited familial segregation, NPY1R L53P and PPY P63L with leanness and NPY2R D42G and PYY Q62P with obesity. Functional analysis of the obese variants revealed NPY2R D42G to have reduced cell surface expression, while previous cell culture based studies indicated variant PYY Q62P to have altered receptor binding selectivity and we show that it fails to reduce food intake through mouse peptide injection experiments. These results support that rare non-synonymous variants within these genes can alter susceptibility to human body mass index extremes.

Schizosaccharomyces pombe Mms1 channels repair of perturbed replication into Rhp51 independent homologous recombination

DEFF Research Database (Denmark)

Vejrup-Hansen, Rasmus; Mizuno, Ken'Ichi; Miyabe, Izumi

2011-01-01

-like protein, Rtt101/Cul8, a potential paralog of Cullin 4. We performed epistasis analysis between ¿mms1 and mutants of pathways with known functions in genome integrity, and measured the recruitment of homologous recombination proteins to blocked replication forks and recombination frequencies. We show that......-specific replication fork barrier and that, in a ¿mms1 strain, Rad22(Rad52) and RPA recruitment to blocked forks are reduced, whereas Rhp51 recruitment is unaffected. In addition, Mms1 appears to specifically promote chromosomal rearrangements in a recombination assay. These observations suggest that Mms1 acts...... is particularly important when a single strand break is converted into a double strand break during replication. Genetic data connect Mms1 to a Mus81 and Rad22(Rad52) dependent, but Rhp51 independent, branch of homologous recombination. This is supported by results demonstrating that Mms1 is recruited to a site...

Molecular Pathways

Science.gov (United States)

Lok, Benjamin H.; Powell, Simon N.

2012-01-01

The Rad52 protein was largely ignored in humans and other mammals when the mouse knockout revealed a largely “no-effect” phenotype. However, using synthetic lethal approaches to investigate context dependent function, new studies have shown that Rad52 plays a key survival role in cells lacking the function of the BRCA1-BRCA2 pathway of homologous recombination. Biochemical studies also showed significant differences between yeast and human Rad52, in which yeast Rad52 can promote strand invasion of RPA-coated single-stranded DNA in the presence of Rad51, but human Rad52 cannot. This results in the paradox of how is human Rad52 providing Rad51 function: presumably there is something missing in the biochemical assays that exists in-vivo, but the nature of this missing factor is currently unknown. Recent studies have suggested that Rad52 provides back-up Rad51 function for all members of the BRCA1-BRCA2 pathway, suggesting that Rad52 may be a target for therapy in BRCA pathway deficient cancers. Screening for ways to inhibit Rad52 would potentially provide a complementary strategy for targeting BRCA-deficient cancers in addition to PARP inhibitors. PMID:23071261

RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

DEFF Research Database (Denmark)

Sleeth, Kate M; Sørensen, Claus Storgaard; Issaeva, Natalia

2007-01-01

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited...... the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation...... and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role...

Human activated protein C variants in a rat model of arterial thrombosis

Directory of Open Access Journals (Sweden)

Dahlbäck Björn

2008-10-01

Full Text Available Abstract Background Activated protein C (APC inhibits coagulation by degrading activated factor V (FVa and factor VIII (FVIIIa, protein S (PS functioning as a cofactor to APC. Methods By mutagenesis of the vitamin K-dependent Gla domain of APC, we have recently created an APC variant having enhanced anticoagulant activity due to increased affinity for negatively charged phospholipid membranes. In the present study, the potential antithrombotic effects of this APC variant, and of a variant APC that is additionally mutated in the serine protease domain, have been evaluated in a blind randomized study in a rat model of arterial thrombosis. In this model, we have previously found the combination of bovine APC and PS to be highly antithrombotic. Four treatment groups each containing 10 rats were, in a blind random fashion, given intravenous bolus injections of wild-type or mutant variants of APC (0.8 mg/kg together with human PS (0.6 mg/kg or human PS (0.6 mg/kg alone. A control group with 20 animals where given vehicle only. Results A trend to increased patency rates was noted in a group receiving one of the APC variants, but it did not reach statistical significance. Conclusion In conclusion, administration of human APC variants having enhanced anticoagulant efficacy together with human PS in a rat model of arterial thrombosis did not give an efficient antithrombotic effect. The lack of effect may be due to species-specific differences between the human protein C system and the rat hemostatic system.

Structural Basis of Human CYP51 Inhibition by Antifungal Azoles

Energy Technology Data Exchange (ETDEWEB)

Strushkevich, Natallia; Usanov, Sergey A.; Park, Hee-Won (Toronto); (IBC-Belarus)

2010-09-22

The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (sterol 14-alpha demethylase) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, meiosis-activating sterols, are produced by CYP51. Three crystal structures of human CYP51, ligand-free and complexed with antifungal drugs ketoconazole and econazole, were determined, allowing analysis of the molecular basis for functional conservation within the CYP51 family. Azole binding occurs mostly through hydrophobic interactions with conservative residues of the active site. The substantial conformational changes in the B{prime} helix and F-G loop regions are induced upon ligand binding, consistent with the membrane nature of the protein and its substrate. The access channel is typical for mammalian sterol-metabolizing P450 enzymes, but is different from that observed in Mycobacterium tuberculosis CYP51. Comparison of the azole-bound structures provides insight into the relative binding affinities of human and bacterial P450 enzymes to ketoconazole and fluconazole, which can be useful for the rational design of antifungal compounds and specific modulators of human CYP51.

Hb variants in Korea: effect on HbA1c using five routine methods.

Science.gov (United States)

Yun, Yeo-Min; Ji, Misuk; Ko, Dae-Hyun; Chun, Sail; Kwon, Gye Cheol; Lee, Kyunghoon; Song, Sang Hoon; Seong, Moon Woo; Park, Sung Sup; Song, Junghan

2017-07-26

Quantification of glycated hemoglobin (HbA1c) is a challenge in patients with hemoglobin (Hb) variants. We evaluated the impact of various Hb variants on five routine HbA1c assays by comparing with the IFCC reference measurement procedure (RMP). Whole blood samples showing warning flags or no results on routine HPLC HbA1c assays were confirmed for Hb variants and were submitted to HbA1c quantification using Sebia Capillarys 2 Flex Piercing, Roche Tina-quant HbA1c Gen. 2, Bio-Rad Variant II Turbo 2.0, ADAMS HA-8180, Tosoh G8 standard mode, and IFCC RMP using LC-MS. Among 114 samples, the most common variants were Hb G-Coushatta (n=47), Queens (n=41), Ube-4 (n=11), Chad (n=4), Yamagata (n=4), G-His-Tsou (n=2), G-Taipei (n=1), Fort de France (n=1), Hoshida (n=1), and two novel variants (Hb α-globin, HBA 52 Gly>Cys and Hb β-globin, HBB 146 His>Asn). In terms of control samples, all the result of HbA1c were "acceptable", within the criteria of ±7% compared to IFCC RMP target values. However, percentage of "unacceptable" results of samples with Hb variants were 16% for Capillarys 2, 7% for Tina-quant, 51% for Variant II Turbo 2.0, 95% for G8 standard mode, and 89% for HA-8180. The Capillarys 2 and HA-8180 assay did not provide the results in 5 and 40 samples with Hb variants, respectively. HbA1c results from five routine assays in patients with relatively common Hb variants in Korea showed various degrees of bias compared to those of IFCC RMP. Therefore, laboratories should be aware of the limitation of their methods with respect to interference from Hb variants found commonly in their local population and suggest an alternative HbA1c quantification method.

Hundreds of variants clustered in genomic loci and biological pathways affect human height

DEFF Research Database (Denmark)

Lango Allen, Hana; Estrada, Karol; Lettre, Guillaume

2010-01-01

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions...

Annotating DNA variants is the next major goal for human genetics.

Science.gov (United States)

Cutting, Garry R

2014-01-02

Clinical genetic testing has undergone a dramatic transformation in the past two decades. Diagnostic laboratories that previously tested for well-established disease-causing DNA variants in a handful of genes have evolved into sequencing factories identifying thousands of variants of known and unknown medical consequence. Sorting out what does and does not cause disease in our genomes is the next great challenge in making genetics a central feature of healthcare. I propose that closing the gap in our ability to interpret variation responsible for Mendelian disorders provides a grand and unprecedented opportunity for geneticists. Human geneticists are well placed to coordinate a systematic evaluation of variants in collaboration with basic scientists and clinicians. Sharing of knowledge, data, methods, and tools will aid both researchers and healthcare workers in achieving their common goal of defining the pathogenic potential of variants. Generation of variant annotations will inform genetic testing and will deepen our understanding of gene and protein function, thereby aiding the search for molecular targeted therapies. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Hundreds of variants clustered in genomic loci and biological pathways affect human height

NARCIS (Netherlands)

H.L. Allen; K. Estrada Gil (Karol); G. Lettre (Guillaume); S.I. Berndt (Sonja); F. Rivadeneira Ramirez (Fernando); C.J. Willer (Cristen); A.U. Jackson (Anne); S. Vedantam (Sailaja); S. Raychaudhuri (Soumya); T. Ferreira (Teresa); A.R. Wood (Andrew); R.J. Weyant (Robert); A.V. Segrè (Ayellet); E.K. Speliotes (Elizabeth); E. Wheeler (Eleanor); N. Soranzo (Nicole); J.H. Park; J. Yang (Joanna); D.F. Gudbjartsson (Daniel); N.L. Heard-Costa (Nancy); J.C. Randall (Joshua); L. Qi (Lu); A.V. Smith (Albert Vernon); R. Mägi (Reedik); T. Pastinen (Tomi); L. Liang (Liming); I.M. Heid (Iris); J. Luan; G. Thorleifsson (Gudmar); T.W. Winkler (Thomas); M.E. Goddard (Michael); K.S. Lo; C. Palmer (Cameron); T. Workalemahu (Tsegaselassie); Y.S. Aulchenko (Yurii); A. Johansson (Åsa); M.C. Zillikens (Carola); M.F. Feitosa (Mary Furlan); T. Esko (Tõnu); T. Johnson (Toby); S. Ketkar (Shamika); P. Kraft (Peter); M. Mangino (Massimo); I. Prokopenko (Inga); D. Absher (Devin); E. Albrecht (Eva); F.D.J. Ernst (Florian); N.L. Glazer (Nicole); C. Hayward (Caroline); J.J. Hottenga (Jouke Jan); K.B. Jacobs (Kevin); J.W. Knowles (Joshua); Z. Kutalik (Zoltán); K.L. Monda (Keri); O. Polasek (Ozren); M. Preuss (Michael); N.W. Rayner (Nigel William); N.R. Robertson (Neil); V. Steinthorsdottir (Valgerdur); J.P. Tyrer (Jonathan); B.F. Voight (Benjamin); F. Wiklund (Fredrik); J. Xu (Jianfeng); J.H. Zhao (Jing Hua); D.R. Nyholt (Dale); N. Pellikka (Niina); M. Perola (Markus); J.R.B. Perry (John); I. Surakka (Ida); M.L. Tammesoo; E.L. Altmaier (Elizabeth); N. Amin (Najaf); T. Aspelund (Thor); T. Bhangale (Tushar); G. Boucher (Gabrielle); D.I. Chasman (Daniel); C. Chen (Constance); L. Coin (Lachlan); M.N. Cooper (Matthew); A.L. Dixon (Anna); Q. Gibson (Quince); E. Grundberg (Elin); K. Hao (Ke); M.J. Junttila (Juhani); R.C. Kaplan (Robert); J. Kettunen (Johannes); I.R. König (Inke); T. Kwan (Tony); R.W. Lawrence (Robert); D.F. Levinson (Douglas); M. Lorentzon (Mattias); B. McKnight (Barbara); A.D. Morris (Andrew); M. Müller (Martina); J.S. Ngwa; S. Purcell (Shaun); S. Rafelt (Suzanne); R.M. Salem (Rany); E. Salvi (Erika); S. Sanna (Serena); J. Shi (Jianxin); U. Sovio (Ulla); J.R. Thompson (John); M.C. Turchin (Michael); L. Vandenput (Liesbeth); D.J. Verlaan (Dominique); V. Vitart (Veronique); C.C. White (Charles); A. Ziegler (Andreas); P. Almgren (Peter); A.J. Balmforth (Anthony); H. Campbell (Harry); L. Citterio (Lorena); A. de Grandi (Alessandro); A. Dominiczak (Anna); J. Duan (Jubao); P. Elliott (Paul); R. Elosua (Roberto); J.G. Eriksson (Johan); N.B. Freimer (Nelson); E.J.C. de Geus (Eco); N. Glorioso (Nicola); S. Haiqing (Shen); A.L. Hartikainen; A.S. Havulinna (Aki); A.A. Hicks (Andrew); J. Hui (Jennie); W. Igl (Wilmar); T. Illig (Thomas); A. Jula (Antti); E. Kajantie (Eero); T.O. Kilpeläinen (Tuomas); M. Koiranen (Markku); I. Kolcic (Ivana); S. Koskinen (Seppo); P. Kovacs (Peter); J. Laitinen (Jaana); J. Liu (Jianjun); M.L. Lokki; A. Marusic (Ana); A. Maschio; T. Meitinger (Thomas); A. Mulas (Antonella); G. Paré (Guillaume); A.N. Parker (Alex); J. Peden (John); A. Petersmann (Astrid); I. Pichler (Irene); K.H. Pietilainen (Kirsi Hannele); A. Pouta (Anneli); M. Ridderstråle (Martin); J.I. Rotter (Jerome); J.G. Sambrook (Jennifer); A.R. Sanders (Alan); C.O. Schmidt (Carsten Oliver); J. Sinisalo (Juha); J.H. Smit (Jan); H.M. Stringham (Heather); G.B. Walters (Bragi); E. Widen (Elisabeth); S.H. Wild (Sarah); G.A.H.M. Willemsen (Gonneke); L. Zagato (Laura); L. Zgaga (Lina); P. Zitting (Paavo); H. Alavere (Helene); M. Farrall (Martin); W.L. McArdle (Wendy); M. Nelis (Mari); M.J. Peters (Marjolein); S. Ripatti (Samuli); J.B.J. van Meurs (Joyce); K.K.H. Aben (Katja); J.S. Beckmann (Jacques); J.P. Beilby (John); R.N. Bergman (Richard); S.M. Bergmann (Sven); F.S. Collins (Francis); D. Cusi (Daniele); M. den Heijer (Martin); G. Eiriksdottir (Gudny); P.V. Gejman (Pablo); A.S. Hall (Alistair); A. Hamsten (Anders); H.V. Huikuri (Heikki); C. Iribarren (Carlos); M. Kähönen (Mika); J. Kaprio (Jaakko); S. Kathiresan (Sekar); L.A.L.M. Kiemeney (Bart); T. Kocher (Thomas); L.J. Launer (Lenore); T. Lehtimäki (Terho); O. Melander (Olle); T.H. Mosley (Thomas); A.W. Musk (Arthur); M.S. Nieminen (Markku); C.J. O'Donnell (Christopher); C. Ohlsson (Claes); B.A. Oostra (Ben); O. Raitakari (Olli); P.M. Ridker (Paul); J.D. Rioux (John); A. Rissanen (Aila); C. Rivolta (Carlo); H. Schunkert (Heribert); A.R. Shuldiner (Alan); D.S. Siscovick (David); M. Stumvoll (Michael); A. Tönjes (Anke); J. Tuomilehto (Jaakko); G.J. van Ommen (Gert); J. Viikari (Jorma); A.C. Heath (Andrew); N.G. Martin (Nicholas); G.W. Montgomery (Grant); M.A. Province (Mike); M.H. Kayser (Manfred); A.M. Arnold (Alice); L.D. Atwood (Larry); E.A. Boerwinkle (Eric); S.J. Chanock (Stephen); P. Deloukas (Panagiotis); C. Gieger (Christian); H. Grönberg (Henrik); A.T. Hattersley (Andrew); C. Hengstenberg (Christian); W. Hoffman (Wolfgang); G.M. Lathrop (Mark); V. Salomaa (Veikko); S. Schreiber (Stefan); M. Uda (Manuela); D. Waterworth (Dawn); A.F. Wright (Alan); T.L. Assimes (Themistocles); I.E. Barroso (Inês); A. Hofman (Albert); K.L. Mohlke (Karen); D.I. Boomsma (Dorret); M. Caulfield (Mark); L.A. Cupples (Adrienne); C.S. Fox (Caroline); V. Gudnason (Vilmundur); U. Gyllensten (Ulf); T.B. Harris (Tamara); R.B. Hayes (Richard); M.R. Järvelin; V. Mooser (Vincent); P. Munroe (Patricia); W.H. Ouwehand (Willem); B.W.J.H. Penninx (Brenda); P.P. Pramstaller (Peter Paul); T. Quertermous (Thomas); I. Rudan (Igor); N.J. Samani (Nilesh); T.D. Spector (Timothy); H. Völzke (Henry); H. Watkins (Hugh); J.F. Wilson (James); L. Groop (Leif); T. Haritunians (Talin); F.B. Hu (Frank); A. Metspalu (Andres); K.E. North (Kari); D. Schlessinger; N.J. Wareham (Nick); D.J. Hunter (David); J.R. O´Connell; D.P. Strachan (David); H.E. Wichmann (Heinz Erich); I.B. Borecki (Ingrid); C.M. van Duijn (Cornelia); E.E. Schadt (Eric); U. Thorsteinsdottir (Unnur); L. Peltonen (Leena Johanna); A.G. Uitterlinden (André); P.M. Visscher (Peter); N. Chatterjee (Nilanjan); J. Erdmann (Jeanette); R.J.F. Loos (Ruth); M. Boehnke (Michael); M.I. McCarthy (Mark); E. Ingelsson (Erik); C.M. Lindgren (Cecilia); G.R. Abecasis (Gonçalo); K. Stefansson (Kari); T.M. Frayling (Timothy); J.N. Hirschhorn (Joel); K.G. Ardlie (Kristin); M.N. Weedon (Michael)

2010-01-01

textabstractMost common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits1, but these typically explain small

Hundreds of variants clustered in genomic loci and biological pathways affect human height

NARCIS (Netherlands)

Allen, Hana Lango; Estrada, Karol; Lettre, Guillaume; Berndt, Sonja I.; Weedon, Michael N.; Rivadeneira, Fernando; Willer, Cristen J.; Jackson, Anne U.; Vedantam, Sailaja; Raychaudhuri, Soumya; Ferreira, Teresa; Wood, Andrew R.; Weyant, Robert J.; Segre, Ayellet V.; Speliotes, Elizabeth K.; Wheeler, Eleanor; Soranzo, Nicole; Park, Ju-Hyun; Yang, Jian; Gudbjartsson, Daniel; Heard-Costa, Nancy L.; Randall, Joshua C.; Qi, Lu; Smith, Albert Vernon; Maegi, Reedik; Pastinen, Tomi; Liang, Liming; Heid, Iris M.; Luan, Jian'an; Thorleifsson, Gudmar; Winkler, Thomas W.; Goddard, Michael E.; Lo, Ken Sin; Palmer, Cameron; Workalemahu, Tsegaselassie; Aulchenko, Yurii S.; Johansson, Asa; Zillikens, M. Carola; Feitosa, Mary F.; Esko, Tonu; Johnson, Toby; Ketkar, Shamika; Kraft, Peter; Mangino, Massimo; Prokopenko, Inga; Absher, Devin; Albrecht, Eva; Ernst, Florian; Zhao, Jing Hua; Chen, Constance

2010-01-01

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits(1), but these typically explain small fractions

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

Directory of Open Access Journals (Sweden)

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.

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

Radioimmunological activity of 22K variant of human growth hormone

International Nuclear Information System (INIS)

Camillo, M.A.P.; Ribela, M.T.C.P.; Rogero, J.R.

1986-01-01

From a preparation of human growth hormone its integral variant (hGH-22K) was isolated by isoelectric focusing, having a pI of 5,20 and relative mobility (Rm) of 0,621 in the polyacrylamide gel electrophoresis. Several experiments for the characterization of the isolated variant were carried out. The immunological properties was tested by radioimmunoassay (RIE), in which the activity of the isolated variant and the activity of the total preparation were compared. The dose response-curves obtained by RIE were found to be considered parallels (p [pt

Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system.

Science.gov (United States)

Takayama, Kazuo; Igai, Keisuke; Hagihara, Yasuko; Hashimoto, Rina; Hanawa, Morifumi; Sakuma, Tetsushi; Tachibana, Masashi; Sakurai, Fuminori; Yamamoto, Takashi; Mizuguchi, Hiroyuki

2017-05-19

Genome editing research of human ES/iPS cells has been accelerated by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALEN) technologies. However, the efficiency of biallelic genetic engineering in transcriptionally inactive genes is still low, unlike that in transcriptionally active genes. To enhance the biallelic homologous recombination efficiency in human ES/iPS cells, we performed screenings of accessorial genes and compounds. We found that RAD51 overexpression and valproic acid treatment enhanced biallelic-targeting efficiency in human ES/iPS cells regardless of the transcriptional activity of the targeted locus. Importantly, RAD51 overexpression and valproic acid treatment synergistically increased the biallelic homologous recombination efficiency. Our findings would facilitate genome editing study using human ES/iPS cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Intraobserver interpretation of breast ultrasonography following the BI-RADS classification

International Nuclear Information System (INIS)

Calas, M.J.G.; Almeida, R.M.V.R.; Gutfilen, B.; Pereira, W.C.A.

2010-01-01

Purpose: To use the BI-RADS ultrasound classification in an intraobserver retrospective study of the interpretation of breast images. Materials and Methods: The study used 40 breast ultrasound images recorded in orthogonal planes, obtained from patients with an indication for surgery. Eight professionals experienced in breast imaging analysis retrospectively reviewed these lesions, in three rounds of image interpretation (with a 3-6 months interval between rounds). Observers had no access to information from medical records or histopathological results, and, without their knowledge, in each new round were assigned the same images previously interpreted by them. Fleiss-modified Kappa measures were the study main concordance index. Besides the BI-RADS, a scale grouping its categories 2-3 and 4-5 was also used. The statistical analysis concerned the intraobserver agreement. Results: Kappa values ranged from 0.37 to 0.75 (original categories) and from 0.73 to 0.87 (grouped categories). Overall, out of the 8 observers, 7 presented moderate to substantial concordance (Kappa values 0.51 to 0.74). Conclusion: The BI-RADS is a reporting tool that provides a standardized terminology for US exams. In this study, moderate to substantial concordance in Kappa values was found, in agreement with other studies of the literature.

Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination.

NARCIS (Netherlands)

D.F.R. Muris; O.Y. Bezzubova (Olga); J-M. Buerstedde; K. Vreeken; A.S. Balajee; C.J. Osgood; C. Troelstra (Christine); J.H.J. Hoeijmakers (Jan); K. Ostermann; H. Schmidt (Henning); A.T. Natarajan; J.C.J. Eeken; P.H.M. Lohmann (Paul); A. Pastink (Albert)

1994-01-01

textabstractThe RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were

Comparison of the BioRad Variant and Primus Ultra2 high-pressure liquid chromatography (HPLC) instruments for the detection of variant hemoglobins.

Science.gov (United States)

Gosselin, R C; Carlin, A C; Dwyre, D M

2011-04-01

Hemoglobin variants are a result of genetic changes resulting in abnormal or dys-synchronous hemoglobin chain production (thalassemia) or the generation of hemoglobin chain variants such as hemoglobin S. Automated high-pressure liquid chromatography (HPLC) systems have become the method of choice for the evaluation of patients suspected with hemoglobinopathies. In this study, we evaluated the performance of two HPLC methods used in the detection of common hemoglobin variants: Variant and Ultra2. There were 377 samples tested, 26% (99/377) with HbS, 8.5% (32/377) with HbC, 20.7% (78/377) with other hemoglobin variant or thalassemia, and 2.9% with increased hemoglobin A(1) c. The interpretations of each chromatograph were compared. There were no differences noted for hemoglobins A(0), S, or C. There were significant differences between HPLC methods for hemoglobins F, A(2), and A(1) c. However, there was good concordance between normal and abnormal interpretations (97.9% and 96.2%, respectively). Both Variant and Ultra2 HPLC methods were able to detect most common hemoglobin variants. There was better discrimination for fast hemoglobins, between hemoglobins E and A(2), and between hemoglobins S and F using the Ultra2 HPLC method. © 2010 Blackwell Publishing Ltd.

Sensitization of Tumor to 212Pb Radioimmunotherapy by Gemcitabine Involves Initial Abrogation of G2 Arrest and Blocked DNA Damage Repair by Interference With Rad51

International Nuclear Information System (INIS)

Yong, Kwon Joong; Milenic, Diane E.; Baidoo, Kwamena E.; Brechbiel, Martin W.

2013-01-01

Purpose: To elucidate the mechanism of the therapeutic efficacy of targeted α-particle radiation therapy using 212 Pb-TCMC-trastuzumab together with gemcitabine for treatment of disseminated peritoneal cancers. Methods and Materials: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pretreated with gemcitabine, followed by 212 Pb-TCMC-trastuzumab and compared with controls. Results: Treatment with 212 Pb-TCMC-trastuzumab increased the apoptotic rate in the S-phase-arrested tumors induced by gemcitabine at earlier time points (6 to 24 hours). 212 Pb-TCMC-trastuzumab after gemcitabine pretreatment abrogated G2/M arrest at the same time points, which may be associated with the inhibition of Chk1 phosphorylation and, in turn, cell cycle perturbation, resulting in apoptosis. 212 Pb-TCMC-trastuzumab treatment after gemcitabine pretreatment caused depression of DNA synthesis, DNA double-strand breaks, accumulation of unrepaired DNA, and down-regulation of Rad51 protein, indicating that DNA damage repair was blocked. In addition, modification in the chromatin structure of p21 may be associated with transcriptionally repressed chromatin states, indicating that the open structure was delayed at earlier time points. Conclusion: These findings suggest that the cell-killing efficacy of 212 Pb-TCMC-trastuzumab after gemcitabine pretreatment may be associated with abrogation of the G2/M checkpoint, inhibition of DNA damage repair, and chromatin remodeling

Expression of the human growth hormone variant gene in cultured fibroblasts and transgenic mice

International Nuclear Information System (INIS)

Selden, R.F.; Wagner, T.E.; Blethen, S.; Yun, J.S.; Rowe, M.E.; Goodman, H.M.

1988-01-01

The nucleotide sequence of the human growth hormone variant gene, one of the five members of the growth hormone gene family, predicts that it encodes a growth hormone-like protein. As a first step in determining whether this gene is functional in humans, the authors have expressed a mouse methallothionein I/human growth hormone variant fusion gene in mouse L cells and in transgenic mice. The growth hormone variant protein expressed in transiently transfected L cells is distinct from growth hormone itself with respect to reactivity with anti-growth hormone monoclonal antibodies, behavior during column chromatography, and isoelectric point. Transgenic mice expressing the growth hormone variant protein are 1.4- to 1.9-fold larger than nontransgenic controls, suggesting that the protein has growth-promoting properties

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

Locus Reference Genomic sequences: An improved basis for describing human DNA variants

KAUST Repository

Dalgleish, Raymond; Flicek, Paul; Cunningham, Fiona; Astashyn, Alex; Tully, Raymond E; Proctor, Glenn; Chen, Yuan; McLaren, William M; Larsson, Pontus; Vaughan, Brendan W; Bé roud, Christophe; Dobson, Glen; Lehvä slaiho, Heikki; Taschner, Peter EM; den Dunnen, Johan T; Devereau, Andrew; Birney, Ewan; Brookes, Anthony J; Maglott, Donna R

2010-01-01

As our knowledge of the complexity of gene architecture grows, and we increase our understanding of the subtleties of gene expression, the process of accurately describing disease-causing gene variants has become increasingly problematic. In part, this is due to current reference DNA sequence formats that do not fully meet present needs. Here we present the Locus Reference Genomic (LRG) sequence format, which has been designed for the specifi c purpose of gene variant reporting. The format builds on the successful National Center for Biotechnology Information (NCBI) RefSeqGene project and provides a single-fi le record containing a uniquely stable reference DNA sequence along with all relevant transcript and protein sequences essential to the description of gene variants. In principle, LRGs can be created for any organism, not just human. In addition, we recognize the need to respect legacy numbering systems for exons and amino acids and the LRG format takes account of these. We hope that widespread adoption of LRGs - which will be created and maintained by the NCBI and the European Bioinformatics Institute (EBI) - along with consistent use of the Human Genome Variation Society (HGVS)- approved variant nomenclature will reduce errors in the reporting of variants in the literature and improve communication about variants aff ecting human health. Further information can be found on the LRG web site (http://www.lrg-sequence.org). 2010 Dalgleish et al.; licensee BioMed Central Ltd.

Locus Reference Genomic sequences: An improved basis for describing human DNA variants

KAUST Repository

Dalgleish, Raymond

2010-04-15

As our knowledge of the complexity of gene architecture grows, and we increase our understanding of the subtleties of gene expression, the process of accurately describing disease-causing gene variants has become increasingly problematic. In part, this is due to current reference DNA sequence formats that do not fully meet present needs. Here we present the Locus Reference Genomic (LRG) sequence format, which has been designed for the specifi c purpose of gene variant reporting. The format builds on the successful National Center for Biotechnology Information (NCBI) RefSeqGene project and provides a single-fi le record containing a uniquely stable reference DNA sequence along with all relevant transcript and protein sequences essential to the description of gene variants. In principle, LRGs can be created for any organism, not just human. In addition, we recognize the need to respect legacy numbering systems for exons and amino acids and the LRG format takes account of these. We hope that widespread adoption of LRGs - which will be created and maintained by the NCBI and the European Bioinformatics Institute (EBI) - along with consistent use of the Human Genome Variation Society (HGVS)- approved variant nomenclature will reduce errors in the reporting of variants in the literature and improve communication about variants aff ecting human health. Further information can be found on the LRG web site (http://www.lrg-sequence.org). 2010 Dalgleish et al.; licensee BioMed Central Ltd.

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.

Rare human papillomavirus 16 E6 variants reveal significant oncogenic potential

Directory of Open Access Journals (Sweden)

Tommasino Massimo

2011-06-01

Full Text Available Abstract The aim of this study was to determine whether low prevalence human papillomavirus (HPV 16 E6 variants differ from high prevalence types in their functional abilities. We evaluated functions relevant to carcinogenesis for the rarely-detected European variants R8Q, R10G and R48W as compared to the commonly detected L83V. Human immortalized keratinocytes (NIKS stably transduced with the E6 variants were used in most functional assays. Low and high prevalence E6 variants displayed similar abilities in abrogation of growth arrest and inhibition of p53 elevation induced by actinomycin D. Differences were detected in the abilities to dysregulate stratification and differentiation of NIKS in organotypic raft cultures, modulate detachment induced apoptosis (anoikis and hyperactivate Wnt signaling. No distinctive phenotype could be assigned to include all rare variants. Like L83V, raft cultures derived from variants R10G and R48W similarly induced hyperplasia and aberrantly expressed keratin 5 in the suprabasal compartment with significantly lower expression of keratin 10. Unlike L83V, both variants, and particularly R48W, induced increased levels of anoikis upon suspension in semisolid medium. R8Q induced a unique phenotype characterized by thin organotypic raft cultures, low expression of keratin 10, and high expression of keratins 5 and 14 throughout all raft layers. Interestingly, in a reporter based assay R8Q exhibited a higher ability to augment TCF/β-catenin transcription. The data suggests that differences in E6 variant prevalence in cervical carcinoma may not be related to the carcinogenic potential of the E6 protein.

Common genetic variants influence human subcortical brain structures

Science.gov (United States)

Hibar, Derrek P.; Stein, Jason L.; Renteria, Miguel E.; Arias-Vasquez, Alejandro; Desrivières, Sylvane; Jahanshad, Neda; Toro, Roberto; Wittfeld, Katharina; Abramovic, Lucija; Andersson, Micael; Aribisala, Benjamin S.; Armstrong, Nicola J.; Bernard, Manon; Bohlken, Marc M.; Boks, Marco P.; Bralten, Janita; Brown, Andrew A.; Chakravarty, M. Mallar; Chen, Qiang; Ching, Christopher R. K.; Cuellar-Partida, Gabriel; den Braber, Anouk; Giddaluru, Sudheer; Goldman, Aaron L.; Grimm, Oliver; Guadalupe, Tulio; Hass, Johanna; Woldehawariat, Girma; Holmes, Avram J.; Hoogman, Martine; Janowitz, Deborah; Jia, Tianye; Kim, Sungeun; Klein, Marieke; Kraemer, Bernd; Lee, Phil H.; Olde Loohuis, Loes M.; Luciano, Michelle; Macare, Christine; Mather, Karen A.; Mattheisen, Manuel; Milaneschi, Yuri; Nho, Kwangsik; Papmeyer, Martina; Ramasamy, Adaikalavan; Risacher, Shannon L.; Roiz-Santiañez, Roberto; Rose, Emma J.; Salami, Alireza; Sämann, Philipp G.; Schmaal, Lianne; Schork, Andrew J.; Shin, Jean; Strike, Lachlan T.; Teumer, Alexander; van Donkelaar, Marjolein M. J.; van Eijk, Kristel R.; Walters, Raymond K.; Westlye, Lars T.; Whelan, Christopher D.; Winkler, Anderson M.; Zwiers, Marcel P.; Alhusaini, Saud; Athanasiu, Lavinia; Ehrlich, Stefan; Hakobjan, Marina M. H.; Hartberg, Cecilie B.; Haukvik, Unn K.; Heister, Angelien J. G. A. M.; Hoehn, David; Kasperaviciute, Dalia; Liewald, David C. M.; Lopez, Lorna M.; Makkinje, Remco R. R.; Matarin, Mar; Naber, Marlies A. M.; McKay, D. Reese; Needham, Margaret; Nugent, Allison C.; Pütz, Benno; Royle, Natalie A.; Shen, Li; Sprooten, Emma; Trabzuni, Daniah; van der Marel, Saskia S. L.; van Hulzen, Kimm J. E.; Walton, Esther; Wolf, Christiane; Almasy, Laura; Ames, David; Arepalli, Sampath; Assareh, Amelia A.; Bastin, Mark E.; Brodaty, Henry; Bulayeva, Kazima B.; Carless, Melanie A.; Cichon, Sven; Corvin, Aiden; Curran, Joanne E.; Czisch, Michael; de Zubicaray, Greig I.; Dillman, Allissa; Duggirala, Ravi; Dyer, Thomas D.; Erk, Susanne; Fedko, Iryna O.; Ferrucci, Luigi; Foroud, Tatiana M.; Fox, Peter T.; Fukunaga, Masaki; Gibbs, J. Raphael; Göring, Harald H. H.; Green, Robert C.; Guelfi, Sebastian; Hansell, Narelle K.; Hartman, Catharina A.; Hegenscheid, Katrin; Heinz, Andreas; Hernandez, Dena G.; Heslenfeld, Dirk J.; Hoekstra, Pieter J.; Holsboer, Florian; Homuth, Georg; Hottenga, Jouke-Jan; Ikeda, Masashi; Jack, Clifford R.; Jenkinson, Mark; Johnson, Robert; Kanai, Ryota; Keil, Maria; Kent, Jack W.; Kochunov, Peter; Kwok, John B.; Lawrie, Stephen M.; Liu, Xinmin; Longo, Dan L.; McMahon, Katie L.; Meisenzahl, Eva; Melle, Ingrid; Mohnke, Sebastian; Montgomery, Grant W.; Mostert, Jeanette C.; Mühleisen, Thomas W.; Nalls, Michael A.; Nichols, Thomas E.; Nilsson, Lars G.; Nöthen, Markus M.; Ohi, Kazutaka; Olvera, Rene L.; Perez-Iglesias, Rocio; Pike, G. Bruce; Potkin, Steven G.; Reinvang, Ivar; Reppermund, Simone; Rietschel, Marcella; Romanczuk-Seiferth, Nina; Rosen, Glenn D.; Rujescu, Dan; Schnell, Knut; Schofield, Peter R.; Smith, Colin; Steen, Vidar M.; Sussmann, Jessika E.; Thalamuthu, Anbupalam; Toga, Arthur W.; Traynor, Bryan J.; Troncoso, Juan; Turner, Jessica A.; Valdés Hernández, Maria C.; van ’t Ent, Dennis; van der Brug, Marcel; van der Wee, Nic J. A.; van Tol, Marie-Jose; Veltman, Dick J.; Wassink, Thomas H.; Westman, Eric; Zielke, Ronald H.; Zonderman, Alan B.; Ashbrook, David G.; Hager, Reinmar; Lu, Lu; McMahon, Francis J.; Morris, Derek W.; Williams, Robert W.; Brunner, Han G.; Buckner, Randy L.; Buitelaar, Jan K.; Cahn, Wiepke; Calhoun, Vince D.; Cavalleri, Gianpiero L.; Crespo-Facorro, Benedicto; Dale, Anders M.; Davies, Gareth E.; Delanty, Norman; Depondt, Chantal; Djurovic, Srdjan; Drevets, Wayne C.; Espeseth, Thomas; Gollub, Randy L.; Ho, Beng-Choon; Hoffmann, Wolfgang; Hosten, Norbert; Kahn, René S.; Le Hellard, Stephanie; Meyer-Lindenberg, Andreas; Müller-Myhsok, Bertram; Nauck, Matthias; Nyberg, Lars; Pandolfo, Massimo; Penninx, Brenda W. J. H.; Roffman, Joshua L.; Sisodiya, Sanjay M.; Smoller, Jordan W.; van Bokhoven, Hans; van Haren, Neeltje E. M.; Völzke, Henry; Walter, Henrik; Weiner, Michael W.; Wen, Wei; White, Tonya; Agartz, Ingrid; Andreassen, Ole A.; Blangero, John; Boomsma, Dorret I.; Brouwer, Rachel M.; Cannon, Dara M.; Cookson, Mark R.; de Geus, Eco J. C.; Deary, Ian J.; Donohoe, Gary; Fernández, Guillén; Fisher, Simon E.; Francks, Clyde; Glahn, David C.; Grabe, Hans J.; Gruber, Oliver; Hardy, John; Hashimoto, Ryota; Hulshoff Pol, Hilleke E.; Jönsson, Erik G.; Kloszewska, Iwona; Lovestone, Simon; Mattay, Venkata S.; Mecocci, Patrizia; McDonald, Colm; McIntosh, Andrew M.; Ophoff, Roel A.; Paus, Tomas; Pausova, Zdenka; Ryten, Mina; Sachdev, Perminder S.; Saykin, Andrew J.; Simmons, Andy; Singleton, Andrew; Soininen, Hilkka; Wardlaw, Joanna M.; Weale, Michael E.; Weinberger, Daniel R.; Adams, Hieab H. H.; Launer, Lenore J.; Seiler, Stephan; Schmidt, Reinhold; Chauhan, Ganesh; Satizabal, Claudia L.; Becker, James T.; Yanek, Lisa; van der Lee, Sven J.; Ebling, Maritza; Fischl, Bruce; Longstreth, W. T.; Greve, Douglas; Schmidt, Helena; Nyquist, Paul; Vinke, Louis N.; van Duijn, Cornelia M.; Xue, Luting; Mazoyer, Bernard; Bis, Joshua C.; Gudnason, Vilmundur; Seshadri, Sudha; Ikram, M. Arfan; Martin, Nicholas G.; Wright, Margaret J.; Schumann, Gunter; Franke, Barbara; Thompson, Paul M.; Medland, Sarah E.

2015-01-01

The highly complex structure of the human brain is strongly shaped by genetic influences1. Subcortical brain regions form circuits with cortical areas to coordinate movement2, learning, memory3 and motivation4, and altered circuits can lead to abnormal behaviour and disease2. To investigate how common genetic variants affect the structure of these brain regions, here we conduct genome-wide association studies of the volumes of seven subcortical regions and the intracranial volume derived from magnetic resonance images of 30,717 individuals from 50 cohorts. We identify five novel genetic variants influencing the volumes of the putamen and caudate nucleus. We also find stronger evidence for three loci with previously established influences on hippocampal volume5 and intracranial volume6. These variants show specific volumetric effects on brain structures rather than global effects across structures. The strongest effects were found for the putamen, where a novel intergenic locus with replicable influence on volume (rs945270; P = 1.08 × 10−33; 0.52% variance explained) showed evidence of altering the expression of the KTN1 gene in both brain and blood tissue. Variants influencing putamen volume clustered near developmental genes that regulate apoptosis, axon guidance and vesicle transport. Identification of these genetic variants provides insight into the causes of variability inhuman brain development, and may help to determine mechanisms of neuropsychiatric dysfunction. PMID:25607358

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)

Rad9 contribution to radiosensitivity and the G2 checkpoint in a DT40 cell line

Energy Technology Data Exchange (ETDEWEB)

Kumano, Tomoyasu [Kanazawa Univ. (Japan). Graduate School of Medical Science

2002-12-01

In fission yeast, the rad9 (radiation sensitive) gene was cloned from a mutant that is sensitive to ionizing radiation, ultraviolet and hydroxyurea. This gene has also been shown to be required for a DNA damage checkpoint. Orthologues of the rad9 gene have recently been identified in higher eukaryote cells including human. Here we generated Rad9 knockout (Rad9-/-) cells from the chicken B lymphocyte line DT40 to examine the role of Rad9 in higher eukaryotes. First we isolated a part of the chicken Rad9 gene which was 54% identical with human Rad9 at the amino acid sequence level. Next we isolated genomic clones, determined exons and introns, and constructed targeting vectors designed to disrupt exon 1-3 of the chicken Rad9 gene by replacement with a drug-resistant gene. Successful targeted integration was verified by Southern blot analysis and the disruption of the Rad9 gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR). To analyze the radiosensitivity of these Rad9-/- cells, we monitored the clonogenic survival after various degrees of X-ray irradiation. Rad9-/- cells were more sensitive to X-rays than wild type cells at all dosages. However, these cells were less sensitive than ATM knockout (ATM-/-) cells that are known to be X-ray sensitive and that showed a defective checkpoint control. In contrast, Rad9-/- cells were markedly more sensitive to ultraviolet and hydroxyruea. In addition, we assessed the G2 checkpoint by measurement of the mitotic index that is the fraction of the accumulating number of cells in mitosis at various times after X-ray irradiation. While the number of mitotic wild type cells did not increase until 2 hrs after X-ray irradiation, the number of mitotic Rad9-/- cells showed an increase similar to that of ATM-/- cells. These results suggest that just as in fission yeast, in higher eukaryotes Rad9 also contributes to X-ray, ultraviolet and hydroxyurea sensitivity, and plays an important role in the G2 checkpoint

Rad9 contribution to radiosensitivity and the G2 checkpoint in a DT40 cell line

International Nuclear Information System (INIS)

Kumano, Tomoyasu

2002-01-01

In fission yeast, the rad9 (radiation sensitive) gene was cloned from a mutant that is sensitive to ionizing radiation, ultraviolet and hydroxyurea. This gene has also been shown to be required for a DNA damage checkpoint. Orthologues of the rad9 gene have recently been identified in higher eukaryote cells including human. Here we generated Rad9 knockout (Rad9-/-) cells from the chicken B lymphocyte line DT40 to examine the role of Rad9 in higher eukaryotes. First we isolated a part of the chicken Rad9 gene which was 54% identical with human Rad9 at the amino acid sequence level. Next we isolated genomic clones, determined exons and introns, and constructed targeting vectors designed to disrupt exon 1-3 of the chicken Rad9 gene by replacement with a drug-resistant gene. Successful targeted integration was verified by Southern blot analysis and the disruption of the Rad9 gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR). To analyze the radiosensitivity of these Rad9-/- cells, we monitored the clonogenic survival after various degrees of X-ray irradiation. Rad9-/- cells were more sensitive to X-rays than wild type cells at all dosages. However, these cells were less sensitive than ATM knockout (ATM-/-) cells that are known to be X-ray sensitive and that showed a defective checkpoint control. In contrast, Rad9-/- cells were markedly more sensitive to ultraviolet and hydroxyruea. In addition, we assessed the G2 checkpoint by measurement of the mitotic index that is the fraction of the accumulating number of cells in mitosis at various times after X-ray irradiation. While the number of mitotic wild type cells did not increase until 2 hrs after X-ray irradiation, the number of mitotic Rad9-/- cells showed an increase similar to that of ATM-/- cells. These results suggest that just as in fission yeast, in higher eukaryotes Rad9 also contributes to X-ray, ultraviolet and hydroxyurea sensitivity, and plays an important role in the G2 checkpoint

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.

Human papillomavirus variants among Inuit women in northern Quebec, Canada.

Science.gov (United States)

Gauthier, Barbara; Coutlée, Francois; Franco, Eduardo L; Brassard, Paul

2015-01-01

Inuit communities in northern Quebec have high rates of human papillomavirus (HPV) infection, cervical cancer and cervical cancer-related mortality as compared to the Canadian population. HPV types can be further classified as intratypic variants based on the extent of homology in their nucleotide sequences. There is limited information on the distribution of intratypic variants in circumpolar areas. Our goal was to describe the HPV intratypic variants and associated baseline characteristics. We collected cervical cell samples in 2002-2006 from 676 Inuit women between the ages of 15 and 69 years in Nunavik. DNA isolates from high-risk HPVs were sequenced to determine the intratypic variant. There were 149 women that were positive for HPVs 16, 18, 31, 33, 35, 45, 52, 56 or 58 during follow-up. There were 5 different HPV16 variants, all of European lineage, among the 57 women positive for this type. There were 8 different variants of HPV18 present and all were of European lineage (n=21). The majority of samples of HPV31 (n=52) were of lineage B. The number of isolates and diversity of the other HPV types was low. Age was the only covariate associated with HPV16 variant category. These frequencies are similar to what was seen in another circumpolar region of Canada, although there appears to be less diversity as only European variants were detected. This study shows that most variants were clustered in one lineage for each HPV type.

Sensitization of Tumor to {sup 212}Pb Radioimmunotherapy by Gemcitabine Involves Initial Abrogation of G2 Arrest and Blocked DNA Damage Repair by Interference With Rad51

Energy Technology Data Exchange (ETDEWEB)

Yong, Kwon Joong; Milenic, Diane E.; Baidoo, Kwamena E. [Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States); Brechbiel, Martin W., E-mail: martinwb@mail.nih.gov [Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States)

2013-03-15

Purpose: To elucidate the mechanism of the therapeutic efficacy of targeted α-particle radiation therapy using {sup 212}Pb-TCMC-trastuzumab together with gemcitabine for treatment of disseminated peritoneal cancers. Methods and Materials: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pretreated with gemcitabine, followed by {sup 212}Pb-TCMC-trastuzumab and compared with controls. Results: Treatment with {sup 212}Pb-TCMC-trastuzumab increased the apoptotic rate in the S-phase-arrested tumors induced by gemcitabine at earlier time points (6 to 24 hours). {sup 212}Pb-TCMC-trastuzumab after gemcitabine pretreatment abrogated G2/M arrest at the same time points, which may be associated with the inhibition of Chk1 phosphorylation and, in turn, cell cycle perturbation, resulting in apoptosis. {sup 212}Pb-TCMC-trastuzumab treatment after gemcitabine pretreatment caused depression of DNA synthesis, DNA double-strand breaks, accumulation of unrepaired DNA, and down-regulation of Rad51 protein, indicating that DNA damage repair was blocked. In addition, modification in the chromatin structure of p21 may be associated with transcriptionally repressed chromatin states, indicating that the open structure was delayed at earlier time points. Conclusion: These findings suggest that the cell-killing efficacy of {sup 212}Pb-TCMC-trastuzumab after gemcitabine pretreatment may be associated with abrogation of the G2/M checkpoint, inhibition of DNA damage repair, and chromatin remodeling.

RAD50 germline mutations are associated with poor survival in BRCA1/2-negative breast cancer patients.

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Fan, Cong; Zhang, Juan; Ouyang, Tao; Li, Jinfeng; Wang, Tianfeng; Fan, Zhaoqing; Fan, Tie; Lin, Benyao; Xie, Yuntao

2018-05-04

RAD50 is a highly conserved DNA double-strand break (DSB) repair gene. However, the associations between RAD50 germline mutations and the survival and risk of breast cancer have not been fully elucidated. Here, we aimed to investigate the clinical impact of RAD50 germline mutations in a large cohort of unselected breast cancer patients. In this study, RAD50 germline mutations were determined using next-generation sequencing in 7657 consecutive unselected breast cancer patients without BRCA1/2 mutations. We also screened for RAD50 recurrent mutations (L719fs, K994fs, and H1269fs) in 5000 healthy controls using Sanger sequencing. We found that 26 out of 7657 (0.34%) patients had RAD50 pathogenic mutations, and 16 patients carried one of the three recurrent mutations (L719fs, n=6 cases; K994fs, n=5 cases; and H1269fs, n=5 cases); the recurrent mutation rate was 0.21%. The frequency of the three recurrent mutations in the 5000 healthy controls was 0.18% (9/5000). These mutations did not confer an increased risk of breast cancer in the studied patients [odds ratios (OR), 1.16; 95% confidence interval (CI), 0.51-2.63; P = 0.72]. Nevertheless, multivariate analysis revealed that RAD50 pathogenic mutations were an independent unfavourable predictor of recurrence-free survival (RFS) [adjusted hazard ratio (HR) 2.66; 95% CI, 1.18-5.98; P=0.018] and disease-specific survival (DSS) (adjusted HR 4.36; 95% CI, 1.58-12.03; P=0.004) in the entire study cohort. Our study suggested that RAD50 germline mutations are not associated with an increased risk of breast cancer, but patients with RAD50 germline mutations have unfavourable survival compared with patients without these mutations. This article is protected by copyright. All rights reserved. © 2018 UICC.

Delineation of concentration ranges and longitudinal changes of human plasma protein variants.

Directory of Open Access Journals (Sweden)

Olgica Trenchevska

Full Text Available Human protein diversity arises as a result of alternative splicing, single nucleotide polymorphisms (SNPs and posttranslational modifications. Because of these processes, each protein can exists as multiple variants in vivo. Tailored strategies are needed to study these protein variants and understand their role in health and disease. In this work we utilized quantitative mass spectrometric immunoassays to determine the protein variants concentration of beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin, in a population of 500 healthy individuals. Additionally, we determined the longitudinal concentration changes for the protein variants from four individuals over a 6 month period. Along with the native forms of the four proteins, 13 posttranslationally modified variants and 7 SNP-derived variants were detected and their concentration determined. Correlations of the variants concentration with geographical origin, gender, and age of the individuals were also examined. This work represents an important step toward building a catalog of protein variants concentrations and examining their longitudinal changes.

Neanderthal and Denisova tooth protein variants in present-day humans.

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Clément Zanolli

Full Text Available Environment parameters, diet and genetic factors interact to shape tooth morphostructure. In the human lineage, archaic and modern hominins show differences in dental traits, including enamel thickness, but variability also exists among living populations. Several polymorphisms, in particular in the non-collagenous extracellular matrix proteins of the tooth hard tissues, like enamelin, are involved in dental structure variation and defects and may be associated with dental disorders or susceptibility to caries. To gain insights into the relationships between tooth protein polymorphisms and dental structural morphology and defects, we searched for non-synonymous polymorphisms in tooth proteins from Neanderthal and Denisova hominins. The objective was to identify archaic-specific missense variants that may explain the dental morphostructural variability between extinct and modern humans, and to explore their putative impact on present-day dental phenotypes. Thirteen non-collagenous extracellular matrix proteins specific to hard dental tissues have been selected, searched in the publicly available sequence databases of Neanderthal and Denisova individuals and compared with modern human genome data. A total of 16 non-synonymous polymorphisms were identified in 6 proteins (ameloblastin, amelotin, cementum protein 1, dentin matrix acidic phosphoprotein 1, enamelin and matrix Gla protein. Most of them are encoded by dentin and enamel genes located on chromosome 4, previously reported to show signs of archaic introgression within Africa. Among the variants shared with modern humans, two are ancestral (common with apes and one is the derived enamelin major variant, T648I (rs7671281, associated with a thinner enamel and specific to the Homo lineage. All the others are specific to Neanderthals and Denisova, and are found at a very low frequency in modern Africans or East and South Asians, suggesting that they may be related to particular dental traits or

Human GRIN2B variants in neurodevelopmental disorders

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Chun Hu

2016-10-01

Full Text Available The development of whole exome/genome sequencing technologies has given rise to an unprecedented volume of data linking patient genomic variability to brain disorder phenotypes. A surprising number of variants have been found in the N-methyl-d-aspartate receptor (NMDAR gene family, with the GRIN2B gene encoding the GluN2B subunit being implicated in many cases of neurodevelopmental disorders, which are psychiatric conditions originating in childhood and include language, motor, and learning disorders, autism spectrum disorder (ASD, attention deficit hyperactivity disorder (ADHD, developmental delay, epilepsy, and schizophrenia. The GRIN2B gene plays a crucial role in normal neuronal development and is important for learning and memory. Mutations in human GRIN2B were distributed throughout the entire gene in a number of patients with various neuropsychiatric and developmental disorders. Studies that provide functional analysis of variants are still lacking, however current analysis of de novo variants that segregate with disease cases such as intellectual disability, developmental delay, ASD or epileptic encephalopathies reveal altered NMDAR function. Here, we summarize the current reports of disease-associated variants in GRIN2B from patients with multiple neurodevelopmental disorders, and discuss implications, highlighting the importance of functional analysis and precision medicine therapies.

Novel Parvovirus and Related Variant in Human Plasma

Science.gov (United States)

Fryer, Jacqueline F.; Kapoor, Amit; Minor, Philip D.; Delwart, Eric

2006-01-01

We report a novel parvovirus (PARV4) and related variants in pooled human plasma used in the manufacture of plasma-derived medical products. Viral DNA was detected by using highly selective polymerase chain reaction assays; 5% of pools tested positive, and amounts of DNA ranged from 106 copies/mL plasma. PMID:16494735

Population structure of Atlantic Mackerel inferred from RAD-seq derived SNP markers: effects of sequence clustering parameters and hierarchical SNP selection

KAUST Repository

Rodríguez-Ezpeleta, Naiara

2016-03-03

Restriction-site associated DNA sequencing (RAD-seq) and related methods are revolutionizing the field of population genomics in non-model organisms as they allow generating an unprecedented number of single nucleotide polymorphisms (SNPs) even when no genomic information is available. Yet, RAD-seq data analyses rely on assumptions on nature and number of nucleotide variants present in a single locus, the choice of which may lead to an under- or overestimated number of SNPs and/or to incorrectly called genotypes. Using the Atlantic mackerel (Scomber scombrus L.) and a close relative, the Atlantic chub mackerel (Scomber colias), as case study, here we explore the sensitivity of population structure inferences to two crucial aspects in RAD-seq data analysis: the maximum number of mismatches allowed to merge reads into a locus and the relatedness of the individuals used for genotype calling and SNP selection. Our study resolves the population structure of the Atlantic mackerel, but, most importantly, provides insights into the effects of alternative RAD-seq data analysis strategies on population structure inferences that are directly applicable to other species.

Structural comparisons of two allelic variants of human placental alkaline phosphatase.

Science.gov (United States)

Millán, J L; Stigbrand, T; Jörnvall, H

1985-01-01

A simple immunosorbent purification scheme based on monoclonal antibodies has been devised for human placental alkaline phosphatase. The two most common allelic variants, S and F, have similar amino acid compositions with identical N-terminal amino acid sequences through the first 13 residues. Both variants have identical lectin binding properties towards concanavalin A, lentil-lectin, wheat germ agglutinin, phytohemagglutinin and soybean agglutinin, and identical carbohydrate contents as revealed by methylation analysis. CNBr fragments of the variants demonstrate identical high performance liquid chromatography patterns. The carbohydrate containing fragment is different from the 32P-labeled active site fragment and the N-terminal fragment.

An Alternate Splicing Variant of the Human Telomerase Catalytic Subunit Inhibits Telomerase Activity

Directory of Open Access Journals (Sweden)

Xiaoming Yi

2000-09-01

Full Text Available Telomerase, a cellular reverse transcriptase, adds telomeric repeats to chromosome ends. In normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to proliferative senescence. Introduction of the telomerase (hTERT cDNA is sufficient to produce telomerase activity and immortalize normal human cells, suggesting that the repression of telomerase activity is transcriptional. The telomerase transcript has been shown to have at least six alternate splicing sites (four insertion sites and two deletion sites, and variants containing both or either of the deletion sites are present during development and in a panel of cancer cell lines we surveyed. One deletion (β site and all four insertions cause premature translation terminations, whereas the other deletion (α site is 36 by and lies within reverse transcriptase (RT motif A, suggesting that this deletion variant may be a candidate as a dominant-negative inhibitor of telomerase. We have cloned three alternately spliced hTERT variants that contain the α,β or both α and,β deletion sites. These alternate splicing variants along with empty vector and wild-type hTERT were introduced into normal human fibroblasts and several telomerase-positive immortal and tumor cell lines. Expression of the α site deletion variant (hTERT α− construct was confirmed by Western blotting. We found that none of the three alternate splicing variants reconstitutes telomerase activity in fibroblasts. However, hTERT α− inhibits telomerase activities in telomerase-positive cells, causes telomere shortening and eventually cell death. This alternately spliced dominant-negative variant may be important in understanding telomerase regulation during development, differentiation and in cancer progression.

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.

Functional analysis of four naturally occurring variants of human constitutive androstane receptor.

Science.gov (United States)

Ikeda, Shinobu; Kurose, Kouichi; Jinno, Hideto; Sai, Kimie; Ozawa, Shogo; Hasegawa, Ryuichi; Komamura, Kazuo; Kotake, Takeshi; Morishita, Hideki; Kamakura, Shiro; Kitakaze, Masafumi; Tomoike, Hitonobu; Tamura, Tomohide; Yamamoto, Noboru; Kunitoh, Hideo; Yamada, Yasuhide; Ohe, Yuichiro; Shimada, Yasuhiro; Shirao, Kuniaki; Kubota, Kaoru; Minami, Hironobu; Ohtsu, Atsushi; Yoshida, Teruhiko; Saijo, Nagahiro; Saito, Yoshiro; Sawada, Jun-ichi

2005-01-01

The human constitutive androstane receptor (CAR, NR1I3) is a member of the orphan nuclear receptor superfamily that plays an important role in the control of drug metabolism and disposition. In this study, we sequenced all the coding exons of the NR1I3 gene for 334 Japanese subjects. We identified three novel single nucleotide polymorphisms (SNPs) that induce non-synonymous alterations of amino acids (His246Arg, Leu308Pro, and Asn323Ser) residing in the ligand-binding domain of CAR, in addition to the Val133Gly variant, which was another CAR variant identified in our previous study. We performed functional analysis of these four naturally occurring CAR variants in COS-7 cells using a CYP3A4 promoter/enhancer reporter gene that includes the CAR responsive elements. The His246Arg variant caused marked reductions in both transactivation of the reporter gene and in the response to 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), which is a human CAR-specific agonist. The transactivation ability of the Leu308Pro variant was also significantly decreased, but its responsiveness to CITCO was not abrogated. The transactivation ability and CITCO response of the Val133Gly and Asn323Ser variants did not change as compared to the wild-type CAR. These data suggest that the His246Arg and Leu308Pro variants, especially His246Arg, may influence the expression of drug-metabolizing enzymes and transporters that are transactivated by CAR.

Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21p family conserved from fision yeast to humans.

NARCIS (Netherlands)

S. Parisi; M.J. McKay (Michael); M. Molnar; M.A. Thompson (Anne); P.J. van der Spek (Peter); E. van Drunen-Schoenmaker; R. Kanaar (Roland); E. Lehmann; J.H.J. Hoeijmakers (Jan); J. Kohli

1999-01-01

textabstractOur work and that of others defined mitosis-specific (Rad21 subfamily) and meiosis-specific (Rec8 subfamily) proteins involved in sister chromatid cohesion in several eukaryotes, including humans. Mutation of the fission yeast Schizosaccharomyces pombe rec8 gene was previously shown to

Double-digest RAD sequencing using Ion Proton semiconductor platform (ddRADseq-ion) with nonmodel organisms.

Science.gov (United States)

Recknagel, Hans; Jacobs, Arne; Herzyk, Pawel; Elmer, Kathryn R

2015-11-01

Research in evolutionary biology involving nonmodel organisms is rapidly shifting from using traditional molecular markers such as mtDNA and microsatellites to higher throughput SNP genotyping methodologies to address questions in population genetics, phylogenetics and genetic mapping. Restriction site associated DNA sequencing (RAD sequencing or RADseq) has become an established method for SNP genotyping on Illumina sequencing platforms. Here, we developed a protocol and adapters for double-digest RAD sequencing for Ion Torrent (Life Technologies; Ion Proton, Ion PGM) semiconductor sequencing. We sequenced thirteen genomic libraries of three different nonmodel vertebrate species on Ion Proton with PI chips: Arctic charr Salvelinus alpinus, European whitefish Coregonus lavaretus and common lizard Zootoca vivipara. This resulted in ~962 million single-end reads overall and a mean of ~74 million reads per library. We filtered the genomic data using Stacks, a bioinformatic tool to process RAD sequencing data. On average, we obtained ~11,000 polymorphic loci per library of 6-30 individuals. We validate our new method by technical and biological replication, by reconstructing phylogenetic relationships, and using a hybrid genetic cross to track genomic variants. Finally, we discuss the differences between using the different sequencing platforms in the context of RAD sequencing, assessing possible advantages and disadvantages. We show that our protocol can be used for Ion semiconductor sequencing platforms for the rapid and cost-effective generation of variable and reproducible genetic markers. © 2015 John Wiley & Sons Ltd.

T3 Regulates a Human Macrophage-Derived TSH-β Splice Variant: Implications for Human Bone Biology.

Science.gov (United States)

Baliram, R; Latif, R; Morshed, S A; Zaidi, M; Davies, T F

2016-09-01

TSH and thyroid hormones (T3 and T4) are intimately involved in bone biology. We have previously reported the presence of a murine TSH-β splice variant (TSH-βv) expressed specifically in bone marrow-derived macrophages and that exerted an osteoprotective effect by inducing osteoblastogenesis. To extend this observation and its relevance to human bone biology, we set out to identify and characterize a TSH-β variant in human macrophages. Real-time PCR analyses using human TSH-β-specific primers identified a 364-bp product in macrophages, bone marrow, and peripheral blood mononuclear cells that was sequence verified and was homologous to a human TSH-βv previously reported. We then examined TSH-βv regulation using the THP-1 human monocyte cell line matured into macrophages. After 4 days, 46.1% of the THP-1 cells expressed the macrophage markers CD-14 and macrophage colony-stimulating factor and exhibited typical morphological characteristics of macrophages. Real-time PCR analyses of these cells treated in a dose-dependent manner with T3 showed a 14-fold induction of human TSH-βv mRNA and variant protein. Furthermore, these human TSH-βv-positive cells, induced by T3 exposure, had categorized into both M1 and M2 macrophage phenotypes as evidenced by the expression of macrophage colony-stimulating factor for M1 and CCL-22 for M2. These data indicate that in hyperthyroidism, bone marrow resident macrophages have the potential to exert enhanced osteoprotective effects by oversecreting human TSH-βv, which may exert its local osteoprotective role via osteoblast and osteoclast TSH receptors.

Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

International Nuclear Information System (INIS)

Chu, Xiufeng; Zhang, Ting; Wang, Jie; Li, Meng; Zhang, Xiaolei; Tu, Jing; Sun, Shiqin; Chen, Xiangmei; Lu, Fengmin

2014-01-01

Highlights: • The expression of Fbx4 was significantly lower in HCC tissues. • Novel splicing variants of Fbx4 were identified. • These novel variants are much more abundant in human cancer tissues and cells. • The novel Fbx4 isoforms could promote cell proliferation and migration in vitro. • These isoforms showed less capability for cyclin D1 binding and degradation. - Abstract: Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant

Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

Energy Technology Data Exchange (ETDEWEB)

Chu, Xiufeng; Zhang, Ting; Wang, Jie; Li, Meng; Zhang, Xiaolei; Tu, Jing [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Sun, Shiqin [College of Pharmacy, Harbin Medical University-Daqing, Daqing, Heilongjiang 163319 (China); Chen, Xiangmei, E-mail: xm_chen6176@bjmu.edu.cn [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Lu, Fengmin [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China)

2014-04-25

Highlights: • The expression of Fbx4 was significantly lower in HCC tissues. • Novel splicing variants of Fbx4 were identified. • These novel variants are much more abundant in human cancer tissues and cells. • The novel Fbx4 isoforms could promote cell proliferation and migration in vitro. • These isoforms showed less capability for cyclin D1 binding and degradation. - Abstract: Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant

The role of the Mre11–Rad50–Nbs1 complex in double-strand break repair—facts and myths

International Nuclear Information System (INIS)

Takeda, Shunichi; Hoa, Nguyen Ngoc; Sasanuma, Hiroyuki

2016-01-01

Homologous recombination (HR) initiates double-strand break (DSB) repair by digesting 5′-termini at DSBs, the biochemical reaction called DSB resection, during which DSBs are processed by nucleases to generate 3′ single-strand DNA. Rad51 recombinase polymerizes along resected DNA, and the resulting Rad51–DNA complex undergoes homology search. Although DSB resection by the Mre11 nuclease plays a critical role in HR in Saccharomyces cerevisiae, it remains elusive whether DSB resection by Mre11 significantly contributes to HR-dependent DSB repair in mammalian cells. Depletion of Mre11 decreases the efficiency of DSB resection only by 2- to 3-fold in mammalian cells. We show that although Mre11 is required for efficient HR-dependent repair of ionizing-radiation–induced DSBs, Mre11 is largely dispensable for DSB resection in both chicken DT40 and human TK6 B cell lines. Moreover, a 2- to 3-fold decrease in DSB resection has virtually no impact on the efficiency of HR. Thus, although a large number of researchers have reported the vital role of Mre11-mediated DSB resection in HR, the role may not explain the very severe defect in HR in Mre11-deficient cells, including their lethality. We here show experimental evidence for the additional roles of Mre11 in (i) elimination of chemical adducts from DSB ends for subsequent DSB repair, and (ii) maintaining HR intermediates for their proper resolution

Dose Calibration of the ISS-RAD Fast Neutron Detector

Science.gov (United States)

Zeitlin, C.

2015-01-01

The ISS-RAD instrument has been fabricated by Southwest Research Institute and delivered to NASA for flight to the ISS in late 2015 or early 2016. ISS-RAD is essentially two instruments that share a common interface to ISS. The two instruments are the Charged Particle Detector (CPD), which is very similar to the MSL-RAD detector on Mars, and the Fast Neutron Detector (FND), which is a boron-loaded plastic scintillator with readout optimized for the 0.5 to 10 MeV energy range. As the FND is completely new, it has been necessary to develop methodology to allow it to be used to measure the neutron dose and dose equivalent. This talk will focus on the methods developed and their implementation using calibration data obtained in quasi-monoenergetic (QMN) neutron fields at the PTB facility in Braunschweig, Germany. The QMN data allow us to determine an approximate response function, from which we estimate dose and dose equivalent contributions per detected neutron as a function of the pulse height. We refer to these as the "pSv per count" curves for dose equivalent and the "pGy per count" curves for dose. The FND is required to provide a dose equivalent measurement with an accuracy of ?10% of the known value in a calibrated AmBe field. Four variants of the analysis method were developed, corresponding to two different approximations of the pSv per count curve, and two different implementations, one for real-time analysis onboard ISS and one for ground analysis. We will show that the preferred method, when applied in either real-time or ground analysis, yields good accuracy for the AmBe field. We find that the real-time algorithm is more susceptible to chance-coincidence background than is the algorithm used in ground analysis, so that the best estimates will come from the latter.

Aging Effects of Caenorhabditis elegans Ryanodine Receptor Variants Corresponding to Human Myopathic Mutations

Directory of Open Access Journals (Sweden)

Katie Nicoll Baines

2017-05-01

Full Text Available Delaying the decline in skeletal muscle function will be critical to better maintenance of an active lifestyle in old age. The skeletal muscle ryanodine receptor, the major intracellular membrane channel through which calcium ions pass to elicit muscle contraction, is central to calcium ion balance and is hypothesized to be a significant factor for age-related decline in muscle function. The nematode Caenorhabditis elegans is a key model system for the study of human aging, and strains were generated with modified C. elegans ryanodine receptors corresponding to human myopathic variants linked with malignant hyperthermia and related conditions. The altered response of these strains to pharmacological agents reflected results of human diagnostic tests for individuals with these pathogenic variants. Involvement of nerve cells in the C. elegans responses may relate to rare medical symptoms concerning the central nervous system that have been associated with ryanodine receptor variants. These single amino acid modifications in C. elegans also conferred a reduction in lifespan and an accelerated decline in muscle integrity with age, supporting the significance of ryanodine receptor function for human aging.

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

Differentiation of prostatitis and prostate cancer using the Prostate Imaging-Reporting and Data System (PI-RADS).

Science.gov (United States)

Meier-Schroers, Michael; Kukuk, Guido; Wolter, Karsten; Decker, Georges; Fischer, Stefan; Marx, Christian; Traeber, Frank; Sprinkart, Alois Martin; Block, Wolfgang; Schild, Hans Heinz; Willinek, Winfried

2016-07-01

To determine if prostate cancer (PCa) and prostatitis can be differentiated by using PI-RADS. 3T MR images of 68 patients with 85 cancer suspicious lesions were analyzed. The findings were correlated with histopathology. T2w imaging (T2WI), diffusion weighted imaging (DWI), dynamic contrast enhancement (DCE), and MR-Spectroscopy (MRS) were acquired. Every lesion was given a single PI-RADS score for each parameter, as well as a sum score and a PI-RADS v2 score. Furthermore, T2-morphology, ADC-value, perfusion type, citrate/choline-level, and localization were evaluated. 44 of 85 lesions showed PCa (51.8%), 21 chronic prostatitis (24.7%), and 20 other benign tissue such as hyperplasia or fibromuscular tissue (23.5%). The single PI-RADS score for T2WI, DWI, DCE, as well as the aggregated score including and not including MRS, and the PI-RADS v2-score were all significantly higher for PCa than for prostatitis or other tissue (pprostatitis than for other tissue (p=0.029 and p=0.020), whereas the other parameters were not different. Prostatitis usually presented borderline pathological PI-RADS scores, showed restricted diffusion with ADC≥900mm(2)/s in 100% of cases, was more often indistinctly hypointense on T2WI (66.7%), and localized in the transitional zone (57.1%). An ADC≥900mm(2)/s achieved the highest predictive value for prostatitis (AUC=0.859). Prostatitis can be differentiated from PCa using PI-RADS, since all available parameters are more distinct in cases of cancer. However, there is significant overlap between prostatitis and other benign findings, thus PI-RADS is only suitable to a limited extent for the primary assessment of prostatitis. Restricted diffusion with ADC≥900mm(2)/s is believed to be a good indicator for prostatitis. MRS can help to distinguish between prostatitis and other tissue. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Mars science laboratory radiation assessment detector (MSL/RAD) modeling workshop proceedings

Science.gov (United States)

Hassler, Donald M.; Norbury, John W.; Reitz, Günther

2017-08-01

The Radiation Assessment Detector (RAD) (Hassler et al., 2012; Zeitlin et al., 2016) onboard the Mars Science Laboratory (MSL) Curiosity rover (Grotzinger et al., 2012) is a sophisticated charged and neutral particle radiation analyzer developed by an international team of scientists and engineers from Southwest Research Institute in Boulder, Colorado as the leading institution, the University of Kiel and the German Aerospace Center in Cologne, Germany. RAD is a compact, powerful instrument capable of distinguishing between ionizing particles and neutral particles and providing neutron, gamma, and charged particle spectra from protons to iron as well as absorbed dose measurements in tissue-equivalent material. During the 6 month cruise to Mars, inside the MSL spacecraft, RAD served as a proxy to validate models of the radiation levels expected inside a spacecraft that future astronauts might experience (Zeitlin et al., 2013). RAD was turned on one day after the landing on August 7, 2012, exactly 100 years to the day after the discovery of cosmic rays on Earth by Victor Hess. These measurements are the first of their kind on the surface of another planet (Hassler et al., 2014), and the radiation data collected by RAD on the surface of Mars will inform projections of crew health risks and the design of protective surface habitats and other countermeasures for future human missions in the coming decades.

Expression of CD44 splice variants in human primary brain tumors

NARCIS (Netherlands)

Kaaijk, P.; Troost, D.; Morsink, F.; Keehnen, R. M.; Leenstra, S.; Bosch, D. A.; Pals, S. T.

1995-01-01

Expression of CD44, particularly of certain splice variants, has been linked to tumor progression and metastatic potential in a number of different animal and human cancers. Although differential expression of CD44 standard epitopes (CD44s) in human brain tumors has been reported, the expression of

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

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

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

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.

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

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.

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.

The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

Directory of Open Access Journals (Sweden)

Anita Collavoli

2011-01-01

Full Text Available By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB. This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

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

A genetic electrophoretic variant of high-sulfur hair proteins for forensic hair comparisons. I. Characterization of variant high-sulfur proteins of human hair.

Science.gov (United States)

Miyake, B

1989-02-01

In a survey of the proteins from human hair, a genetic electrophoretic variant has been observed in the high-sulfur protein region. S-carboxymethylated proteins were examined by 15% polyacrylamide gel electrophoresis at pH 8.9. Out of 150 unrelated samples of Japanese head hairs analyzed, 107 showed 6 major high-sulfur protein bands (normal) and the remaining 43 samples showed an additional high-sulfur protein band (variant). Of 21 Caucasian samples analyzed only one variant sample was found. Characterization of the proteins by two-dimensional electrophoresis evidenced a variant protein spot which showed an apparent molecular weight of 30 k Da. Isoelectric points of the high-sulfur proteins ranged from 3.25-3.55 and that of variant protein band from 3.3-3.4. Family studies of 21 matings resulting in 49 children indicated that this variant was inherited in an autosomal fashion.

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...

Identification and characterization of two functional variants in the human longevity gene FOXO3

DEFF Research Database (Denmark)

Flachsbart, Friederike; Dose, Janina; Gentschew, Liljana

2017-01-01

FOXO3 is consistently annotated as a human longevity gene. However, functional variants and underlying mechanisms for the association remain unknown. Here, we perform resequencing of the FOXO3 locus and single-nucleotide variant (SNV) genotyping in three European populations. We find two FOXO3 SN...

Importance of non-synonymous OCA2 variants in human eye colour prediction

DEFF Research Database (Denmark)

Andersen, Jeppe Dyrberg; Pietroni, Carlotta; Johansen, Peter

2016-01-01

in the promotor region of OCA2 (OMIM #611409). Nevertheless, many eye colors cannot be explained by only considering rs12913832:A>G. Methods: In this study, we searched for additional variants in OCA2 to explain human eye color by sequencing a 500 kbp region, encompassing OCA2 and its promotor region. Results: We...... identified three nonsynonymous OCA2 variants as important for eye color, including rs1800407:G>A (p.Arg419Gln) and two variants, rs74653330:A>T (p.Ala481Thr) and rs121918166:G>A (p.Val443Ile), not previously described as important for eye color variation. It was shown that estimated haplotypes consisting...

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

Comparative analysis of cytokeratin 15, TDAG51, cytokeratin 20 and androgen receptor in sclerosing adnexal neoplasms and variants of basal cell carcinoma.

Science.gov (United States)

Evangelista, Mara Therese P; North, Jeffrey P

2015-11-01

Desmoplastic trichoepithelioma (DTE), morpheaform basal cell carcinoma (BCC) and microcystic adnexal carcinoma (MAC) are sclerosing adnexal neoplasms with overlapping histopathologic features. We compared cytokeratin 15, (CK15), T-cell death-associated gene 51 (TDAG51), cytokeratin 20 (CK20) and androgen receptor (AR) in differentiating these tumors and assessed their expression in BCC subtypes. Fifteen DTE, 15 infundibulocystic BCC, 18 micronodular BCC, 18 morpheaform BCC and 6 MAC were assessed for CK15, TDAG51, CK20 and AR expression. Quantitative CK15 staining was higher in DTE compared with BCC (p < 0.0001) and MAC (p = 0.02). Quantitative TDAG51 staining was higher in DTE than BCC (p < 0.0001). The CK20+AR- immunophenotype was 100% sensitive and specific in diagnosing DTE. The CK20-AR+ immunophenotype was 95.24% specific and 83.33% sensitive for BCC. The CK20-AR- immunophenotype was 83.33% sensitive and 90.91% specific for MAC. CK15, CK20 and AR were positive in 87, 53 and 67% of infundibulocystic BCC cases, respectively. Combination of CK20 and AR best differentiated these sclerosing adnexal neoplasms. Greater positivity for CK15 and TDAG51 generally favors benign lesions. Infundibulocystic BCC has higher CK20 and lower AR immunopositivity than other BCC variants and a high degree of CK15 and TDAG51 positivity. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

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

Energy Technology Data Exchange (ETDEWEB)

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

CDKL5 variants: Improving our understanding of a rare neurologic disorder.

Science.gov (United States)

Hector, Ralph D; Kalscheuer, Vera M; Hennig, Friederike; Leonard, Helen; Downs, Jenny; Clarke, Angus; Benke, Tim A; Armstrong, Judith; Pineda, Mercedes; Bailey, Mark E S; Cobb, Stuart R

2017-12-01

To provide new insights into the interpretation of genetic variants in a rare neurologic disorder, CDKL5 deficiency, in the contexts of population sequencing data and an updated characterization of the CDKL5 gene. We analyzed all known potentially pathogenic CDKL5 variants by combining data from large-scale population sequencing studies with CDKL5 variants from new and all available clinical cohorts and combined this with computational methods to predict pathogenicity. The study has identified several variants that can be reclassified as benign or likely benign. With the addition of novel CDKL5 variants, we confirm that pathogenic missense variants cluster in the catalytic domain of CDKL5 and reclassify a purported missense variant as having a splicing consequence. We provide further evidence that missense variants in the final 3 exons are likely to be benign and not important to disease pathology. We also describe benign splicing and nonsense variants within these exons, suggesting that isoform hCDKL5_5 is likely to have little or no neurologic significance. We also use the available data to make a preliminary estimate of minimum incidence of CDKL5 deficiency. These findings have implications for genetic diagnosis, providing evidence for the reclassification of specific variants previously thought to result in CDKL5 deficiency. Together, these analyses support the view that the predominant brain isoform in humans (hCDKL5_1) is crucial for normal neurodevelopment and that the catalytic domain is the primary functional domain.

Significance of functional disease-causal/susceptible variants identified by whole-genome analyses for the understanding of human diseases.

Science.gov (United States)

Hitomi, Yuki; Tokunaga, Katsushi

2017-01-01

Human genome variation may cause differences in traits and disease risks. Disease-causal/susceptible genes and variants for both common and rare diseases can be detected by comprehensive whole-genome analyses, such as whole-genome sequencing (WGS), using next-generation sequencing (NGS) technology and genome-wide association studies (GWAS). Here, in addition to the application of an NGS as a whole-genome analysis method, we summarize approaches for the identification of functional disease-causal/susceptible variants from abundant genetic variants in the human genome and methods for evaluating their functional effects in human diseases, using an NGS and in silico and in vitro functional analyses. We also discuss the clinical applications of the functional disease causal/susceptible variants to personalized medicine.

Epidermal growth factor receptor (EGFR mutation status and Rad51 determine the response of glioblastoma (GBM to multimodality therapy with cetuximab, temozolomide and radiation

Directory of Open Access Journals (Sweden)

Phyllis Rachelle Wachsberger

2013-02-01

Full Text Available Purpose: EGFR amplification and mutation (i.e., EGFRvIII are found in 40% of primary GBM tumors and are believed to contribute to tumor development and therapeutic resistance. This study was designed to investigate how EGFR mutational status modulates response to multimodality treatment with cetuximab, an anti-EGFR inhibitor, the chemotherapeutic agent, temozolamide (TMZ and radiation therapy (RT Methods and Materials: In vitro and in vivo experiments were performed on two isogenic U87 GBM cell lines: one overexpressing wildtype EGFR (U87wtEGFR and the other overexpressing EGFRvIII (U87EGFRvIII. Results: Xenografts harboring EGFRvIII were more sensitive to TMZ alone and TMZ in combination with RT and/or cetuximab than xenografts expressing wtEGFR. In vitro experiments demonstrated that U87EGFRvIII-expressing tumors appear to harbor defective DNA homologous recombination repair in the form of Rad51 processing, Conclusions: The difference in sensitivity between EGFR-expressing and EGFRvIII-expressing tumors to combined modality treatment may help in the future tailoring of GBM therapy to subsets of patients expressing more or less of the EGFR mutant.

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...

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.

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...

Radio-ligand immunoassay for human hemoglobin variants

International Nuclear Information System (INIS)

Javid, J.; Pettis, P.K.; Miller, J.E.

1981-01-01

A quantitative method is described for the individual assay of human hemoglobin variants occurring singly or in mixture. The hemoglobin to be assayed is bound to specific antibody; the immune complex is attached to protein A-containing S. aureus and removed from the mixture. The hemoglobin thus isolated is quantified by its ability to bind radiolabeled haptoglobin. The technique is accurate and distinguishes among the 4 hemoglobins tested, namely Hb A, S, C and F. It has the advantage over conventional radioimmunoassay that a single probe, radiolabeled haptoglobin, is needed for the specific assay of any hemoglobin. (Auth.)

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

Directory of Open Access Journals (Sweden)

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.

Rare Variant Analysis of Human and Rodent Obesity Genes in Individuals with Severe Childhood Obesity

NARCIS (Netherlands)

Hendricks, Audrey E.; Bochukova, Elena G.; Marenne, Gaëlle; Keogh, Julia M.; Atanassova, Neli; Bounds, Rebecca; Wheeler, Eleanor; Mistry, Vanisha; Henning, Elana; Körner, Antje; Muddyman, Dawn; McCarthy, Shane; Hinney, Anke; Hebebrand, Johannes; Scott, Robert A.; Langenberg, Claudia; Wareham, Nick J.; Surendran, Praveen; Howson, Joanna M M; Butterworth, Adam S.; Danesh, John; Nordestgaard, Børge G.; Nielsen, Sune F.; Afzal, Shoaib; Papadia, Sofia; Ashford, Sofie; Garg, Sumedha; Millhauser, Glenn L.; Palomino, Rafael I.; Kwasniewska, Alexandra; Tachmazidou, Ioanna; O'Rahilly, Stephen; Zeggini, Eleftheria; Barroso, Inês; Farooqi, I. Sadaf; Benzeval, Michaela; Burton, Jonathan; Buck, Nicholas; Jäckle, Annette; Kumari, Meena; Laurie, Heather; Lynn, Peter; Pudney, Stephen; Rabe, Birgitta; Wolke, Dieter; Overvad, Kim; Tjønneland, Anne; Clavel-Chapelon, Francoise; Kaaks, Rudolf; Boeing, Heiner; Trichopoulou, Antonia; Ferrari, Pietro; Palli, Domenico; Krogha, Vittorio; Panico, Salvatore; Tuminoa, Rosario; Matullo, Giuseppe; Boer, Jolanda Ma; Van Der Schouw, Yvonne; Weiderpass, Elisabete; Quiros, J. Ramon; Sánchez, María José; Navarro, Carmen; Moreno-Iribas, Conchi; Arriola, Larraitz; Melander, Olle; Wennberg, Patrik; Key, Timothy J.; Riboli, Elio; Al-Turki, Saeed; Anderson, Carl A; Anney, Richard; Antony, Dinu; Soler Artigas, María; Ayub, Muhammad; Bala, Senduran; Barrett, Jeffrey C; Beales, Phil; Bentham, Jamie; Bhattacharyaa, Shoumo; Birney, Ewan; Blackwooda, Douglas; Bobrow, Martin; Bolton, Patrick F.; Boustred, Chris; Breen, Gerome; Calissanoa, Mattia; Carss, Keren; Charlton, Ruth; Chatterjee, Krishna; Chen, Lu; Ciampia, Antonio; Cirak, Sebahattin; Clapham, Peter; Clement, Gail; Coates, Guy; Coccaa, Massimiliano; Collier, David A; Cosgrove, Catherine; Coxa, Tony; Craddock, Nick; Crooks, Lucy; Curran, Sarah; Curtis, David; Daly, Allan; Danecek, Petr; Day, Ian N M; Day-Williams, Aaron G; Dominiczak, Anna; Down, Thomas; Du, Yuanping; Dunham, Ian; Durbin, Richard; Edkins, Sarah; Ekong, Rosemary; Ellis, Peter; Evansa, David M.; FitzPatrick, David R.; Flicek, Paul; Floyd, James S.; Foley, A. Reghan; Franklin, Christopher S.; Futema, Marta; Gallagher, Louise; Gaunt, Tom R.; Geihs, Matthias; Geschwind, Daniel H.; Greenwood, Celia M.T.; Griffin, Heather; Grozeva, Detelina; Guo, Xiaosen; Guo, Xueqin; Gurling, Hugh; Hart, Deborah J.; Holmans, Peter A; Howie, Bryan; Huang, Jie; Huang, Liren; Hubbard, Tim; Humphries, Steve E.; Hurles, Matthew E.; Hysi, Pirro G.; Iotchkova, Valentina; Jackson, David K.; Jamshidi, Yalda; Joyce, Chris; Karczewski, Konrad J.; Kaye, Jane; Keane, Thomas; Kemp, John P.; Kennedy, Karen; Kent, Alastair; Khawaja, Farrah; Van Kogelenberg, Margriet; Kolb-Kokocinski, Anja; Lachance, Genevieve; Langford, Cordelia; Lawson, Daniel; Lee, Irene; Lek, Monkol; Li, Rui; Li, Yingrui; Liang, Jieqin; Lin, Hong; Liu, Ryan; Lönnqvist, Jouko; Lopes, Luis R.; Lopes, Margarida; MacArthur, Daniel G.; Mangino, Massimo; Marchini, Jonathan; Maslen, John; Mathieson, Iain; McGuffin, Peter; McIntosh, Andrew M.; McKechanie, Andrew G.; McQuillin, Andrew; Memari, Yasin; Metrustry, Sarah; Migone, Nicola; Min, Josine L.; Mitchison, Hannah M; Moayyeri, Alireza; Morris, Andrew D.; Morris, James; Muntoni, Francesco; Northstone, Kate; O'Donovan, Michael C.; Onoufriadis, Alexandros; Oualkacha, Karim; Owen, Michael J; Palotie, Aarno; Panoutsopoulou, Kalliope; Parker, Victoria; Parr, Jeremy R.; Paternoster, Lavinia; Paunio, Tiina; Payne, Felicity; Payne, Stewart J.; Perry, John R. B.; Pietilainen, Olli; Plagnol, Vincent; Pollitt, Rebecca C.; Porteous, David J.; Povey, Sue; Quail, Michael A.; Quaye, Lydia; Raymond, F. Lucy; Rehnström, Karola; Richards, J Brent; Ridout, Cheryl K.; Ring, Susan M.; Ritchie, Graham R.S.; Roberts, Nicola; Robinson, Rachel L.; Savage, David B.; Scambler, Peter; Schiffels, Stephan; Schmidts, Miriam; Schoenmakers, Nadia; Scott, Richard H.; Semple, Robert K.; Serra, Eva; Sharp, Sally I.; Shaw, Adam; Shihab, Hashem A.; Shin, So Youn; Skuse, David; Small, Kerrin S; Smee, Carol; Smith, Blair H.; Davey Smith, George; Soranzo, Nicole; Southam, Lorraine; Spasic-Boskovic, Olivera; Spector, Timothy D; St Clair, David; St Pourcain, Beate; Stalker, Jim; Stevens, Elizabeth; Sun, Jianping; Surdulescu, Gabriela L; Suvisaari, Jaana; Syrris, Petros; Taylor, Rohan; Tian, Jing; Timpson, Nicholas J.; Tobin, Martin D; Valdes, Ana M.; Vandersteen, Anthony M.; Vijayarangakannan, Parthiban; Visscher, Peter M.; Wain, Louise V.; Walter, Klaudia; Walters, James T.R.; Wang, Guangbiao; Wang, Jun; Wang, Nai-Yu; Ward, Kirsten; Whyte, Tamieka; Williams, Hywel J.; Williamson, Kathleen A.; Wilson, Crispian; Wilson, Scott G.; Wong, Kim; Xu, Changjiang; Yang, Jian; Zhang, Feng; Zhang, Pingbo; Zheng, Hou Feng

2017-01-01

Obesity is a genetically heterogeneous disorder. Using targeted and whole-exome sequencing, we studied 32 human and 87 rodent obesity genes in 2,548 severely obese children and 1,117 controls. We identified 52 variants contributing to obesity in 2% of cases including multiple novel variants in GNAS,

Rare variant analysis of human and rodent obesity genes in individuals with severe childhood obesity

DEFF Research Database (Denmark)

Hendricks, Audrey E.; Bochukova, Elena G.; Marenne, Gaëlle

2017-01-01

Obesity is a genetically heterogeneous disorder. Using targeted and whole-exome sequencing, we studied 32 human and 87 rodent obesity genes in 2,548 severely obese children and 1,117 controls. We identified 52 variants contributing to obesity in 2% of cases including multiple novel variants in GN...

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,...

Species association of hepatitis B virus (HBV in non-human apes; evidence for recombination between gorilla and chimpanzee variants.

Directory of Open Access Journals (Sweden)

Sinéad Lyons

Full Text Available Hepatitis B virus (HBV infections are widely distributed in humans, infecting approximately one third of the world's population. HBV variants have also been detected and genetically characterised from Old World apes; Gorilla gorilla (gorilla, Pan troglodytes (chimpanzee, Pongo pygmaeus (orang-utan, Nomascus nastusus and Hylobates pileatus (gibbons and from the New World monkey, Lagothrix lagotricha (woolly monkey. To investigate species-specificity and potential for cross species transmission of HBV between sympatric species of apes (such as gorillas and chimpanzees in Central Africa or between humans and chimpanzees or gorillas, variants of HBV infecting captive wild-born non-human primates were genetically characterised. 9 of 62 chimpanzees (11.3% and two from 11 gorillas (18% were HBV-infected (15% combined frequency, while other Old world monkey species were negative. Complete genome sequences were obtained from six of the infected chimpanzee and both gorillas; those from P. t .ellioti grouped with previously characterised variants from this subspecies. However, variants recovered from P. t. troglodytes HBV variants also grouped within this clade, indicative of transmission between sub-species, forming a paraphyletic clade. The two gorilla viruses were phylogenetically distinct from chimpanzee and human variants although one showed evidence for a recombination event with a P.t.e.-derived HBV variant in the partial X and core gene region. Both of these observations provide evidence for circulation of HBV between different species and sub-species of non-human primates, a conclusion that differs from the hypothesis if of strict host specificity of HBV genotypes.

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

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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

New polymorphic variants of human blood clotting factor IX

Energy Technology Data Exchange (ETDEWEB)

Surin, V.L.; Luk`yanenko, A.V.; Tagiev, A.F.; Smirnova, O.V. [Hematological Research Center, Moscow (Russian Federation); Plutalov, O.V.; Berlin, Yu.A. [Shemyakin Institute of Bioorganic Chemistry, Moscow (Russian Federation)

1995-04-01

The polymorphism of Alu-repeats, which are located in the introns of the human factor IX gene (copies 1-3), was studied. To identify polymorphic variants, direct sequencing of PCR products that contained appropriate repeats was used. In each case, 20 unrelated X chromosomes were studied. A polymorphic Dra I site was found near the 3{prime}-end of Alu copy 3 within the region of the polyA tract. A PCR-based testing system with internal control of restriction hydrolysis was suggested. Testing 81 unrelated X chromosomes revealed that the frequency of the polymorphic Dra I site is 0.23. Taq I polymorphism, which was revealed in Alu copy 4 of factor IX gene in our previous work, was found to be closely linked to Dra I polymorphism. Studies in linkage between different types of polymorphisms of the factor IX gene revealed the presence of a rare polymorphism in intron a that was located within the same minisatellite region as the known polymorphic insertion 50 bp/Dde I. However, the size of the insertion in our case was 26 bp. Only one polymorphic variant was found among over 150 unrelated X chromosomes derived from humans from Moscow and its vicinity. 10 refs., 4 figs., 1 tab.

Analysis of 6,515 exomes reveals the recent origin of most human protein-coding variants.

Science.gov (United States)

Fu, Wenqing; O'Connor, Timothy D; Jun, Goo; Kang, Hyun Min; Abecasis, Goncalo; Leal, Suzanne M; Gabriel, Stacey; Rieder, Mark J; Altshuler, David; Shendure, Jay; Nickerson, Deborah A; Bamshad, Michael J; Akey, Joshua M

2013-01-10

Establishing the age of each mutation segregating in contemporary human populations is important to fully understand our evolutionary history and will help to facilitate the development of new approaches for disease-gene discovery. Large-scale surveys of human genetic variation have reported signatures of recent explosive population growth, notable for an excess of rare genetic variants, suggesting that many mutations arose recently. To more quantitatively assess the distribution of mutation ages, we resequenced 15,336 genes in 6,515 individuals of European American and African American ancestry and inferred the age of 1,146,401 autosomal single nucleotide variants (SNVs). We estimate that approximately 73% of all protein-coding SNVs and approximately 86% of SNVs predicted to be deleterious arose in the past 5,000-10,000 years. The average age of deleterious SNVs varied significantly across molecular pathways, and disease genes contained a significantly higher proportion of recently arisen deleterious SNVs than other genes. Furthermore, European Americans had an excess of deleterious variants in essential and Mendelian disease genes compared to African Americans, consistent with weaker purifying selection due to the Out-of-Africa dispersal. Our results better delimit the historical details of human protein-coding variation, show the profound effect of recent human history on the burden of deleterious SNVs segregating in contemporary populations, and provide important practical information that can be used to prioritize variants in disease-gene discovery.

A naturally occurring variant of the human prion protein completely prevents prion disease.

Science.gov (United States)

Asante, Emmanuel A; Smidak, Michelle; Grimshaw, Andrew; Houghton, Richard; Tomlinson, Andrew; Jeelani, Asif; Jakubcova, Tatiana; Hamdan, Shyma; Richard-Londt, Angela; Linehan, Jacqueline M; Brandner, Sebastian; Alpers, Michael; Whitfield, Jerome; Mead, Simon; Wadsworth, Jonathan D F; Collinge, John

2015-06-25

Mammalian prions, transmissible agents causing lethal neurodegenerative diseases, are composed of assemblies of misfolded cellular prion protein (PrP). A novel PrP variant, G127V, was under positive evolutionary selection during the epidemic of kuru--an acquired prion disease epidemic of the Fore population in Papua New Guinea--and appeared to provide strong protection against disease in the heterozygous state. Here we have investigated the protective role of this variant and its interaction with the common, worldwide M129V PrP polymorphism. V127 was seen exclusively on a M129 PRNP allele. We demonstrate that transgenic mice expressing both variant and wild-type human PrP are completely resistant to both kuru and classical Creutzfeldt-Jakob disease (CJD) prions (which are closely similar) but can be infected with variant CJD prions, a human prion strain resulting from exposure to bovine spongiform encephalopathy prions to which the Fore were not exposed. Notably, mice expressing only PrP V127 were completely resistant to all prion strains, demonstrating a different molecular mechanism to M129V, which provides its relative protection against classical CJD and kuru in the heterozygous state. Indeed, this single amino acid substitution (G→V) at a residue invariant in vertebrate evolution is as protective as deletion of the protein. Further study in transgenic mice expressing different ratios of variant and wild-type PrP indicates that not only is PrP V127 completely refractory to prion conversion but acts as a potent dose-dependent inhibitor of wild-type prion propagation.

Immunochip analysis identifies association of the RAD50/IL13 region with human longevity

DEFF Research Database (Denmark)

Flachsbart, Friederike; Ellinghaus, David; Gentschew, Liljana

2016-01-01

Human longevity is characterized by a remarkable lack of confirmed genetic associations. Here, we report on the identification of a novel locus for longevity in the RAD50/IL13 region on chromosome 5q31.1 using a combined European sample of 3208 long-lived individuals (LLI) and 8919 younger controls....... First, we performed a large-scale association study on 1458 German LLI (mean age 99.0 years) and 6368 controls (mean age 57.2 years) by targeting known immune-associated loci covered by the Immunochip. The analysis of 142 136 autosomal single nucleotide polymorphisms (SNPs) revealed an Immunochip...... (1257 LLI, mean age 102.4 years; 1811 controls, mean age 49.1 years) and Denmark (493 LLI, mean age 96.2 years; 740 controls, mean age 63.1 years). The association at SNP rs2706372 replicated in the French study collection and showed a similar trend in the Danish participants and was also significant...

Flavonoids as Inhibitors of Human Butyrylcholinesterase Variants

Directory of Open Access Journals (Sweden)

Maja Katalinić

2014-01-01

Full Text Available The inhibition of butyrylcholinesterase (BChE, EC 3.1.1.8 appears to be of interest in treating diseases with symptoms of reduced neurotransmitter levels, such as Alzheimer’s disease. However, BCHE gene polymorphism should not be neglected in research since it could have an effect on the expected outcome. Several well-known cholinergic drugs (e.g. galantamine, huperzine and rivastigmine originating from plants, or synthesised as derivatives of plant compounds, have shown that herbs could serve as a source of novel target-directed compounds. We focused our research on flavonoids, biologically active polyphenolic compounds found in many plants and plant-derived products, as BChE inhibitors. All of the tested flavonoids: galangin, quercetin, fisetin and luteolin reversibly inhibited usual, atypical, and fluoride-resistant variants of human BChE. The inhibition potency increased in the following order, identically for all three BChE variants: luteolin

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

Directory of Open Access Journals (Sweden)

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

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.

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

Genetic variation in the NBS1, MRE11, RAD50 and BLM genes and susceptibility to non-Hodgkin lymphoma

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Gascoyne Randy D

2009-11-01

Full Text Available Abstract Background Translocations are hallmarks of non-Hodgkin lymphoma (NHL genomes. Because lymphoid cell development processes require the creation and repair of double stranded breaks, it is not surprising that disruption of this type of DNA repair can cause cancer. The members of the MRE11-RAD50-NBS1 (MRN complex and BLM have central roles in maintenance of DNA integrity. Severe mutations in any of these genes cause genetic disorders, some of which are characterized by increased risk of lymphoma. Methods We surveyed the genetic variation in these genes in constitutional DNA of NHL patients by means of gene re-sequencing, then conducted genetic association tests for susceptibility to NHL in a population-based collection of 797 NHL cases and 793 controls. Results 114 SNPs were discovered in our sequenced samples, 61% of which were novel and not previously reported in dbSNP. Although four variants, two in RAD50 and two in NBS1, showed association results suggestive of an effect on NHL, they were not significant after correction for multiple tests. Conclusion These results suggest an influence of RAD50 and NBS1 on susceptibility to diffuse large B-cell lymphoma and marginal zone lymphoma. Larger association and functional studies could confirm such a role.

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...

Analysis of Maxi-K alpha subunit splice variants in human myometrium

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Morrison John J

2004-09-01

Full Text Available Abstract Background Large-conductance, calcium-activated potassium (Maxi-K channels are implicated in the modulation of human uterine contractions and myometrial Ca2+ homeostasis. However, the regulatory mechanism(s governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset. Methods Myometrial biopsies were obtained at hysterectomy (non-pregnant, NP, and at Caesarean section, at elective (pregnant not-in-labour, PNL and intrapartum (pregnant in-labour, PL procedures. RNA was extracted from all biopsies and quantitative real-time RT-PCR was used to investigate for possible differential expression of the Maxi-K alpha subunit, and that of its splice variants, between these functionally-distinct myometrial tissue sets. Results RT-PCR analysis identified the presence of a 132 bp and an 87 bp spliced exon of the Maxi-K alpha subunit in all three myometrial tissue sets. Quantitative real-time PCR indicated a decrease in the expression of the Maxi-K alpha subunit with labour onset. While there was no change in the proportion of Maxi-K alpha subunits expressing the 87 bp spliced exon, the proportion of alpha subunits expressing the 132 bp spliced exon was significantly increased with labour onset, compared to both non-pregnant and pregnant not-in-labour tissues. An increased proportion of 132 bp exon-containing alpha subunit variants with labour onset is of interest, as channels expressing this spliced exon have decreased calcium and voltage sensitivities. Conclusions Our findings suggest that decreased Maxi-K alpha subunit mRNA expression in human myometrium at

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.

Cloning and expression of a novel human profilin variant, profilin II

DEFF Research Database (Denmark)

Honoré, B; Madsen, Peder; Andersen, A H

1993-01-01

We have isolated a 1.7 kbp cDNA encoding a 140 amino acid protein (15.1 kDa, pI 5.91) with a high sequence similarity (62%) to human profilin (profilin I). We have termed this variant profilin II. Northern blot analysis showed that profilin II is highly expressed in brain, skeletal muscle and kid...

A Low Density Microarray Method for the Identification of Human Papillomavirus Type 18 Variants

Science.gov (United States)

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B.; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C.

2013-01-01

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings. PMID:24077317

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,...

KinMutRF: a random forest classifier of sequence variants in the human protein kinase superfamily

DEFF Research Database (Denmark)

Pons, Tirso; Vazquez, Miguel; Matey-Hernandez, María Luisa

2016-01-01

annotations from UniProt, Phospho.ELM and FireDB. KinMutRF identifies disease-associated variants satisfactorily (Acc: 0.88, Prec:0.82, Rec:0.75, F-score:0.78, MCC:0.68) when trained and cross-validated with the 3689 human kinase variants from UniProt that have been annotated as neutral or pathogenic. All...

Human nucleolus organizers on nonhomologous chromosomes can share the same ribosomal gene variants.

Science.gov (United States)

Krystal, M; D'Eustachio, P; Ruddle, F H; Arnheim, N

1981-01-01

The distributions of three human ribosomal gene polymorphisms among individual chromosomes containing nucleolus organizers were analyzed by using mouse--human hybrid cells. Different nucleolus organizers can contain the same variant, suggesting the occurrence of genetic exchanges among ribosomal gene clusters on nonhomologous chromosomes. Such exchanges appear to occur less frequently in mice. This difference is discussed in terms of the nucleolar organization and chromosomal location of ribosomal gene clusters in humans and mice. Images PMID:6272316

Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections.

Science.gov (United States)

van der Weele, Pascal; Meijer, Chris J L M; King, Audrey J

2017-10-01

Human papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially ( P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related. IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is

Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells

International Nuclear Information System (INIS)

Wang, Jing; Yang, Qifeng; Haffty, Bruce G.; Li, Xiaoyan; Moran, Meena S.

2013-01-01

Highlights: ► Fulvestrant radiosensitizes MCF-7 cells. ► Fulvestrant increases G1 arrest and decreases S phase in MCF-7 cells. ► Fulvestrant down-regulates DNA-PKcs and RAD51 in MCF-7 cells. -- Abstract: The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F + RT). This study was conducted to assess the effects of fulvestrant alone vs. F + RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F + RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy ± fulvestrant. The effects of F + RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F + RT was 0.885 ± 0.013 vs. 0.622 ± 0.029 @2 Gy, 0.599 ± 0.045 vs. 0.475 ± 0.054 @4 Gy, and 0.472 ± 0.021 vs. 0.380 ± 0.018 @6 Gy RT (p = 0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F + RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p < 0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F + RT compared with irradiation alone. F + RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F + RT

Characterization of iminothiosulfine-type ions [HNCS 2] rad +/ rad - and their neutral counterparts by mass spectrometry and computational chemistry

Science.gov (United States)

Vivekananda, S.; Raghunath, P.; Bhanuprakash, K.; Srinivas, R.; Trikoupis, Moschoula A.; Terlouw, Johan K.

2000-12-01

Electron ionization of rhodanine yields iminothiosulfine ions H- N C- S- Srad + , 1brad + , which readily communicate with the higher energy cyclic isomer H- N CS2rad + , 1arad + . CBS-QB3 and G AUSSIAN-2 model chemistries predict that one electron reduction reverses the stability order but that the (singlet) neutrals remain connected via a negligible energy barrier. Neutralization-reionization (NR) experiments demonstrate that singlet 1a and its heterocumulene isomer 1b are stable species in the gas-phase. However, the co-generated triplet species readily dissociate into 3S2rad + + HNC. Confirmatory experimental evidence comes from charge reversal (CR) and NR experiments on the cyclic anion H- N CS2rad - , 1arad - .

Radiological information management system (RadIMS)

International Nuclear Information System (INIS)

Oesterling, R.G.; Marko, S.A.; Tschaeche, A.N.

1991-01-01

Westinghouse Idaho Nuclear Company, Inc. (WINCO) is developing and implementing an information management system, known as RadIMS, to track and record personnel exposure to ionizing radiation. RadIMS has been designed to fulfill all the requirements of US Department of Energy (USDOE) Order 5480.11, ''Radiation Protection for Occupational Workers.'' This Order requires the contractor to maintain detailed radiation exposure records on all individuals who work at the facility. These records must be retrievable for the entire working life of the individual and be available to other USDOE contractors on request. To meet these general needs, RadIMS provides for retrieval of detailed, comprehensive individual exposure histories as well as the usual online interactions to accomplish day-today radiation protection operations. These two extremes of functionality require different approaches in the WINCO computing environment. The exposure histories include database text, paper, microfilm, and electronic bitmaps

RadCat 3.0 user guide.

Energy Technology Data Exchange (ETDEWEB)

Hinojosa, Daniel; Penisten, Janelle J.; Dennis, Matthew L.; Osborn, Douglas M.; Weiner, Ruth F.; Heames, Terence John; Marincel, Michelle K.

2009-05-01

RADTRAN is an internationally accepted program and code for calculating the risks of transporting radioactive materials. The first versions of the program, RADTRAN I and II, were developed for NUREG-0170 (USNRC, 1977), the first environmental statement on transportation of radioactive materials. RADTRAN and its associated software have undergone a number of improvements and advances consistent with improvements in both available data and computer technology. The version of RADTRAN currently bundled with RadCat is RADTRAN 6.0. This document provides a detailed discussion and a guide for the use of the RadCat 3.0 Graphical User Interface input file generator for the RADTRAN code. RadCat 3.0 integrates the newest analysis capabilities of RADTRAN 6.0 which includes an economic model, updated loss-of-lead shielding model, and unit conversion. As of this writing, the RADTRAN version in use is RADTRAN 6.0.

Radiological information management system (RadIMS)

Energy Technology Data Exchange (ETDEWEB)

Oesterling, R.G.; Marko, S.A.; Tschaeche, A.N.

1991-08-19

Westinghouse Idaho Nuclear Company, Inc. (WINCO) is developing and implementing an information management system, known as RadIMS, to track and record personnel exposure to ionizing radiation. RadIMS has been designed to fulfill all the requirements of US Department of Energy (USDOE) Order 5480.11, Radiation Protection for Occupational Workers.'' This Order requires the contractor to maintain detailed radiation exposure records on all individuals who work at the facility. These records must be retrievable for the entire working life of the individual and be available to other USDOE contractors on request. To meet these general needs, RadIMS provides for retrieval of detailed, comprehensive individual exposure histories as well as the usual online interactions to accomplish day-today radiation protection operations. These two extremes of functionality require different approaches in the WINCO computing environment. The exposure histories include database text, paper, microfilm, and electronic bitmaps.

Radiological information management system (RadIMS)

Energy Technology Data Exchange (ETDEWEB)

Oesterling, R.G.; Marko, S.A.; Tschaeche, A.N.

1991-08-19

Westinghouse Idaho Nuclear Company, Inc. (WINCO) is developing and implementing an information management system, known as RadIMS, to track and record personnel exposure to ionizing radiation. RadIMS has been designed to fulfill all the requirements of US Department of Energy (USDOE) Order 5480.11, ``Radiation Protection for Occupational Workers.`` This Order requires the contractor to maintain detailed radiation exposure records on all individuals who work at the facility. These records must be retrievable for the entire working life of the individual and be available to other USDOE contractors on request. To meet these general needs, RadIMS provides for retrieval of detailed, comprehensive individual exposure histories as well as the usual online interactions to accomplish day-today radiation protection operations. These two extremes of functionality require different approaches in the WINCO computing environment. The exposure histories include database text, paper, microfilm, and electronic bitmaps.

Effects of common hemoglobin variants on HbA1c measurements in China: results for α- and β-globin variants measured by six methods.

Science.gov (United States)

Xu, Anping; Chen, Weidong; Xia, Yong; Zhou, Yu; Ji, Ling

2018-04-07

HbA1c is a widely used biomarker for diabetes mellitus management. Here, we evaluated the accuracy of six methods for determining HbA1c values in Chinese patients with common α- and β-globin chains variants in China. Blood samples from normal subjects and individuals exhibiting hemoglobin variants were analyzed for HbA1c, using Sebia Capillarys 2 Flex Piercing (C2FP), Bio-Rad Variant II Turbo 2.0, Tosoh HLC-723 G8 (ver. 5.24), Arkray ADAMS A1c HA-8180V fast mode, Cobas c501 and Trinity Ultra2 systems. DNA sequencing revealed five common β-globin chain variants and three common α-globin chain variants. The most common variant was Hb E, followed by Hb New York, Hb J-Bangkok, Hb G-Coushatta, Hb Q-Thailand, Hb G-Honolulu, Hb Ube-2 and Hb G-Taipei. Variant II Turbo 2.0, Ultra2 and Cobas c501 showed good agreement with C2FP for most samples with variants. HLC-723 G8 yielded no HbA1c values for Hb J-Bangkok, Hb Q-Thailand and Hb G-Honolulu. Samples with Hb E, Hb G-Coushatta, Hb G-Taipei and Hb Ube-2 produced significant negative biases for HLC-723 G8. HA-8180V showed statistically significant differences for Hb E, Hb G-Coushatta, Hb G-Taipei, Hb Q-Thailand and Hb G-Honolulu. HA-8180V yielded no HbA1c values for Hb J-Bangkok. All methods showed good agreement for samples with Hb New York. Some common hemoglobin variants can interfere with HbA1c determination by the most popular methods in China.

RadNet Radiological Air Monitoring Network

International Nuclear Information System (INIS)

Scott Telofski, J.; Askren, D.R.; Petko, Ch.M.; Fraass, R.G.

2010-01-01

The United States Environmental Protection Agency operates a national environmental radiation monitoring program called RadNet. RadNet monitors airborne particulates, precipitation, milk, and drinking water for radiation levels. The primary purpose of the original program in the 1950's and 1960's was to collect and analyze samples in various media to assess the effects of radioactive fallout from above-ground nuclear weapon testing. As above-ground testing diminished in the 1970's, the program, especially the air network, became critical in evaluating effects of other types of nuclear incidents, such as the nuclear reactor accident at Chernobyl, as well as monitoring trends in environmental radioactive contamination. The value of rapid data collection subsequent to such incidents led to the consideration of developing air monitors with radiation detectors and telecommunication equipment for real-time radiation measurement. The strengthened United States homeland security posture after 2001 led to production and installation of the current real-time RadNet air monitors. There are now 118 stationary, continuously operating air monitoring stations and 40 mobile air monitors for site specific monitoring. The stationary air monitors include radiation detectors, meteorological sensors, a high-volume air sampler, and communication devices for hourly data transfers. When unusual levels are detected, scientists download a full sodium iodide detector spectrum for analysis. The real-time data collected by RadNet stationary systems permit rapid identification and quantification of airborne nuclides with sufficient sensitivity to provide critical information to help determine protective actions. The data also may help to rapidly refine long-range radioactive plume models and estimate exposure to the population. This paper provides an overview of the airborne particulate monitoring conducted during above-ground nuclear weapon testing, summarizes the uses of data from the program

A new splice variant of the major subunit of human asialoglycoprotein receptor encodes a secreted form in hepatocytes.

Directory of Open Access Journals (Sweden)

Jia Liu

Full Text Available BACKGROUND: The human asialoglycoprotein receptor (ASGPR is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.

Protective effect of the LevRad on treat of paracoccidioidomycosis

International Nuclear Information System (INIS)

Martins, Estefania M.N.; Andrade, Antero S.R.; Fernandes, Viviane Cristina; Morais, Elis Araujo; Goes, Alfredo M.; Resende, Maria Aparecida de

2011-01-01

Paracoccidioides brasiliensis is the agent of Paracoccidioidomycosis (PCM), the most prevalent deep mycosis of Latin America. The period of treat depend on the chemotherapeutic and the severity of disease and its administration not ensure the complete destruction of the fungus. The search for new alternatives is necessary. The aim of this study was to evaluate the protective effect of yeast cells of P. brasiliensis attenuated by gamma irradiation (LevRad) on therapeutic vaccination of BALB/c. The therapeutic potential of LevRad with or without fluconazole was assessed for the first time, intraperitoneally, in BALB/c, 60 days after intratracheal infection with a highly virulent non-irradiated P.brasiliensis isolate. The animals were divided in five experimental groups: uninfected (C-), infected (C+), infected treated with fluconazole (Inmed), infected treated with LevRad (InRad) and infected treated with fluconazole + LevRad (InRadMed). The organs (lungs, spleen and liver) were collected to analyze CFU (colony forming units) and histology. The sera were used to evaluate the immunization efficacy, and to assess IgG subtypes (IgG1, IgG2a, IgG2b, IgG3) and total IgG levels. There was significant decrease in the CFU counts of the lungs of InMed, InRadMed and InRad. No were visualized histopathological alterations in the organs of these groups, except in InRad there was granulomatous lesions unifocal, little and discrete. The levels of IgG and its subtypes IgG2a, IgG2b increased, probably due to the increase of cytokines that promote switching to these isotypes. These preliminary results can provide new prospect for immunotherapy of PCM, but it will be necessary new studies to evaluate administration dose and period treatment. (author)

Complexity of mechanisms among human proprotein convertase subtilisin-kexin type 9 variants.

Science.gov (United States)

Dron, Jacqueline S; Hegele, Robert A

2017-04-01

There are many reports of human variants in proprotein convertase subtilisin-kexin type 9 (PCSK9) that are either gain-of-function (GOF) or loss-of-function (LOF), with downstream effects on LDL cholesterol and cardiovascular disease (CVD) risk. However, data on particular mechanisms have only been minimally curated. GOF variants are individually ultrarare, affect all domains of the protein, act to reduce LDL receptor expression through several mechanisms, are a minor cause of familial hypercholesterolemia, have been reported mainly within families, have variable LDL cholesterol-raising effects, and are associated with increased CVD risk mainly through observational studies in families and small cohorts. In contrast, LOF variants can be either ultrarare mutations or relatively more common polymorphisms seen in populations, affect all domains of the protein, act to increase LDL receptor expression through several mechanisms, have variable LDL cholesterol-lowering effects, and have been associated with decreased CVD risk mainly through Mendelian randomization studies in epidemiologic populations. There is considerable complexity underlying the clinical concept of both LOF and GOF variants of PCSK9. But despite the underlying mechanistic heterogeneity, altered PCSK9 secretion or function is ultimately correlated with plasma LDL cholesterol level, which is also the driver of CVD outcomes.

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

Science.gov (United States)

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.

A reliability-risk modelling of nuclear rad-waste facilities

International Nuclear Information System (INIS)

Lehmann, P.H.; El-Bassioni, A.A.

1975-01-01

Rad-waste disposal systems of nuclear power sites are designed and operated to collect, delay, contain, and concentrate radioactive wastes from reactor plant processes such that on-site and off-site exposures to radiation are well below permissible limits. To assist the designer in achieving minimum release/exposure goals, a computerized reliability-risk model has been developed to simulate the rad-waste system. The objectives of the model are to furnish a practical tool for quantifying the effects of changes in system configuration, operation, and equipment, and for the identification of weak segments in the system design. Primarily, the model comprises a marriage of system analysis, reliability analysis, and release-risk assessment. Provisions have been included in the model to permit the optimization of the system design subject to constraints on cost and rad-releases. The system analysis phase involves the preparation of a physical and functional description of the rad-waste facility accompanied by the formation of a system tree diagram. The reliability analysis phase embodies the formulation of appropriate reliability models and the collection of model parameters. Release-risk assessment constitutes the analytical basis whereupon further system and reliability analyses may be warranted. Release-risk represents the potential for release of radioactivity and is defined as the product of an element's unreliability at time, t, and the radioactivity available for release in time interval, Δt. A computer code (RARISK) has been written to simulate the tree diagram of the rad-waste system. Reliability and release-risk results have been generated for cases which examined the process flow paths of typical rad-waste systems, the effects of repair and standby, the variations of equipment failure and repair rates, and changes in system configurations. The essential feature of this model is that a complex system like the rad-waste facility can be easily decomposed into its

A Novel Human Ghrelin Variant (In1-Ghrelin) and Ghrelin-O-Acyltransferase Are Overexpressed in Breast Cancer: Potential Pathophysiological Relevance

Science.gov (United States)

Gahete, Manuel D.; Córdoba-Chacón, José; Hergueta-Redondo, Marta; Martínez-Fuentes, Antonio J.; Kineman, Rhonda D.; Moreno-Bueno, Gema

2011-01-01

The human ghrelin gene, which encodes the ghrelin and obestatin peptides, contains 5 exons (Ex), with Ex1-Ex4 encoding a 117 amino-acid (aa) preproprotein that is known to be processed to yield a 28-aa (ghrelin) and/or a 23-aa (obestatin) mature peptides, which possess biological activities in multiple tissues. However, the ghrelin gene also encodes additional peptides through alternative splicing or post-translational modifications. Indeed, we previously identified a spliced mRNA ghrelin variant in mouse (In2-ghrelin-variant), which is regulated in a tissue-dependent manner by metabolic status and may thus be of biological relevance. Here, we have characterized a new human ghrelin variant that contains Ex0-1, intron (In) 1, and Ex2 and lacks Ex3-4. This human In1-ghrelin variant would encode a new prepropeptide that conserves the first 12aa of native-ghrelin (including the Ser3-potential octanoylation site) but has a different C-terminal tail. Expression of In1-variant was detected in 22 human tissues and its levels were positively correlated with those of ghrelin-O-acyltransferase (GOAT; p = 0.0001) but not with native-ghrelin expression, suggesting that In1-ghrelin could be a primary substrate for GOAT in human tissues. Interestingly, levels of In1-ghrelin variant expression in breast cancer samples were 8-times higher than those of normal mammary tissue, and showed a strong correlation in breast tumors with GOAT (p = 0.0001), ghrelin receptor-type 1b (GHSR1b; p = 0.049) and cyclin-D3 (a cell-cycle inducer/proliferation marker; p = 0.009), but not with native-ghrelin or GHSR1a expression. Interestingly, In1-ghrelin variant overexpression increased basal proliferation of MDA-MB-231 breast cancer cells. Taken together, our results provide evidence that In1-ghrelin is a novel element of the ghrelin family with a potential pathophysiological role in breast cancer. PMID:21829727

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

Science.gov (United States)

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.

Integrative Annotation of Variants from 1092 Humans: Application to Cancer Genomics

DEFF Research Database (Denmark)

Khurana, Ekta; Fu, Yao; Colonna, Vincenza

2013-01-01

Identifying Important Identifiers Each of us has millions of sequence variations in our genomes. Signatures of purifying or negative selection should help identify which of those variations is functionally important. Khurana et al. (1235587) used sequence polymorphisms from 1092 humans across 14...... sites tended to occur in network hub promoters. Many recurrent somatic cancer variants occurred in noncoding regulatory regions and thus might indicate mutations that drive cancer....

CAD-RADS - a new clinical decision support tool for coronary computed tomography angiography.

Science.gov (United States)

Foldyna, Borek; Szilveszter, Bálint; Scholtz, Jan-Erik; Banerji, Dahlia; Maurovich-Horvat, Pál; Hoffmann, Udo

2018-04-01

Coronary computed tomography angiography (CTA) has been established as an accurate method to non-invasively assess coronary artery disease (CAD). The proposed 'Coronary Artery Disease Reporting and Data System' (CAD-RADS) may enable standardised reporting of the broad spectrum of coronary CTA findings related to the presence, extent and composition of coronary atherosclerosis. The CAD-RADS classification is a comprehensive tool for summarising findings on a per-patient-basis dependent on the highest-grade coronary artery lesion, ranging from CAD-RADS 0 (absence of CAD) to CAD-RADS 5 (total occlusion of a coronary artery). In addition, it provides suggestions for clinical management for each classification, including further testing and therapeutic options. Despite some limitations, CAD-RADS may facilitate improved communication between imagers and patient caregivers. As such, CAD-RADS may enable a more efficient use of coronary CTA leading to more accurate utilisation of invasive coronary angiograms. Furthermore, widespread use of CAD-RADS may facilitate registry-based research of diagnostic and prognostic aspects of CTA. • CAD-RADS is a tool for standardising coronary CTA reports. • CAD-RADS includes clinical treatment recommendations based on CTA findings. • CAD-RADS has the potential to reduce variability of CTA reports.

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

Directory of Open Access Journals (Sweden)

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.

Nucleotide sequence, transcript mapping, and regulation of the RAD2 gene of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Madura, K.; Prakash, S.

1986-01-01

The authors determined the nucleotide sequence, mapped the 5' and 3' nRNA termini, and examined the regulation of the RAD2 gene of Saccharomyces cerevisiae. A long open reading frame within the RAD2 transcribed region encodes a protein of 1031 amino acids with a calculated molecular weight of 117,847. A disruption of the RAD2 gene that deletes the 78 carboxyl terminal codons results in loss of RAD2 function. The 5' ends of RAD2 mRNA show considerable heterogeneity, mapping 5 to 62 nucleotides upstream of the first ATG codon of the long RAD2 open reading frame. The longest RAD2 transcripts also contain a short open reading frame of 37 codons that precedes and overlaps the 5' end of the long RAD2 open reading frame. The RAD2 3' nRNA end maps 171 nucleotides downstream of the TAA termination codon and 20 nucleotides downstream from a 12-base-pair inverted repeat that might function in transcript termination. Northern blot analysis showed a ninefold increase in steady-state levels of RAD2 mRNA after treatment of yeast cells with UV light. The 5' flanking region of the RAD2 gene contains several direct and inverted repeats and a 44-nuclotide-long purine-rich tract. The sequence T G G A G G C A T T A A found at position - 167 to -156 in the RAD2 gene is similar to at sequence present in the 5' flanking regions of the RAD7 and RAD10 genes

RADTRAN 6/RadCat 6 user guide.

Energy Technology Data Exchange (ETDEWEB)

Weiner, Ruth F.; Hinojosa, Daniel; Heames, Terence John; Farnum, Cathy Ottinger; Kalinina, Elena Arkadievna

2013-09-01

This document provides a detailed discussion and a guide for the use of the RadCat 6.0 Graphical User Interface input file generator for the RADTRAN code, Version 6. RadCat 6.0 integrates the newest analysis capabilities of RADTRAN 6.0, including an economic model, updated loss-of-lead shielding model, a new ingestion dose model, and unit conversion. As of this writing, the RADTRAN version in use is RADTRAN 6.02.

The tumor suppressor homolog in fission yeast, myh1+, displays a strong interaction with the checkpoint gene rad1+

International Nuclear Information System (INIS)

Jansson, Kristina; Warringer, Jonas; Farewell, Anne; Park, Han-Oh; Hoe, Kwang-Lae; Kim, Dong-Uk; Hayles, Jacqueline; Sunnerhagen, Per

2008-01-01

The DNA glycosylase MutY is strongly conserved in evolution, and homologs are found in most eukaryotes and prokaryotes examined. This protein is implicated in repair of oxidative DNA damage, in particular adenine mispaired opposite 7,8-dihydro-8-oxoguanine. Previous investigations in Escherichia coli, fission yeast, and mammalian cells show an association of mutations in MutY homologs with a mutator phenotype and carcinogenesis. Eukaryotic MutY homologs physically associate with several proteins with a role in replication, DNA repair, and checkpoint signaling, specifically the trimeric 9-1-1 complex. In a genetic investigation of the fission yeast MutY homolog, myh1 + , we show that the myh1 mutation confers a moderately increased UV sensitivity alone and in combination with mutations in several DNA repair genes. The myh1 rad1, and to a lesser degree myh1 rad9, double mutants display a synthetic interaction resulting in enhanced sensitivity to DNA damaging agents and hydroxyurea. UV irradiation of myh1 rad1 double mutants results in severe chromosome segregation defects and visible DNA fragmentation, and a failure to activate the checkpoint. Additionally, myh1 rad1 double mutants exhibit morphological defects in the absence of DNA damaging agents. We also found a moderate suppression of the slow growth and UV sensitivity of rhp51 mutants by the myh1 mutation. Our results implicate fission yeast Myh1 in repair of a wider range of DNA damage than previously thought, and functionally link it to the checkpoint pathway

Discovery of a novel dual fungal CYP51/human 5-lipoxygenase inhibitor: implications for anti-fungal therapy.

Directory of Open Access Journals (Sweden)

Eric K Hoobler

Full Text Available We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11 and human 5-lipoxygenase (5-LOX with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM and CYP51 (IC50 = 43 nM in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.

Handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins in transgenic mice

DEFF Research Database (Denmark)

Kragh, Peter M; Pedersen, Christina B; Schmidt, Stine P

2007-01-01

Abstract To investigate the in vivo handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins, three transgenic mouse lines were produced by pronuclear injection of cDNA encoding the wild-type, hSCAD-wt, and two disease causing folding variants hSCAD-319C > T and hSCAD-625G > A....... The transgenic mice were mated with an SCAD-deficient mouse strain (BALB/cByJ) and, in the second generation, three mouse lines were obtained without endogenous SCAD expression but harboring hSCAD-wt, hSCAD-319C > T, and hSCAD-625G > A transgenes, respectively. All three lines had expression of the transgene...... developed for any of the lines transgenic for the hSCAD folding variants. The indicated remarkable efficiency of the mouse protein quality control system in the degradation of SCAD folding variants should be further substantiated and investigated, since it might indicate ways to prevent disease...

BI-RADS: Use in the French radiologic community

International Nuclear Information System (INIS)

Stines, Joseph

2007-01-01

In the United States, BI-RADS TM (Breast Imaging Reporting and Data System) has been set up as a quality assurance system for better communication between professionals and for the follow-up of breast screening programs. It has become a reference in the field of mammographic imaging and has been adopted by several countries throughout the world. It has been translated in French. The aim of this article is to discuss the difficulties in using it in the French radiologic communities. There are few problems with vocabulary excepted for microcalcifications. BI-RADS TM includes a guidance chapter giving some recommendations for using properly the lexicon. Classification of normal breast remains of concern, as it is difficult to evaluate precisely the content of fat and as the final image is also dependant of technical factors. The main difficulties are related to final classification in BI-RADS TM categories as the lexicon does not explicit which mammographic features should be included in the categories from three to five. In France, a table concerning the classification of mammographic abnormalities has been established by the HAS (former ANAES) which represents the highest scientific health authority in France. There are no major problems for using the BI-RADS TM for US and MRI. BI-RADS TM is suitable for different categories of women and for male and training has an important impact on acceptance and proper use of the lexicon

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.

Purification of human growth hormone. Isolation of the 22k variant. Assays of biological and radioimmunological activities

International Nuclear Information System (INIS)

Camillo, M.A.P.

1985-01-01

An extraction method for hGH from frozen human pituitaries with high yield: 8,7 mg per processed gland, is described. Isoelectric focusing studies made it possible to isolate and integral variant of the total preparation of hGH. After liophylization the protein was analysed in polyacrylamide gel electrophoresis showing only one protein band that corresponds to the 22K variant. (M.A.C.) [pt

Interdependence of the rad50 hook and globular domain functions.

Science.gov (United States)

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.

Differential hRad17 expression by histologic subtype of ovarian cancer

Directory of Open Access Journals (Sweden)

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.

Reorganization of Damaged Chromatin by the Exchange of Histone Variant H2A.Z-2

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Nishibuchi, Ikuno [Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima (Japan); Department of Radiation Oncology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima (Japan); Department of Radiation Oncology, Hiroshima Prefectural Hospital, Hiroshima (Japan); Suzuki, Hidekazu; Kinomura, Aiko; Sun, Jiying; Liu, Ning-Ang [Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima (Japan); Horikoshi, Yasunori [Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima (Japan); Research Center for Mathematics of Chromatin Live Dynamics, Hiroshima University, Hiroshima (Japan); Shima, Hiroki [Department of Biochemistry, Graduate School of Medical Sciences, Tohoku University, Sendai (Japan); Kusakabe, Masayuki; Harata, Masahiko [Laboratory of Molecular Biology, Graduate School of Agricultural Science, Tohoku University, Sendai (Japan); Fukagawa, Tatsuo [Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima (Japan); Ikura, Tsuyoshi [Laboratory of Chromatin Regulatory Network, Department of Mutagenesis, Radiation Biology Center, Kyoto University, Kyoto (Japan); Ishida, Takafumi [Department of Cardiovascular Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima (Japan); Nagata, Yasushi [Department of Radiation Oncology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima (Japan); Tashiro, Satoshi, E-mail: ktashiro@hiroshima-u.ac.jp [Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima (Japan); Research Center for Mathematics of Chromatin Live Dynamics, Hiroshima University, Hiroshima (Japan)

2014-07-15

Purpose: The reorganization of damaged chromatin plays an important role in the regulation of the DNA damage response. A recent study revealed the presence of 2 vertebrate H2A.Z isoforms, H2A.Z-1 and H2A.Z-2. However, the roles of the vertebrate H2A.Z isoforms are still unclear. Thus, in this study we examined the roles of the vertebrate H2A.Z isoforms in chromatin reorganization after the induction of DNA double-strand breaks (DSBs). Methods and Materials: To examine the dynamics of H2A.Z isoforms at damaged sites, we constructed GM0637 cells stably expressing each of the green fluorescent protein (GFP)-labeled H2A.Z isoforms, and performed fluorescence recovery after photobleaching (FRAP) analysis and inverted FRAP analysis in combination with microirradiation. Immunofluorescence staining using an anti-RAD51 antibody was performed to study the kinetics of RAD51 foci formation after 2-Gy irradiation of wild-type (WT), H2A.Z-1- and H2A.Z-2-deficient DT40 cells. Colony-forming assays were also performed to compare the survival rates of WT, H2A.Z-1-, and H2A.Z-2-deficient DT40 cells with control, and H2A.Z-1- and H2A.Z-2-depleted U2OS cells after irradiation. Results: FRAP analysis revealed that H2A.Z-2 was incorporated into damaged chromatin just after the induction of DSBs, whereas H2A.Z-1 remained essentially unchanged. Inverted FRAP analysis showed that H2A.Z-2 was released from damaged chromatin. These findings indicated that H2A.Z-2 was exchanged at DSB sites immediately after the induction of DSBs. RAD51 focus formation after ionizing irradiation was disturbed in H2A.Z-2-deficient DT40 cells but not in H2A.Z-1-deficient cells. The survival rate of H2A.Z-2-deficient cells after irradiation was lower than those of WT and H2A.Z-1- DT40 cells. Similar to DT40 cells, H2A.Z-2-depleted U2OS cells were also radiation-sensitive compared to control and H2A.Z-1-depleted cells. Conclusions: We found that vertebrate H2A.Z-2 is involved in the regulation of the DNA

Reorganization of Damaged Chromatin by the Exchange of Histone Variant H2A.Z-2

International Nuclear Information System (INIS)

Nishibuchi, Ikuno; Suzuki, Hidekazu; Kinomura, Aiko; Sun, Jiying; Liu, Ning-Ang; Horikoshi, Yasunori; Shima, Hiroki; Kusakabe, Masayuki; Harata, Masahiko; Fukagawa, Tatsuo; Ikura, Tsuyoshi; Ishida, Takafumi; Nagata, Yasushi; Tashiro, Satoshi

2014-01-01

Purpose: The reorganization of damaged chromatin plays an important role in the regulation of the DNA damage response. A recent study revealed the presence of 2 vertebrate H2A.Z isoforms, H2A.Z-1 and H2A.Z-2. However, the roles of the vertebrate H2A.Z isoforms are still unclear. Thus, in this study we examined the roles of the vertebrate H2A.Z isoforms in chromatin reorganization after the induction of DNA double-strand breaks (DSBs). Methods and Materials: To examine the dynamics of H2A.Z isoforms at damaged sites, we constructed GM0637 cells stably expressing each of the green fluorescent protein (GFP)-labeled H2A.Z isoforms, and performed fluorescence recovery after photobleaching (FRAP) analysis and inverted FRAP analysis in combination with microirradiation. Immunofluorescence staining using an anti-RAD51 antibody was performed to study the kinetics of RAD51 foci formation after 2-Gy irradiation of wild-type (WT), H2A.Z-1- and H2A.Z-2-deficient DT40 cells. Colony-forming assays were also performed to compare the survival rates of WT, H2A.Z-1-, and H2A.Z-2-deficient DT40 cells with control, and H2A.Z-1- and H2A.Z-2-depleted U2OS cells after irradiation. Results: FRAP analysis revealed that H2A.Z-2 was incorporated into damaged chromatin just after the induction of DSBs, whereas H2A.Z-1 remained essentially unchanged. Inverted FRAP analysis showed that H2A.Z-2 was released from damaged chromatin. These findings indicated that H2A.Z-2 was exchanged at DSB sites immediately after the induction of DSBs. RAD51 focus formation after ionizing irradiation was disturbed in H2A.Z-2-deficient DT40 cells but not in H2A.Z-1-deficient cells. The survival rate of H2A.Z-2-deficient cells after irradiation was lower than those of WT and H2A.Z-1- DT40 cells. Similar to DT40 cells, H2A.Z-2-depleted U2OS cells were also radiation-sensitive compared to control and H2A.Z-1-depleted cells. Conclusions: We found that vertebrate H2A.Z-2 is involved in the regulation of the DNA

Expression of Human CAR Splicing Variants in BAC-Transgenic Mice

OpenAIRE

Zhang, Yu-Kun Jennifer; Lu, Hong; Klaassen, Curtis D.

2012-01-01

The nuclear receptor constitutive androstane receptor (CAR) is a key regulator for drug metabolism in liver. Human CAR (hCAR) transcripts are subjected to alternative splicing. Some hCAR splicing variants (SVs) have been shown to encode functional proteins by reporter assays. However, in vivo research on the activity of these hCAR SVs has been impeded by the absence of a valid model. This study engineered an hCAR-BAC-transgenic (hCAR-TG) mouse model by integrating the 8.5-kbp hCAR gene as wel...

NF-κB and androgen receptor variant expression correlate with human BPH progression.

Science.gov (United States)

Austin, David C; Strand, Douglas W; Love, Harold L; Franco, Omar E; Jang, Alex; Grabowska, Magdalena M; Miller, Nicole L; Hameed, Omar; Clark, Peter E; Fowke, Jay H; Matusik, Robert J; Jin, Ren J; Hayward, Simon W

2016-04-01

Benign prostatic hyperplasia (BPH) is a common, chronic progressive disease. Inflammation is associated with prostatic enlargement and resistance to 5α-reductase inhibitor (5ARI) therapy. Activation of the nuclear factor-kappa B (NF-κB) pathway is linked to both inflammation and ligand-independent prostate cancer progression. NF-κB activation and androgen receptor variant (AR-V) expression were quantified in transition zone tissue samples from patients with a wide range of AUASS from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-κB activity. The effects on AR full length (AR-FL) and androgen-independent AR-V expression as well as cellular growth and differentiation were assessed. Canonical NF-κB signaling was found to be upregulated in late versus early stage BPH, and to be strongly associated with non-insulin dependent diabetes mellitus. Elevated expression of AR-variant 7 (AR-V7), but not other AR variants, was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS and TRUS volume. Forced activation of canonical NF-κB in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-κB and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment. Activation of NF-κB and AR-V7 in the prostate is associated with increased disease severity. AR-V7 expression is inducible in human prostate cells by forced activation of NF-κB resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may become resistant to 5ARI therapy. �

γ radiation dosimetry in Mega rad range using sugar solution

International Nuclear Information System (INIS)

Venkataramani, R.; Mehta, S.K.; Soman, S.D.

1976-01-01

The formation of malonaldehyde under γ irradiation of solid sucrose and aqueous sucrose, fructose and arabinose solutions has been studied in the Mega rad range. Malonaldehyde (MA) concentration was estimated spectrophotometrically after complexing with 2-thio-barbituric acid. The effect of free radical scavengers (KI and N 2 O) on the yield of MA was investigated. Of the systems studied a 5% aqueous sucrose solution gave a proportional response of MA formation with dose in 0.2 to 5 Mega rad range. A 5% aqueous solution of sucrose prepared from sucrose irradiated in solid state also gave a smooth response of MA yield with dose from 8 to 30 Mega rad. The aqueous and solid sucrose systems together can be conveniently used for dosimetry in the range of 0.2 30 Mega rad. (author)

gamma. radiation dosimetry in Mega rad range using sugar solution

Energy Technology Data Exchange (ETDEWEB)

Venkataramani, R; Mehta, S K; Soman, S D [Bhabha Atomic Research Centre, Bombay (India). Health Physics Div.

1976-09-01

The formation of malonaldehyde under ..gamma.. irradiation of solid sucrose and aqueous sucrose, fructose and arabinose solutions has been studied in the Mega rad range. Malonaldehyde (MA) concentration was estimated spectrophotometrically after complexing with 2-thio-barbituric acid. The effect of free radical scavengers (KI and N/sub 2/O) on the yield of MA was investigated. Of the systems studied a 5% aqueous sucrose solution gave a proportional response of MA formation with dose in 0.2 to 5 Mega rad range. A 5% aqueous solution of sucrose prepared from sucrose irradiated in solid state also gave a smooth response of MA yield with dose from 8 to 30 Mega rad. The aqueous and solid sucrose systems together can be conveniently used for dosimetry in the range of 0.2 30 Mega rad.

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.)

Characterization of new radiation-sensitive mutant, Escherichia coli K-12 radC102

International Nuclear Information System (INIS)

Felzenszwalb, I.; Sargentini, N.J.; Smith, K.C.

1984-01-01

A new radiation-sensitive mutant, radC, has been isolated. The radC gene is located at 81.0 min on the Escherichia coli K-12 linkage map. The radC mutation sensitized cells to uv radiation, but unlike most DNA repair mutations, sensitization to X rays was observed only for rich medium-grown cells. For cells grown in rich medium, the radC mutant was normal for γ radiation mutagenesis, but showed less uv-radiation mutagenesis than the wild-type strain; it showed normal amount of X- and uv-radiation-induced DNA degradation, and it wasapprox. =60% deficient in recombination ability. The radC strain was normal for host cell reactivation of γ and uv-irradiated bacteriophage the radC mutation did not sensitize a recA strain, but did sensitize a radA and a polA strain to X and uv radiation and a uvrA strain to uv radiation. Therefore, it is suggested that the radC gene product plays a role in the growth medium-dependent, recA gene-dependent repair of DNA single-strand breaks after X irradiation, and in postreplication repair after uv irradiation

Comparative analysis of metastasis variants derived from human prostate carcinoma cells: roles in intravasation of VEGF-mediated angiogenesis and uPA-mediated invasion

DEFF Research Database (Denmark)

Conn, Erin M; Bøtkjær, Kenneth Alrø; Kupriyanova, Tatyana A

2009-01-01

To analyze the process of tumor cell intravasation, we used the human tumor-chick embryo spontaneous metastasis model to select in vivo high (PC-hi/diss) and low (PC-lo/diss) disseminating variants from the human PC-3 prostate carcinoma cell line. These variants dramatically differed in their int...

Coordination of BRCA1/BARD1- and MRE11/RAD50/NBS1-Dependent DNA Transactions in Breast Tumor Suppression

Science.gov (United States)

2011-07-01

human DNA repair proteins at a unique double-strand break in vivo, EMBO J 25, 222-231. 19. Berkovich, E., Monnat, R. J., Jr., and Kastan, M. B...structures around DNA as SMC complexes do. Rad50 exhibits ATPase activity in vitro, which is required for DNA repair and meiosis (3, 57). The rad50S...151). Exo1 expression is induced dur- ing meiosis , suggesting a role in meiotic DSB resection (149). Studies in the dmc1 mutant, which exhibits hyper

Investigations into the binding affinities of different human 5-HT4 receptor splice variants.

Science.gov (United States)

Irving, Helen R; Tochon-Danguy, Nathalie; Chinkwo, Kenneth A; Li, Jian G; Grabbe, Carmen; Shapiro, Marina; Pouton, Colin W; Coupar, Ian M

2010-01-01

This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT(4) receptor splice variants [h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g)] display preferential affinities towards agonists. The agonists selected on the basis of chemical diversity and clinical relevance were: 5-HT4 benzamides, renzapride, zacopride and prucalopride; the benzimidazolones, DAU 6236 and BIMU 1; the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pKi: 7.38-7.91) > or = Y-36912 (pKi: 7.03-7.85) = BIMU 1 (pKi: 6.92-7.78) > or = DAU 6236 (pKi: 6.79-7.99) > or = 5-HT (pKi: 5.82-7.29) > or = 5-MeOT (pKi: 5.64-6.83) > or = renzapride (pKi: 4.85-5.56). We obtained affinity values for the 5-HT4(b), (d) and (g) variants for RS67333 (pKi: 7:48-8.29), prucalopride (pKi: 6.86-7.37) and zacopride (pKi: 5.88-7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically. 2010 S. Karger AG, Basel.

The rem mutations in the ATP-binding groove of the Rad3/XPD helicase lead to Xeroderma pigmentosum-Cockayne syndrome-like phenotypes.

Science.gov (United States)

Herrera-Moyano, Emilia; Moriel-Carretero, María; Montelone, Beth A; Aguilera, Andrés

2014-12-01

The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes). We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them. Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient. Interestingly, the mutations analyzed reside at the ATP-binding groove of Rad3 and in vivo experiments reveal a gain of DNA affinity upon damage of the mutant Rad3 proteins. Since mutations at the ATP-binding groove of XPD in humans are present in the Xeroderma pigmentosum-Cockayne Syndrome (XP-CS), we recreated rem mutations in human cells, and found that these are XP-CS-like. We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters. This conditions NER efficiency and transcription resumption after damage, which in human cells would explain the XP-CS phenotype, opening new perspectives to understand the molecular basis of the role of XPD in human disease.

Evidence for a Rad18-independent frameshift mutagenesis pathway in human cell-free extracts.

Directory of Open Access Journals (Sweden)

Régine Janel-Bintz

Full Text Available Bypass of replication blocks by specialized DNA polymerases is crucial for cell survival but may promote mutagenesis and genome instability. To gain insight into mutagenic sub-pathways that coexist in mammalian cells, we examined N-2-acetylaminofluorene (AAF-induced frameshift mutagenesis by means of SV40-based shuttle vectors containing a single adduct. We found that in mammalian cells, as previously observed in E. coli, modification of the third guanine of two target sequences, 5'-GGG-3' (3G and 5'-GGCGCC-3' (NarI site, induces -1 and -2 frameshift mutations, respectively. Using an in vitro assay for translesion synthesis, we investigated the biochemical control of these events. We showed that Pol eta, but neither Pol iota nor Pol zeta, plays a major role in the frameshift bypass of the AAF adduct located in the 3G sequence. By complementing PCNA-depleted extracts with either a wild-type or a non-ubiquitinatable form of PCNA, we found that this Pol eta-mediated pathway requires Rad18 and ubiquitination of PCNA. In contrast, when the AAF adduct is located within the NarI site, TLS is only partially dependent upon Pol eta and Rad18, unravelling the existence of alternative pathways that concurrently bypass this lesion.

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

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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.

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

DEFF Research Database (Denmark)

Sotiriou, Sotirios K; Kamileri, Irene; Lugli, Natalia

2016-01-01

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...... RAD52 facilitates repair of collapsed DNA replication forks in cancer cells....

A uniform system for mammographic reporting BI-RADS

International Nuclear Information System (INIS)

Masroor, I.; Ahmad, M. N.; Sheikh, M. Y.

2001-01-01

Breast image reporting and data system (BI-RADS) is a new system of categorizing and reporting mammographs and mammographic findings recommended by American College of Radiology. The importance of BI-RADS and final assessment categories are discussed. The purpose is to introduce the above-mentioned mammographic reporting system so that it becomes a standard terminology among the medical personnel, involved in the diagnosis and management of breast diseases. (author)

Split dose recovery studies using homologous recombination deficient gene knockout chicken B lymphocyte cells

International Nuclear Information System (INIS)

Rao, B.S.S.; Tano, Kaori; Utsumi, Hiroshi; Takeda, Shunichi

2007-01-01

To understand the role of proteins involved in double strand breaks (DSB) repair modulating sublethal damage (SLD) recovery, chicken B lymphoma (DT 40) cell lines either proficient or deficient in RAD52, XRCC2, XRCC3, RAD51C and RAD51D were subjected to fractionated irradiation and their survival curves charted. Survival curves of both WT DT40 and RAD52 -/- cells had a big shoulder while all the other cells exhibited small shoulders. However, at the higher doses of radiation, RAD51C -/- cells displayed hypersensitivity comparable to the data obtained for the homologous recombination deficient RAD54 -/- cells. Repair of SLD was measured as an increase in survival after a split dose irradiation with an interval of incubation between the radiation doses. All the cell lines (parental DT40 and genetic knockout cell lines viz., RAD52 -/- , XRCC2 -/- XRCC3 -/- RAD51C -/- and RAD51D -/- ) used in this study demonstrated a typical split-dose recovery capacity with a specific peak, which varied depending on the cell type. The maximum survival of WT DT40 and RAD52 -/- was reached at about 1-2 hours after the first dose of radiation and then decreased to a minimum thereafter (5 h). The increase in the survival peaked once again by about 8 hours. The survival trends observed in XRCC2 -/- , XRCC3 -/- , RAD51C -/- and RAD51D -/- knockout cells were also similar, except for the difference in the initial delay of a peak survival for RAD51D -/- and lower survival ratios. The second phase of increase in the survival in these cell lines was much slower in XRCC2 -/- , XRCC3 -/- , RAD51C -/- nd RAD51D -/- and further delayed when compared with that of RAD52 -/- and parental DT40 cells suggesting a dependence on their cell cycle kinetics. This study demonstrates that the participation of RAD52, XRCC2, XRCC3, RAD51C and RAD51D in the DSB repair via homologous recombination is of less importance in comparison to RAD54, as RAD54 deficient cells demonstrated complete absence of SLD recovery

Effect of 680 rad whole-body irradiation of a human fetus, 27 years afterwards

International Nuclear Information System (INIS)

Kallinger, W.; Granninger, W.

1982-01-01

In 1953 a 38-year-old pregnant woman was given radiation treatment with Ra-226 due to a vaginal carcinoma. The radiation field was reproduced by means of a computer program. It was proved that the fetus was exposed to radiation doses of 50-2000 rad. Despite this high dose no abnormalities were found during clinical examination of the now 27-year-old man who has already fathered a healthy son. (orig.) [de

Dihydrocoumarin, an HDAC Inhibitor, Increases DNA Damage Sensitivity by Inhibiting Rad52

Directory of Open Access Journals (Sweden)

Chin-Chuan Chen

2017-12-01

Full Text Available Effective DNA repair enables cancer cells to survive DNA damage induced by chemotherapeutic or radiotherapeutic treatments. Therefore, inhibiting DNA repair pathways is a promising therapeutic strategy for increasing the efficacy of such treatments. In this study, we found that dihydrocoumarin (DHC, a flavoring agent, causes deficiencies in double-stand break (DSB repair and prolonged DNA damage checkpoint recovery in yeast. Following DNA damage, Rad52 recombinase was revealed to be inhibited by DHC, which results in deficiencies in DSB repair and prolonged DNA damage checkpoint recovery. The deletion of RPD3, a class I histone deacetylase (HDAC, was found to mimic DHC-induced suppression of Rad52 expression, suggesting that the HDAC inhibitor activity of DHC is critical to DSB repair and DNA damage sensitivity. Overall, our findings delineate the regulatory mechanisms of DHC in DSB repair and suggest that it might potentially be used as an inhibitor of the DNA repair pathway in human cells.

Validation of the fifth edition BI-RADS ultrasound lexicon with comparison of fourth and fifth edition diagnostic performance using video clips

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Jung Hyun Yoon

2016-10-01

Full Text Available Purpose The aim of this study was to evaluate the positive predictive value (PPV and the diagnostic performance of the ultrasonographic descriptors in the fifth edition of BI-RADS, comparing with the fourth edition using video clips. Methods From September 2013 to July 2014, 80 breast masses in 74 women (mean age, 47.5±10.7 years from five institutions of the Korean Society of Breast Imaging were included. Two radiologists individually reviewed the static and video images and analyzed the images according to the fourth and fifth edition of BI-RADS. The PPV of each descriptor was calculated and diagnostic performances between the fourth and fifth editions were compared. Results Of the 80 breast masses, 51 (63.8% were benign and 29 (36.2% were malignant. Suspicious ultrasonographic features such as irregular shape, non-parallel orientation, angular or spiculated margins, and combined posterior features showed higher PPV in both editions (all P0.05. The area under the receiver operating characteristics curve was higher in the fourth edition (0.708 to 0.690, without significance (P=0.416. Conclusion The fifth edition of the BI-RADS ultrasound lexicon showed comparable performance to the fourth edition and can be useful in the differential diagnosis of breast masses using ultrasonography.

Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

Energy Technology Data Exchange (ETDEWEB)

Vogelsang, Matjaz; Comino, Aleksandra; Zupanec, Neja [Department for Biosynthesis and Biotransformation, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia); Hudler, Petra [Medical Center for Molecular Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana (Slovenia); Komel, Radovan [Department for Biosynthesis and Biotransformation, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia); Medical Center for Molecular Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana (Slovenia)

2009-10-28

Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells. Yeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested. The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic. Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.

Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

Directory of Open Access Journals (Sweden)

Hudler Petra

2009-10-01

Full Text Available Abstract Background Loss of DNA mismatch repair (MMR in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC. Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells. Methods Yeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested. Results The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic. Conclusion Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.

Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

International Nuclear Information System (INIS)

Vogelsang, Matjaz; Comino, Aleksandra; Zupanec, Neja; Hudler, Petra; Komel, Radovan

2009-01-01

Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells. Yeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested. The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic. Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene

CLEVER: Clique-Enumerating Variant Finder

NARCIS (Netherlands)

Marschall, T.; Costa, I.; Canzar, S.; bauer, m; Klau, G.W.; Schliep, A.; Schönhuth, A.

2012-01-01

Motivation: Next-generation sequencing techniques have facilitated a large-scale analysis of human genetic variation. Despite the advances in sequencing speed, the computational discovery of structural variants is not yet standard. It is likely that many variants have remained undiscovered in most

4-channel rad-hard delay generation ASIC with 1ns timing resolution for LHC

International Nuclear Information System (INIS)

Toifl, T.; Moreira, P.; Marchioro, A.; Vari, R.

1999-01-01

An ASIC was developed to precisely delay digital signals within the range of 0--24ns in steps of 1ns. To obtain well defined delay values independent of variations in process, supply voltage and temperature, four independent delay channels are controlled by a common control voltage derived from a delay-locked loop (DLL), which is synchronized to an external 40 MHz clock signal. The delay values of the four signal channels and the clock channel can be individually programmed via an I 2 C interface. Due to an automatic reset logic the chip does not need an external reset signal. A first version of the chip was developed in a non-rad-hard 0.8 microm technology and the successful prototype was then transferred to a radiation hard process (DMILL). Measurement results for both chip variants will be presented

The tumor suppressor homolog in fission yeast, myh1{sup +}, displays a strong interaction with the checkpoint gene rad1{sup +}

Energy Technology Data Exchange (ETDEWEB)

Jansson, Kristina; Warringer, Jonas; Farewell, Anne [Department of Cell and Molecular Biology, Lundberg Laboratory, Goeteborg University, P.O. Box 462, Goeteborg SE-405 30 (Sweden); Park, Han-Oh [Bioneer Corporation, 49-3, Munpyeong-dong, Daedeok-gu, Daejon 306-220 (Korea, Republic of); Hoe, Kwang-Lae; Kim, Dong-Uk [Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Daejeon (Korea, Republic of); Hayles, Jacqueline [Cell Cycle Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln' s Inn Fields, London WC2A 3PX (United Kingdom); Sunnerhagen, Per [Department of Cell and Molecular Biology, Lundberg Laboratory, Goeteborg University, P.O. Box 462, Goeteborg SE-405 30 (Sweden)], E-mail: per.sunnerhagen@cmb.gu.se

2008-09-26

The DNA glycosylase MutY is strongly conserved in evolution, and homologs are found in most eukaryotes and prokaryotes examined. This protein is implicated in repair of oxidative DNA damage, in particular adenine mispaired opposite 7,8-dihydro-8-oxoguanine. Previous investigations in Escherichia coli, fission yeast, and mammalian cells show an association of mutations in MutY homologs with a mutator phenotype and carcinogenesis. Eukaryotic MutY homologs physically associate with several proteins with a role in replication, DNA repair, and checkpoint signaling, specifically the trimeric 9-1-1 complex. In a genetic investigation of the fission yeast MutY homolog, myh1{sup +}, we show that the myh1 mutation confers a moderately increased UV sensitivity alone and in combination with mutations in several DNA repair genes. The myh1 rad1, and to a lesser degree myh1 rad9, double mutants display a synthetic interaction resulting in enhanced sensitivity to DNA damaging agents and hydroxyurea. UV irradiation of myh1 rad1 double mutants results in severe chromosome segregation defects and visible DNA fragmentation, and a failure to activate the checkpoint. Additionally, myh1 rad1 double mutants exhibit morphological defects in the absence of DNA damaging agents. We also found a moderate suppression of the slow growth and UV sensitivity of rhp51 mutants by the myh1 mutation. Our results implicate fission yeast Myh1 in repair of a wider range of DNA damage than previously thought, and functionally link it to the checkpoint pathway.

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

Science.gov (United States)

Williams, Lindsey N; Marjavaara, Lisette; Knowels, Gary M; Schultz, Eric M; Fox, Edward J; Chabes, Andrei; Herr, Alan J

2015-05-12

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

Extracting BI-RADS Features from Portuguese Clinical Texts.

Science.gov (United States)

Nassif, Houssam; Cunha, Filipe; Moreira, Inês C; Cruz-Correia, Ricardo; Sousa, Eliana; Page, David; Burnside, Elizabeth; Dutra, Inês

2012-01-01

In this work we build the first BI-RADS parser for Portuguese free texts, modeled after existing approaches to extract BI-RADS features from English medical records. Our concept finder uses a semantic grammar based on the BIRADS lexicon and on iterative transferred expert knowledge. We compare the performance of our algorithm to manual annotation by a specialist in mammography. Our results show that our parser's performance is comparable to the manual method.

El radón: ¿riesgo para la salud?

Directory of Open Access Journals (Sweden)

Juan Miguel Barros Dios

2011-12-01

Full Text Available El radón (Rn222 es un gas noble radiactivo que procede directamente del radio (Ra226 cuando este emite una partícula alfa (dos protones y dos neutrones o núcleo de helio, y que a su vez se transforma en otro elemento radiactivo (Po218 al desprenderse de otra partícula alfa. Desde hace varias décadas se conoce su efecto como factor de riesgo del cáncer primario pulmonar, primero en mineros del uranio y posteriormente en la población general expuesta al radón residencial en hogares construidos sobre suelos de rocas ricas en uranio (U238, elemento inicial de la cadena de degradación radiactiva de la que procede el radón. Áreas geológicamente constituidas por granitos o pizarras, como son las de gran parte de Galicia y todo el noroeste y oeste de la península ibérica, han sido catalogadas como de alto riesgo de exhalación de radón al interior de edificios y domicilios. En numerosos países de América y Europa existen desde hace varios lustros, políticas de prevención del cáncer pulmonar en aquellas zonas de riesgo basadas en programas de reducción de radón en los domicilios y edificios públicos. Desde finales de los años 80, la radiación alfa procedente del radón y sus descen- dientes de vida media corta han sido clasificados como agentes cancerígenos por la Internacional Agency of Research on Cancer (Lyon, 1988 y el Nacional Research Council (BEIR IV, 1988, constituyendo la segunda causa de cáncer pulmonar después del tabaco, y responsable del 10 al 15 % de todas las muertes por esa neoplasia. Estudios realizados en Galicia confirman esta evidencia, con riesgos de 2 a 3 en expuestos a concentraciones del gas en domicilios y la responsabilidad directa del 9% de todos los casos de cáncer pulmonar del área estudiada y una interacción radón/tabaco que multiplica por 45 el riesgo.

Contribution of Germline Mutations in the RAD51B>, RAD51C, and RAD51D Genes to Ovarian Cancer in the Population

DEFF Research Database (Denmark)

Song, Honglin; Dicks, Ed; Ramus, Susan J

2015-01-01

Screening Study (UK_FOCSS) after quality-control analysis. RESULTS: In the case-control study, we identified predicted deleterious mutations in 28 EOC cases (0.82%) compared with three controls (0.11%; P

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.

A robust approach to identifying tissue-specific gene expression regulatory variants using personalized human induced pluripotent stem cells.

Directory of Open Access Journals (Sweden)

Je-Hyuk Lee

2009-11-01

Full Text Available Normal variation in gene expression due to regulatory polymorphisms is often masked by biological and experimental noise. In addition, some regulatory polymorphisms may become apparent only in specific tissues. We derived human induced pluripotent stem (iPS cells from adult skin primary fibroblasts and attempted to detect tissue-specific cis-regulatory variants using in vitro cell differentiation. We used padlock probes and high-throughput sequencing for digital RNA allelotyping and measured allele-specific gene expression in primary fibroblasts, lymphoblastoid cells, iPS cells, and their differentiated derivatives. We show that allele-specific expression is both cell type and genotype-dependent, but the majority of detectable allele-specific expression loci remains consistent despite large changes in the cell type or the experimental condition following iPS reprogramming, except on the X-chromosome. We show that our approach to mapping cis-regulatory variants reduces in vitro experimental noise and reveals additional tissue-specific variants using skin-derived human iPS cells.

Radiomic modeling of BI-RADS density categories

Science.gov (United States)

Wei, Jun; Chan, Heang-Ping; Helvie, Mark A.; Roubidoux, Marilyn A.; Zhou, Chuan; Hadjiiski, Lubomir

2017-03-01

Screening mammography is the most effective and low-cost method to date for early cancer detection. Mammographic breast density has been shown to be highly correlated with breast cancer risk. We are developing a radiomic model for BI-RADS density categorization on digital mammography (FFDM) with a supervised machine learning approach. With IRB approval, we retrospectively collected 478 FFDMs from 478 women. As a gold standard, breast density was assessed by an MQSA radiologist based on BI-RADS categories. The raw FFDMs were used for computerized density assessment. The raw FFDM first underwent log-transform to approximate the x-ray sensitometric response, followed by multiscale processing to enhance the fibroglandular densities and parenchymal patterns. Three ROIs were automatically identified based on the keypoint distribution, where the keypoints were obtained as the extrema in the image Gaussian scale-space. A total of 73 features, including intensity and texture features that describe the density and the parenchymal pattern, were extracted from each breast. Our BI-RADS density estimator was constructed by using a random forest classifier. We used a 10-fold cross validation resampling approach to estimate the errors. With the random forest classifier, computerized density categories for 412 of the 478 cases agree with radiologist's assessment (weighted kappa = 0.93). The machine learning method with radiomic features as predictors demonstrated a high accuracy in classifying FFDMs into BI-RADS density categories. Further work is underway to improve our system performance as well as to perform an independent testing using a large unseen FFDM set.

Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

Directory of Open Access Journals (Sweden)

O Rickard Nilsson

Full Text Available Recent studies indicate that defective activity of complement factor H (FH is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

RadCon: Parameter report focusing on Tropical and Subtropical environments in Australia

International Nuclear Information System (INIS)

Domel, R.U.; Crawford, J; Harris, F.; Twining, J.

2000-09-01

Models to simulate the transfer of radionuclides through air, water and terrestrial ecosystems have been developed and used regularly over the last 20 years. RadCon was developed as a simple model to assess the radiological consequences, as dose, to humans of short-term depositions of radionuclides. Internal exposure via inhalation and ingestion are included in this model as well as external exposure from the passing cloud (cloud shine) and from radioactivity deposited on the ground (ground shine).Initially, the RadCon model will deal with the Australian and South East Asian region but flexibility has been incorporated into the design to allow application in other regions. In a manner similar to a geographic information system, the display of input and output data allows quick access to the results, both numerically and graphically. Coloured concentration gradients, stepping through time, are superimposed on the area of interest to present atmospheric and ground concentrations of the radionuclides and, after the calculations, human dose. The model has portability across computer platforms.This report summarises the parameters and some of the transfer factors underlying the calculations. While the focus was on the tropical and subtropical regions in Australia, for many parameters the values may only have been available for temperate regions, in which case this has been used as the default value. The parameter flexibility is a major aspect of this model and a report to describe the editor has been written (Crawford and Domel, ANSTO/M-129). The aim of this document is to chronologically expand on the formulae and parameter tables presented in the Technical Guide (Crawford et al., ANSTO/E-744, May 2000). The two documents should be read together with the Technical Guide presenting the mathematical computations and this report presenting some of the input values used to calculate the end-point results from the computations. A User Guide to assist in the implementation

A high-quality human reference panel reveals the complexity and distribution of genomic structural variants

NARCIS (Netherlands)

Hehir-Kwa, J.Y.; Marschall, T.; Kloosterman, W.P.; Francioli, L.C.; Baaijens, J.A.; Dijkstra, L.J.; Abdellaoui, A.; Koval, V.; Thung, D.T.; Wardenaar, R.; Renkens, I.; Coe, B.P.; Deelen, P.; de Ligt, J.; Lameijer, E.W.; Dijk, F.; Hormozdiari, F.; Uitterlinden, A.G.; van Duijn, C.M.; Eichler, E.E.; Bakker, P.I.W.; Swertz, M.A.; Wijmenga, C.; van Ommen, G.J.B; Slagboom, P.E.; Boomsma, D.I.; Schönhuth, A.; Ye, K.; Guryev, V.

2016-01-01

Structural variation (SV) represents a major source of differences between individual human genomes and has been linked to disease phenotypes. However, the majority of studies provide neither a global view of the full spectrum of these variants nor integrate them into reference panels of genetic

RadNet Air Quality (Deployable) Data

Data.gov (United States)

U.S. Environmental Protection Agency — RadNet Deployable Monitoring is designed to collect radiological and meteorological information and data asset needed to establish the impact of radiation levels on...

The AtRAD21.1 and AtRAD21.3 Arabidopsis cohesins play a synergistic role in somatic DNA double strand break damage repair

Czech Academy of Sciences Publication Activity Database

da Costa-Nunes, J.A.; Capitao, C.; Kozák, Jaroslav; Costa-Nunes, P.; Ducasa, G.M.; Pontes, O.; Angelis, Karel

2014-01-01

Roč. 14, DEC 16 2014 (2014) ISSN 1471-2229 R&D Projects: GA MŠk(CZ) LD13006; GA ČR GA13-06595S Institutional support: RVO:61389030 Keywords : Arabidopsis * AtRAD21.1 * AtRAD21.3 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.813, year: 2014

Genetic variants in DNA double-strand break repair genes and risk of salivary gland carcinoma: a case-control study.

Directory of Open Access Journals (Sweden)

Li Xu

Full Text Available DNA double strand break (DSB repair is the primary defense mechanism against ionizing radiation-induced DNA damage. Ionizing radiation is the only established risk factor for salivary gland carcinoma (SGC. We hypothesized that genetic variants in DSB repair genes contribute to individual variation in susceptibility to SGC. To test this hypothesis, we conducted a case-control study in which we analyzed 415 single nucleotide polymorphisms (SNPs in 45 DSB repair genes in 352 SGC cases and 598 controls. Multivariate logistic regression analysis was performed to calculate odds ratios (ORs and 95% confidence intervals (CIs. Rs3748522 in RAD52 and rs13180356 in XRCC4 were significantly associated with SGC after Bonferroni adjustment; ORs (95% CIs for the variant alleles of these SNPs were 1.71 (1.40-2.09, P = 1.70 × 10(-7 and 0.58 (0.45-0.74, P = 2.00 × 10(-5 respectively. The genetic effects were modulated by histological subtype. The association of RAD52-rs3748522 with SGC was strongest for mucoepidermoid carcinoma (OR = 2.21, 95% CI: 1.55-3.15, P = 1.25 × 10(-5, n = 74, and the association of XRCC4-rs13180356 with SGC was strongest for adenoid cystic carcinoma (OR = 0.60, 95% CI: 0.42-0.87, P = 6.91 × 10(-3, n = 123. Gene-level association analysis revealed one gene, PRKDC, with a marginally significant association with SGC risk in non-Hispanic whites. To our knowledge, this study is the first to comprehensively evaluate the genetic effect of DSB repair genes on SGC risk. Our results indicate that genetic variants in the DSB repair pathways contribute to inter-individual differences in susceptibility to SGC and show that the impact of genetic variants differs by histological subtype. Independent studies are warranted to confirm these findings.

Tsuruga: 1 ton, some 10 mCi of rad-liquid leaks out to the sea, no environmental and human costs

International Nuclear Information System (INIS)

1981-01-01

In the Tsuruga Nuclear Power Station of Japan Atomic Power Co., the leakage of radioactivity from the site into the sea occurred, as follows. Due to a valve being left open on March 8, 1981, the overflow of liquid waste from the filter sludge storage tank into the laundry waste filtering room took place. A part of it leaked through the floor into the general underground waterway. The amount was estimated to be one ton with some 10 mCi. The incident is reviewed with the following topics: nobody expected rad-material in the waterway with the leakage from a filter sludge tank; construction, human and management mistakes were revealed in the survey; no environmental impact, with the defect peculiar to Tsuruga; JAPCO executives assume the responsibility; JAPCO and regulatory authorities need review. (Mori, K.)

A Novel Variant of Narrow-Spectrum Antifungal Bacterial Lipopeptides That Strongly Inhibit Ganoderma boninense.

Science.gov (United States)

Pramudito, Theodorus Eko; Agustina, Delia; Nguyen, Thi Kim Ngan; Suwanto, Antonius

2018-03-01

Bacterial antifungal cyclic lipopeptides (ACLs) have become a promising alternative to synthetic fungicide to control pathogenic fungi. Bacillus sp. is known to produce three families of ACL, namely iturin, surfactin, and fengycin. In this paper, we characterized the ACLs produced by B. methylotrophicus HC51 (referred as HC51) mainly regarding its composition and effectivity against fungal plant pathogen. HC51 culture was tested against various pathogenic fungi and the ACLs were extracted and analyzed using liquid chromatography-electrospray ionization mass spectrometry. HC51 showed strong antifungal activity against the plant pathogens Ganoderma sp. and Fusarium sp. Cell-free methanol extract of HC51 contains iturin A and various variants of fengycin. C16 fengycin A was present in four fractions which indicates it as a major component of ACL from HC51. Five variants of fengycin were detected, four of which had been previously reported. We found a novel C17 fengycin F that is characterized by a substitution of L-ornithine into lysine. Considering that L-ornithine is an important building block of fengycin, this substitution suggests the possibility of an alternative pathway for fengycin biosynthesis.

Use of deep whole-genome sequencing data to identify structure risk variants in breast cancer susceptibility genes.

Science.gov (United States)

Guo, Xingyi; Shi, Jiajun; Cai, Qiuyin; Shu, Xiao-Ou; He, Jing; Wen, Wanqing; Allen, Jamie; Pharoah, Paul; Dunning, Alison; Hunter, David J; Kraft, Peter; Easton, Douglas F; Zheng, Wei; Long, Jirong

2018-03-01

Functional disruptions of susceptibility genes by large genomic structure variant (SV) deletions in germlines are known to be associated with cancer risk. However, few studies have been conducted to systematically search for SV deletions in breast cancer susceptibility genes. We analysed deep (> 30x) whole-genome sequencing (WGS) data generated in blood samples from 128 breast cancer patients of Asian and European descent with either a strong family history of breast cancer or early cancer onset disease. To identify SV deletions in known or suspected breast cancer susceptibility genes, we used multiple SV calling tools including Genome STRiP, Delly, Manta, BreakDancer and Pindel. SV deletions were detected by at least three of these bioinformatics tools in five genes. Specifically, we identified heterozygous deletions covering a fraction of the coding regions of BRCA1 (with approximately 80kb in two patients), and TP53 genes (with ∼1.6 kb in two patients), and of intronic regions (∼1 kb) of the PALB2 (one patient), PTEN (three patients) and RAD51C genes (one patient). We confirmed the presence of these deletions using real-time quantitative PCR (qPCR). Our study identified novel SV deletions in breast cancer susceptibility genes and the identification of such SV deletions may improve clinical testing.

RadNet: Open network protocol for radiation data

International Nuclear Information System (INIS)

Rees, B.; Olson, K.; Beckes-Talcott, J.; Kadner, S.; Wenderlich, T.; Hoy, M.; Doyle, W.; Koskelo, M.

1998-01-01

Safeguards instrumentation is increasingly being incorporated into remote monitoring applications. In the past, vendors of radiation monitoring instruments typically provided the tools for uploading the monitoring data to a host. However, the proprietary nature of communication protocols lends itself to increased computer support needs and increased installation expenses. As a result, a working group of suppliers and customers of radiation monitoring instruments defined an open network protocol for transferring packets on a local area network from radiation monitoring equipment to network hosts. The protocol was termed RadNet. While it is now primarily used for health physics instruments, RadNet's flexibility and strength make it ideal for remote monitoring of nuclear materials. The incorporation of standard, open protocols ensures that future work will not render present work obsolete; because RadNet utilizes standard Internet protocols, and is itself a non-proprietary standard. The use of industry standards also simplifies the development and implementation of ancillary services, e.g. E-main generation or even pager systems

Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning

International Nuclear Information System (INIS)

Takahashi, N.; Takahashi, Y.; Blumberg, B.S.; Putnam, F.W.

1987-01-01

The structural changes in four genetic variants of human serum albumin were analyzed by tandem high-pressure liquid chromatography (HPLC) of the tryptic peptides, HPLC mapping and isoelectric focusing of the CNBr fragments, and amino acid sequence analysis of the purified peptides. Lysine-372 of normal (common) albumin A was changed to glutamic acid both in albumin Naskapi, a widespread polymorphic variant of North American Indians, and in albumin Mersin found in Eti Turks. The two variants also exhibited anomalous migration in NaDodSO 4 /PAGE, which is attributed to a conformational change. The identity of albumins Naskapi and Mersin may have originated through descent from a common mid-Asiatic founder of the two migrating ethnic groups, or it may represent identical but independent mutations of the albumin gene. In albumin Adana, from Eti Turks, the substitution site was not identified but was localized to the region from positions 447 through 548. The substitution of aspartic acid-550 by glycine was found in albumin Mexico-2 from four individuals of the Pima tribe. Although only single-point substitutions have been found in these and in certain other genetic variants of human albumin, five differences exist in the amino acid sequences inferred from cDNA sequences by workers in three other laboratories. However, our results on albumin A and on 14 different genetic variants accord with the amino acid sequence of albumin deduced from the genomic sequence. The apparent amino acid substitutions inferred from comparison of individual cDNA sequences probably reflect artifacts in cloning or in cDNA sequence analysis rather than polymorphism of the coding sections of the albumin gene

RadBall Technology Testing and MCNP Modeling of the Tungsten Collimator.

Science.gov (United States)

Farfán, Eduardo B; Foley, Trevor Q; Coleman, J Rusty; Jannik, G Timothy; Holmes, Christopher J; Oldham, Mark; Adamovics, John; Stanley, Steven J

2010-01-01

The United Kingdom's National Nuclear Laboratory (NNL) has developed a remote, non-electrical, radiation-mapping device known as RadBall(™), which can locate and quantify radioactive hazards within contaminated areas of the nuclear industry. RadBall(™) consists of a colander-like outer shell that houses a radiation-sensitive polymer sphere. The outer shell works to collimate radiation sources and those areas of the polymer sphere that are exposed react, becoming increasingly more opaque, in proportion to the absorbed dose. The polymer sphere is imaged in an optical-CT scanner, which produces a high resolution 3D map of optical attenuation coefficients. Subsequent analysis of the optical attenuation matrix provides information on the spatial distribution of sources in a given area forming a 3D characterization of the area of interest. RadBall(™) has no power requirements and can be positioned in tight or hard-to reach locations. The RadBall(™) technology has been deployed in a number of technology trials in nuclear waste reprocessing plants at Sellafield in the United Kingdom and facilities of the Savannah River National Laboratory (SRNL). This study focuses on the RadBall(™) testing and modeling accomplished at SRNL.

RadBall™ Technology Testing and MCNP Modeling of the Tungsten Collimator

Science.gov (United States)

Farfán, Eduardo B.; Foley, Trevor Q.; Coleman, J. Rusty; Jannik, G. Timothy; Holmes, Christopher J.; Oldham, Mark; Adamovics, John; Stanley, Steven J.

2010-01-01

The United Kingdom’s National Nuclear Laboratory (NNL) has developed a remote, non-electrical, radiation-mapping device known as RadBall™, which can locate and quantify radioactive hazards within contaminated areas of the nuclear industry. RadBall™ consists of a colander-like outer shell that houses a radiation-sensitive polymer sphere. The outer shell works to collimate radiation sources and those areas of the polymer sphere that are exposed react, becoming increasingly more opaque, in proportion to the absorbed dose. The polymer sphere is imaged in an optical-CT scanner, which produces a high resolution 3D map of optical attenuation coefficients. Subsequent analysis of the optical attenuation matrix provides information on the spatial distribution of sources in a given area forming a 3D characterization of the area of interest. RadBall™ has no power requirements and can be positioned in tight or hard-to reach locations. The RadBall™ technology has been deployed in a number of technology trials in nuclear waste reprocessing plants at Sellafield in the United Kingdom and facilities of the Savannah River National Laboratory (SRNL). This study focuses on the RadBall™ testing and modeling accomplished at SRNL. PMID:21617740

Reduced insulin exocytosis in human pancreatic β-cells with gene variants linked to type 2 diabetes

DEFF Research Database (Denmark)

Rosengren, Anders H; Braun, Matthias; Mahdi, Taman

2012-01-01

The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features ...

Regionally variant collagen alignment correlates with viscoelastic properties of the disc of the human temporomandibular joint.

Science.gov (United States)

Gutman, Shawn; Kim, Daniel; Tarafder, Solaiman; Velez, Sergio; Jeong, Julia; Lee, Chang H

2018-02-01

To determine the regionally variant quality of collagen alignment in human TMJ discs and its statistical correlation with viscoelastic properties. For quantitative analysis of the quality of collagen alignment, horizontal sections of human TMJ discs with Pricrosirius Red staining were imaged under circularly polarized microscopy. Mean angle and angular deviation of collagen fibers in each region were analyzed using a well-established automated image-processing for angular gradient. Instantaneous and relaxation moduli of each disc region were measured under stress-relaxation test both in tensile and compression. Then Spearman correlation analysis was performed between the angular deviation and the moduli. To understand the effect of glycosaminoglycans on the correlation, TMJ disc samples were treated by chondroitinase ABC (C-ABC). Our imaging processing analysis showed the region-variant direction of collagen alignment, consistently with previous findings. Interestingly, the quality of collagen alignment, not only the directions, was significantly different in between the regions. The angular deviation of fiber alignment in the anterior and intermediate regions were significantly smaller than the posterior region. Medial and lateral regions showed significantly bigger angular deviation than all the other regions. The regionally variant angular deviation values showed statistically significant correlation with the tensile instantaneous modulus and the relaxation modulus, partially dependent on C-ABC treatment. Our findings suggest the region-variant degree of collagen fiber alignment is likely attributed to the heterogeneous viscoelastic properties of TMJ disc that may have significant implications in development of regenerative therapy for TMJ disc. Copyright © 2017 Elsevier Ltd. All rights reserved.

The rad-hard readout system of the BaBar silicon vertex tracker

Science.gov (United States)

Re, V.; DeWitt, J.; Dow, S.; Frey, A.; Johnson, R. P.; Kroeger, W.; Kipnis, I.; Leona, A.; Luo, L.; Mandelli, E.; Manfredi, P. F.; Nyman, M.; Pedrali-Noy, M.; Poplevin, P.; Perazzo, A.; Roe, N.; Spencer, N.

1998-02-01

This paper discusses the behaviour of a prototype rad-hard version of the chip developed for the readout of the BaBar silicon vertex tracker. A previous version of the chip, implemented in the 0.8 μm HP rad-soft version has been thoroughly tested in the recent times. It featured outstanding noise characteristics and showed that the specifications assumed as target for the tracker readout were met to a very good extent. The next step was the realization of a chip prototype in the rad-hard process that will be employed in the actual chip production. Such a prototype is structurally and functionally identical to its rad-soft predecessor. However, the process parameters being different, and not fully mastered at the time of design, some deviations in the behaviour were to be expected. The reasons for such deviations have been identified and some of them were removed by acting on the points that were left accessible on the chip. Other required small circuit modifications that will not affect the production schedule. The tests done so far on the rad-hard chip have shown that the noise behaviour is very close to that of the rad-soft version, that is fully adequate for the vertex detector readout.

Validation of the fifth edition BI-RADS ultrasound lexicon with comparison of fourth and fifth edition diagnostic performance using video clips

International Nuclear Information System (INIS)

Yoon, Jung Hyun; Kim, Min Jung; Lee, Hye Sun; Kim, Sung Hun; Youk, Ji Hyun; Jeong, Sun Hye; Kim, You Me

2016-01-01

The aim of this study was to evaluate the positive predictive value (PPV) and the diagnostic performance of the ultrasonographic descriptors in the fifth edition of BI-RADS, comparing with the fourth edition using video clips. From September 2013 to July 2014, 80 breast masses in 74 women (mean age, 47.5±10.7 years) from five institutions of the Korean Society of Breast Imaging were included. Two radiologists individually reviewed the static and video images and analyzed the images according to the fourth and fifth edition of BI-RADS. The PPV of each descriptor was calculated and diagnostic performances between the fourth and fifth editions were compared. Of the 80 breast masses, 51 (63.8%) were benign and 29 (36.2%) were malignant. Suspicious ultrasonographic features such as irregular shape, non-parallel orientation, angular or spiculated margins, and combined posterior features showed higher PPV in both editions (all P<0.05). No significant differences were found in the diagnostic performances between the two editions (all P>0.05). The area under the receiver operating characteristics curve was higher in the fourth edition (0.708 to 0.690), without significance (P=0.416). The fifth edition of the BI-RADS ultrasound lexicon showed comparable performance to the fourth edition and can be useful in the differential diagnosis of breast masses using ultrasonography

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

Co-design of RAD and ETHICS methodologies: a combination of information system development methods

Science.gov (United States)

Nasehi, Arezo; Shahriyari, Salman

2011-12-01

Co-design is a new trend in the social world which tries to capture different ideas in order to use the most appropriate features for a system. In this paper, co-design of two information system methodologies is regarded; rapid application development (RAD) and effective technical and human implementation of computer-based systems (ETHICS). We tried to consider the characteristics of these methodologies to see the possibility of having a co-design or combination of them for developing an information system. To reach this purpose, four different aspects of them are analyzed: social or technical approach, user participation and user involvement, job satisfaction, and overcoming change resistance. Finally, a case study using the quantitative method is analyzed in order to examine the possibility of co-design using these factors. The paper concludes that RAD and ETHICS are appropriate to be co-designed and brings some suggestions for the co-design.

Substantial differences in bias between single-digest and double-digest RAD-seq libraries: A case study.

Science.gov (United States)

Flanagan, Sarah P; Jones, Adam G

2018-03-01

The trade-offs of using single-digest vs. double-digest restriction site-associated DNA sequencing (RAD-seq) protocols have been widely discussed. However, no direct empirical comparisons of the two methods have been conducted. Here, we sampled a single population of Gulf pipefish (Syngnathus scovelli) and genotyped 444 individuals using RAD-seq. Sixty individuals were subjected to single-digest RAD-seq (sdRAD-seq), and the remaining 384 individuals were genotyped using a double-digest RAD-seq (ddRAD-seq) protocol. We analysed the resulting Illumina sequencing data and compared the two genotyping methods when reads were analysed either together or separately. Coverage statistics, observed heterozygosity, and allele frequencies differed significantly between the two protocols, as did the results of selection components analysis. We also performed an in silico digestion of the Gulf pipefish genome and modelled five major sources of bias: PCR duplicates, polymorphic restriction sites, shearing bias, asymmetric sampling (i.e., genotyping fewer individuals with sdRAD-seq than with ddRAD-seq) and higher major allele frequencies. This combination of approaches allowed us to determine that polymorphic restriction sites, an asymmetric sampling scheme, mean allele frequencies and to some extent PCR duplicates all contribute to different estimates of allele frequencies between samples genotyped using sdRAD-seq versus ddRAD-seq. Our finding that sdRAD-seq and ddRAD-seq can result in different allele frequencies has implications for comparisons across studies and techniques that endeavour to identify genomewide signatures of evolutionary processes in natural populations. © 2017 John Wiley & Sons Ltd.

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.)

Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

Science.gov (United States)

Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

2001-06-01

1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

Selection and characterization of T-cell variants lacking molecules involved in T-cell activation (T3 T-cell receptor, T44, and T11): analysis of the functional relationship among different pathways of activation

International Nuclear Information System (INIS)

Moretta, A.; Poggi, A.; Olive, D.; Bottino, C.; Fortis, C.; Pantaleo, G.; Moretta, L.

1987-01-01

A clone of the interleukin 2-producing Jurkat leukemia cell line termed JA3 (surface phenotype, T3 + , Ti + , T44 + , T11 + , T40 + ) has been used to induce and select cell variants lacking surface molecules involved in T-cell activation. Following 200 rad of γ-radiation (1 rad = 0.01 Gy), cells were treated with monoclonal antibodies (mAbs) directed to T3, Ti, T44, or T11 antigen and complement. After growth of the residual cells in culture, negative cells were cloned under limiting conditions. Depending on the specificity of the mAb used for the immunoselection, three groups of variants were obtained. (i) The use of mAbs directed to T3 or Ti resulted in cell variants that expressed the T3 - Ti - T44 + Leu1 + T11 + T40 + 4F2 + HLA class I + surface phenotype. (ii) Immunoselection with anti-T44 mAb resulted in 2 variants that shared the T3 - Ti - T44 - Leu1 - T11 - T40 - 4F2 - HLA class I + phenotype. (iii) Cell treatment with anti-T11 mAb resulted in 15 variants characterized by the lack of T11 antigen expression and of all the other T-cell-specific surface antigens. Therefore, it appears that the different sets of JA3 cell variants, like T cells at discrete stages of intrathymic differentiation, may follow a coordinated expression of surface differentiation antigens. Analysis of the functional responsiveness of the three distinct groups of JA3 cell variants to different stimuli showed that all produced interleukin 2 in response to A23187 calcium ionophore plus phorbol 12-myristate 13-acetate

Issues in assessing multi-institutional performance of BI-RADS-based CAD systems

Science.gov (United States)

Markey, Mia K.; Lo, Joseph Y.

2005-04-01

The purpose of this study was to investigate factors that impact the generalization of breast cancer computer-aided diagnosis (CAD) systems that utilize the Breast Imaging Reporting and Data System (BI-RADS). Data sets from four institutions were analyzed: Duke University Medical Center, University of Pennsylvania Medical Center, Massachusetts General Hospital, and Wake Forest University. The latter two data sets are subsets of the Digital Database for Screening Mammography. Each data set consisted of descriptions of mammographic lesions according to the BI-RADS lexicon, patient age, and pathology status (benign/malignant). Models were developed to predict pathology status from the BI-RADS descriptors and the patient age. Comparisons between the models built on data from the different institutions were made in terms of empirical (non-parametric) receiver operating characteristic (ROC) curves. Results suggest that BI-RADS-based CAD systems focused on specific classes of lesions may be more generally applicable than models that cover several lesion types. However, better generalization was seen in terms of the area under the ROC curve than in the partial area index (>90% sensitivity). Previous studies have illustrated the challenges in translating a BI-RADS-based CAD system from one institution to another. This study provides new insights into possible approaches to improve the generalization of BI-RADS-based CAD systems.

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

Autism as an adaptive common variant pathway for human brain development

Directory of Open Access Journals (Sweden)

Mark H. Johnson

2017-06-01

Full Text Available While research on focal perinatal lesions has provided evidence for recovery of function, much less is known about processes of brain adaptation resulting from mild but widespread disturbances to neural processing over the early years (such as alterations in synaptic efficiency. Rather than being viewed as a direct behavioral consequence of life-long neural dysfunction, I propose that autism is best viewed as the end result of engaging adaptive processes during a sensitive period. From this perspective, autism is not appropriately described as a disorder of neurodevelopment, but rather as an adaptive common variant pathway of human functional brain development.

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...

RadNet (Environmental Radiation Ambient Monitoring System)

Data.gov (United States)

U.S. Environmental Protection Agency — RadNet, formerly Environmental Radiation Ambient Monitoring System (ERAMS), is a national network of monitoring stations that regularly collect air, precipitation,...

VirRAD - The virtual radiopharmacy - a virtual learning community in radiopharmacy

International Nuclear Information System (INIS)

Mather, S.J.

2002-01-01

VirRAD is a new research project funded by the Information Society Technologies programme of the Fifth RTD Framework Programme of the European Union. The aim of VirRAD is to develop an Internet-based virtual learning environment for Radiopharmacy which will facilitate learning and understanding in the speciality at all levels both within and outside the radiopharmaceutical community. The final structure of VirRAD is yet to be decided and will be determined by the needs and desires of its ultimate participants, however it is expected to include multimedia teaching modules for distance learning applications, 3-D simulations of specialised equipment and complex techniques used in radiopharmaceutical preparation and research as well as improved systems of peer-to-peer and student-to-tutor communication and information retrieval. To be as useful and effective as possible VirRAD needs ideas and suggestions from the Nuclear Medicine community. A mechanism for obtaining such input and feedback will be an integral part of the development process

Molecular Evolution of the VP1 Gene in Human Norovirus GII.4 Variants in 1974–2015

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Takumi Motoya

2017-12-01

Full Text Available Human norovirus (HuNoV is a leading cause of viral gastroenteritis worldwide, of which GII.4 is the most predominant genotype. Unlike other genotypes, GII.4 has created various variants that escaped from previously acquired immunity of the host and caused repeated epidemics. However, the molecular evolutionary differences among all GII.4 variants, including recently discovered strains, have not been elucidated. Thus, we conducted a series of bioinformatic analyses using numerous, globally collected, full-length GII.4 major capsid (VP1 gene sequences (466 strains to compare the evolutionary patterns among GII.4 variants. The time-scaled phylogenetic tree constructed using the Bayesian Markov chain Monte Carlo (MCMC method showed that the common ancestor of the GII.4 VP1 gene diverged from GII.20 in 1840. The GII.4 genotype emerged in 1932, and then formed seven clusters including 14 known variants after 1980. The evolutionary rate of GII.4 strains was estimated to be 7.68 × 10−3 substitutions/site/year. The evolutionary rates probably differed among variants as well as domains [protruding 1 (P1, shell, and P2 domains]. The Osaka 2007 variant strains probably contained more nucleotide substitutions than any other variant. Few conformational epitopes were located in the shell and P1 domains, although most were contained in the P2 domain, which, as previously established, is associated with attachment to host factors and antigenicity. We found that positive selection sites for the whole GII.4 genotype existed in the shell and P1 domains, while Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants were under positive selection in the P2 domain. Amino acid substitutions overlapped with putative epitopes or were located around the epitopes in the P2 domain. The effective population sizes of the present strains increased stepwise for Den Haag 2006b, New Orleans 2009, and Sydney 2012 variants. These results suggest that HuNoV GII.4 rapidly

The role of Candida albicans homologous recombination factors Rad54 and Rdh54 in DNA damage sensitivity

Directory of Open Access Journals (Sweden)

White Theodore C

2011-09-01

Full Text Available Abstract Background The fungal pathogen Candida albicans is frequently seen in immune suppressed patients, and resistance to one of the most widely used antifungals, fluconazole (FLC, can evolve rapidly. In recent years it has become clear that plasticity of the Candida albicans genome contributes to drug resistance through loss of heterozygosity (LOH at resistance genes and gross chromosomal rearrangements that amplify gene copy number of resistance associated genes. This study addresses the role of the homologous recombination factors Rad54 and Rdh54 in cell growth, DNA damage and FLC resistance in Candida albicans. Results The data presented here support a role for homologous recombination in cell growth and DNA damage sensitivity, as Candida albicans rad54Δ/rad54Δ mutants were hypersensitive to MMS and menadione, and had an aberrant cell and nuclear morphology. The Candida albicans rad54Δ/rad54Δ mutant was defective in invasion of Spider agar, presumably due to the altered cellular morphology. In contrast, mutation of the related gene RDH54 did not contribute significantly to DNA damage resistance and cell growth, and deletion of either Candida albicans RAD54 or Candida albicans RDH54 did not alter FLC susceptibility. Conclusions Together, these results support a role for homologous recombination in genome stability under nondamaging conditions. The nuclear morphology defects in the rad54Δ/rad54Δ mutants show that Rad54 performs an essential role during mitotic growth and that in its absence, cells arrest in G2. The viability of the single mutant rad54Δ/rad54Δ and the inability to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ suggests that Rdh54 can partially compensate for Rad54 during mitotic growth.

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.)

PDGFRα and CD51 mark human nestin+ sphere-forming mesenchymal stem cells capable of hematopoietic progenitor cell expansion.

Science.gov (United States)

Pinho, Sandra; Lacombe, Julie; Hanoun, Maher; Mizoguchi, Toshihide; Bruns, Ingmar; Kunisaki, Yuya; Frenette, Paul S

2013-07-01

The intermediate filament protein Nestin labels populations of stem/progenitor cells, including self-renewing mesenchymal stem cells (MSCs), a major constituent of the hematopoietic stem cell (HSC) niche. However, the intracellular location of Nestin prevents its use for prospective live cell isolation. Hence it is important to find surface markers specific for Nestin⁺ cells. In this study, we show that the expression of PDGFRα and CD51 among CD45⁻ Ter119⁻ CD31⁻ mouse bone marrow (BM) stromal cells characterizes a large fraction of Nestin⁺ cells, containing most fibroblastic CFUs, mesenspheres, and self-renewal capacity after transplantation. The PDGFRα⁺ CD51 ⁺subset of Nestin⁺ cells is also enriched in major HSC maintenance genes, supporting the notion that niche activity co-segregates with MSC activity. Furthermore, we show that PDGFRα⁺ CD51⁺ cells in the human fetal BM represent a small subset of CD146⁺ cells expressing Nestin and enriched for MSC and HSC niche activities. Importantly, cultured human PDGFRα⁺ CD51⁺ nonadherent mesenspheres can significantly expand multipotent hematopoietic progenitors able to engraft immunodeficient mice. These results thus indicate that the HSC niche is conserved between the murine and human species and suggest that highly purified nonadherent cultures of niche cells may represent a useful novel technology to culture human hematopoietic stem and progenitor cells.

Association of common genetic variants with human skin color variation in Indian populations.

Science.gov (United States)

Sarkar, Anujit; Nandineni, Madhusudan R

2018-01-01

Human skin color is one of the most conspicuously variable physical traits that has attracted the attention of physical anthropologists, social scientists and human geneticists. Although several studies have established the underlying genes and their variants affecting human skin color, they were mostly confined to Europeans and Africans and similar studies in Indian populations have been scanty. Studying the association between candidate genetic variants and skin color will help to validate previous findings and to better understand the molecular mechanism of skin color variation. In this study, 22 candidate SNPs from 12 genes were tested for association with skin color in 299 unrelated samples sourced from nine geographical locations in India. Our study establishes the association of 9 SNPs with the phenotype in Indian populations and could explain ∼31% of the variance in skin color. Haplotype analysis of chromosome 15 revealed a significant association of alleles G, A and C of SNPs rs1426654, rs11070627, and rs12913316, respectively, to the phenotype, and accounted for 17% of the variance. Latitude of the sampling location was also a significant factor, contributing to ∼19% of the variation observed in the samples. These observations support the findings that rs1426654 and rs4775730 located in SLC24A5, and rs11070627 and rs12913316 located in MYEF2 and CTXN2 genes respectively, are major contributors toward skin pigmentation and would aid in further unraveling the genotype-phenotype association in Indian populations. These findings can be utilized in forensic DNA applications for criminal investigations. © 2017 Wiley Periodicals, Inc.

Evaluation of the Sebia Capillarys 3 Tera and the Bio-Rad D-100 Systems for the Measurement of Hemoglobin A1c.

Science.gov (United States)

Herpol, Margaux; Lanckmans, Katrien; Van Neyghem, Stefaan; Clement, Pascale; Crevits, Stefanie; De Crem, Kim; Gorus, Frans K; Weets, Ilse

2016-07-01

We evaluated the Bio-Rad (Irvine, CA) D-100 and the Sebia (Lisses, France) Capillarys 3 Tera for the measurement of hemoglobin A1c (HbA1c) in venous blood samples. Whole-blood samples and control material were analyzed with the D-100 and Capillarys 3 Tera and compared with our routine method, HLC-723G7 (Tosoh, Tokyo, Japan). An evaluation protocol to test precision, trueness, linearity, carryover, and selectivity was set up according to Clinical and Laboratory Standards Institute guidelines. The results were presented in National Glycohemoglobin Standardization Program and International Federation of Clinical Chemistry (IFCC) units. Both systems showed excellent precision (total coefficients of variation hemoglobin (≤0.5 mmol/L potassium cyanate), hemoglobin variants, bilirubin (≤15 mg/dL), and triglycerides (≤3,360 mg/dL). The Bio-Rad D-100 and the Sebia Capillarys 3 Tera instruments performed well for the determination of HbA1c in terms of quality criteria as well as for sample throughput. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Suspicious breast calcifications undergoing stereotactic biopsy in women ages 70 and over: Breast cancer incidence by BI-RADS descriptors.

Science.gov (United States)

Grimm, Lars J; Johnson, David Y; Johnson, Karen S; Baker, Jay A; Soo, Mary Scott; Hwang, E Shelley; Ghate, Sujata V

2017-06-01

To determine the malignancy rate overall and for specific BI-RADS descriptors in women ≥70 years who undergo stereotactic biopsy for calcifications. We retrospectively reviewed 14,577 consecutive mammogram reports in 6839 women ≥70 years to collect 231 stereotactic biopsies of calcifications in 215 women. Cases with missing images or histopathology and calcifications associated with masses, distortion, or asymmetries were excluded. Three breast radiologists determined BI-RADS descriptors by majority. Histology, hormone receptor status, and lymph node status were correlated with BI-RADS descriptors. There were 131 (57 %) benign, 22 (10 %) atypia/lobular carcinomas in situ, 55 (24 %) ductal carcinomas in situ (DCIS), and 23 (10 %) invasive diagnoses. Twenty-seven (51 %) DCIS cases were high-grade. Five (22 %) invasive cases were high-grade, two (9 %) were triple-negative, and three (12 %) were node-positive. Malignancy was found in 49 % (50/103) of fine pleomorphic, 50 % (14/28) of fine linear, 25 % (10/40) of amorphous, 20 % (3/15) of round, 3 % (1/36) of coarse heterogeneous, and 0 % (0/9) of dystrophic calcifications. Among women ≥70 years that underwent stereotactic biopsy for calcifications only, we observed a high rate of malignancy. Additionally, coarse heterogeneous calcifications may warrant a probable benign designation. • Cancer rates of biopsied calcifications in women ≥70 years are high • Radiologists should not dismiss suspicious calcifications in older women • Coarse heterogeneous calcifications may warrant a probable benign designation.

Genetic Variants Contribute to Gene Expression Variability in Humans

Science.gov (United States)

Hulse, Amanda M.; Cai, James J.

2013-01-01

Expression quantitative trait loci (eQTL) studies have established convincing relationships between genetic variants and gene expression. Most of these studies focused on the mean of gene expression level, but not the variance of gene expression level (i.e., gene expression variability). In the present study, we systematically explore genome-wide association between genetic variants and gene expression variability in humans. We adapt the double generalized linear model (dglm) to simultaneously fit the means and the variances of gene expression among the three possible genotypes of a biallelic SNP. The genomic loci showing significant association between the variances of gene expression and the genotypes are termed expression variability QTL (evQTL). Using a data set of gene expression in lymphoblastoid cell lines (LCLs) derived from 210 HapMap individuals, we identify cis-acting evQTL involving 218 distinct genes, among which 8 genes, ADCY1, CTNNA2, DAAM2, FERMT2, IL6, PLOD2, SNX7, and TNFRSF11B, are cross-validated using an extra expression data set of the same LCLs. We also identify ∼300 trans-acting evQTL between >13,000 common SNPs and 500 randomly selected representative genes. We employ two distinct scenarios, emphasizing single-SNP and multiple-SNP effects on expression variability, to explain the formation of evQTL. We argue that detecting evQTL may represent a novel method for effectively screening for genetic interactions, especially when the multiple-SNP influence on expression variability is implied. The implication of our results for revealing genetic mechanisms of gene expression variability is discussed. PMID:23150607