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ABSTRACT: PURPOSE: The purpose of this study was to perform a retrospective clinical and radiographic evaluation after opening-wedge high tibial osteotomy (HTO) using a short spacer plate (Aescula; B. Braun Korea, Seoul, South Korea) and rigid long plate (TomoFix plate; Mathys, Bettlach, Switzerland) at follow-up 2 years postoperatively. METHODS: We performed 94 opening-wedge HTOs with the Aescula plate (group I) and 92 HTOs with the TomoFix plate (group II). Patients underwent clinical and radiographic evaluations preoperatively and at 2 years postoperatively. Clinical evaluations were performed with Knee Society scores. Radiographic analysis included the mechanical tibiofemoral angle (mTFA) and the slope of the tibia angle with preoperative and postoperative full weight-bearing anteroposterior whole-leg views, as well as anteroposterior, lateral, and Merchant views of the knee. We measured the mTFA. In addition, we evaluated the complications in each group. The follow-up period was 2 years. RESULTS: At follow-up 2 years postoperatively, we observed an overall complication rate of 38% in group I and 26% in group II (P = .083). We found plate-related complication rates of 20% in group I and 9% in group II (P = .039). Plate-related complications included loss of correction, fracture of the tibial plateau, screw failure, malunion, and fracture of the lateral cortical bone. The mean mTFA was -6.0° ± 3.2° in group I and -4.6° ± 2.8° in group II preoperatively (P = .262). The mean mTFA was 1.0° ± 3.1° in group I and 1.5° ± 2.3° in group II at the latest follow-up (P = .034). In group I, the mean Knee Society knee score and function score were 60.0 ± 12.9 and 57.9 ± 26.8, respectively, preoperatively. They improved to 92.1 ± 8.1 and 89.0 ± 15.1, respectively, at follow-up (P = .001 and P = .001, respectively). In group II, the mean Knee Society knee score and function score were 57.5 ± 14.8 and 57.4 ± 22.1, respectively, preoperatively. They improved to 95.5 ± 5.4 and 95.0 ± 7.6, respectively, at follow-up (P = .001 and P = .001, respectively). In addition, the mean postoperative knee score and function score in group II were higher than those in group I (P = .001 and P = .001, respectively). CONCLUSIONS: We have shown a high plate-related complication rate and a significant loss of correction during a short-term follow-up period (2 years) after opening-wedge HTO using the new short spacer HTO plate compared with the rigid long plate. LEVEL OF EVIDENCE: Level IV, therapeutic case series.
Arthroscopy The Journal of Arthroscopic and Related Surgery 04/2013; · 3.02 Impact Factor
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ABSTRACT: PURPOSE: The purpose of this prospective randomized study was to compare the visible, hidden, total blood loss and postoperative haemodynamic change of 4-h clamping and nonclamping of the drain after TKA. The hypothesis in the present study was that intermittent drain clamping with injection of diluted epinephrine solution would decrease the visible, hidden blood loss and reduction of postoperative haemoglobin or haematocrit change after TKA. METHODS: From January 2010 to January 2011, 100 TKAs were performed at our hospital. In group I (50 knees), drainage was clamped for the first 4 postoperative hours with injection of diluted epinephrine solution. In group II (50 knees), drainage was not clamped without injection of diluted epinephrine solution. Two drains with an external diameter of 3.2 mm were inserted into the knee joint. We checked the amount of drainage recorded at 6, 12, 24, and 48 h postoperatively. Also, we checked the haemoglobin and haematocrit on the preoperation, first, 5th and 10th postoperative days. We analysed the transfusion rate, the possible adverse issues with clamping drainage, and the range of motion of the knee. RESULTS: The mean total bloody drainage was significantly less in group I than group II (560.7 ± 249.9 mL vs 978.3 ± 327.5 mL) (p < 0.001). The decrease of haemoglobin and haematocrit after surgery was not significant between the two groups (n.s.). The hidden blood loss was significantly more in group I than group II (541.1 ± 439.4 mL vs 32.1 ± 21.9 mL) (p < 0.001). So, total blood loss showed no significant difference between the two groups (1,101.8 ± 373.6 mL vs 1,010.4 ± 385.9 mL) (n.s.). The postoperative range of motion and transfusion rate between the two groups were not significant (n.s.). But immediate wound problem, such as oozing, was significantly more in group I (p < 0.001). CONCLUSION: It is not necessary to perform the intermittent drain clamping with injection of the diluted epinephrine solution in TKA because there is no impact on the postoperative haemoglobin and haematocrit. If anything, the intermittent drain clamping with injection of the diluted epinephrine solution increased the hidden blood loss and immediate wound problem than nonclamping without injection of the diluted epinephrine solution. LEVEL OF EVIDENCE: I.
Knee Surgery Sports Traumatology Arthroscopy 10/2012; · 2.21 Impact Factor
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ABSTRACT: PURPOSE: The intramedullary (IM) femoral alignment system does not alway guarantee accuracy of the component position in the total knee arthroplasty (TKA). In some cases, the extramedullary (EM) femoral alignment system in total knee arthroplasty (TKA) is a useful alternative surgical option to adjust femoral component alignment. In the EM technique, accuracy of the femoral head center location is mandatory. The purpose of this prospective randomized study was to compare the alignment after TKA using two different femoral alignment systems. METHODS: From January 2009 to December 2009, 91 patients (106 knees) with osteoarthritis underwent TKA. The IM femoral alignment system was used in 50 TKAs, and the EM system was used in 56 TKAs. We measured the coronal, sagittal alignment of the femoral component, and overall alignment from full-length standing. Anteroposterior radiographs were taken 1 year after surgery. RESULTS: The overall limb alignment was 0.2° ± 1.9° varus in the EM group and 1.1° ± 1.9° valgus in the IM group (p = 0.001). The coronal alignment of the femoral component was 90.0° ± 1.1° in the EM group and 90.3° ± 1.2° in the IM group, not statistically different (n.s.). The sagittal alignment of the femoral component was 2.3° ± 1.7° in the EM group and 2.5° ± 1.0° in the IM group (n.s.). Clinically acceptable overall limb alignment was achieved in 91.1 % of EM group and 84.0 % of IM group (n.s.). CONCLUSION: The present study suggests that by applying our EM technique that uses a newly designed mechanical axis marker system, the alignment of the femoral component and overall limb alignment is reliable and at least as accurate as the standard IM technique. LEVEL OF EVIDENCE: I.
Knee Surgery Sports Traumatology Arthroscopy 04/2012; · 2.21 Impact Factor
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Ho-Jin Moon,
Jong-Soo Lee,
Young-Ki Choi,
Jie-Yeun Park,
Melbourne R Talactac,
Mohammed Y E Chowdhury,
Haryoung Poo,
Moon-Hee Sung, Ji-Hoon Lee,
Jae U Jung,
Chul-Joong Kim
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ABSTRACT: In addition to development of vaccines and synthetic antiviral drugs, recent studies have advocated the use of natural substances that inhibit or prevent viral infections. High-molecular-weight poly-γ-glutamate (HM-γ-PGA) produced by Bacillus subtilis chungkookjang was evaluated for anti-influenza virus activity. HM-γ-PGA induced type I interferons (IFNs), which in turn stimulated expression of Myxovirus resistant 1 protein and IFN-related proteins in vitro. In the B6.A2G-Mx1 mouse model, which mimics the innate immune system of humans, treatment with HM-γ-PGA enhanced the antiviral state of mice and protected them against highly pathogenic influenza A virus. Naturally synthesized HM-γ-PGA has potent anti-influenza activity and may be a useful means for control of influenza virus.
Antiviral research 02/2012; 94(1):98-102. · 3.61 Impact Factor
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ABSTRACT: Current options for meniscal root repair include repair into trans-osseous bone tunnels, trans-osseous suture passage for surface fixation, and suture anchor fixation. Suture anchor repair techniques have been developed since it eliminates the issue of the suture abrasion, tunnel drilling, and distal fixation inherent to trans-osseous tunnel. We present a description of a new variation in the more vertical suture anchor repair technique for meniscal root tear using a novel medial quadriceptal portal. Level of evidence Therapeutic, Level V.
Knee Surgery Sports Traumatology Arthroscopy 01/2012; · 2.21 Impact Factor
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ABSTRACT: Tel2, a protein conserved from yeast to vertebrates, is an essential regulator of diverse cellular processes including telomere maintenance, DNA damage checkpoints, DNA repair, biological clocks, and cell signaling. The Drosophila Tel2 protein is produced as a translational fusion with EpsinR, a Clathrin adapter that facilitates vesicle trafficking between the Golgi and endosomes. EpsinR and Tel2 are encoded by a Drosophila gene called lqfR. lqfR is required for viability, and its specific roles include cell growth, proliferation, and planar cell polarity. We find that all of these functions of lqfR are attributed entirely to Tel2, not EpsinR. In addition, we find that Drosophila LqfR/Tel2 is a component of one or more protein complexes that contain E-cadherin and Armadillo. Moreover, Tel2 modulates E-cadherin and Armadillo cellular dynamics. We propose that at least one of the functions of Drosophila Tel2 is regulation of Wingless signaling.
PLoS ONE 01/2012; 7(9):e46357. · 4.09 Impact Factor
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ABSTRACT: We present a single cell-gap transflective liquid crystal (LC) device using a homogeneous alignment polyimide (H-PI) mixed with a liquid crystalline reactive monomer that is able to vertically align the LC. We obtain two different pretilt angles in each pixel through the region by region control of the UV exposure time. The smaller pretilt angle is used to obtain a half-wave phase retardation for the transmissive part, whereas the larger pretilt angle is used to obtain a quarter-wave phase retardation for the reflective part.
Optics Express 12/2011; 19(25):25617-22. · 3.59 Impact Factor
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Magtouf Gatei,
Burkhard Jakob,
Philip Chen,
Amanda W. Kijas,
Olivier J. Becherel,
Nuri Gueven,
Geoff Birrell, Ji-Hoon Lee,
Tanya T. Paull,
Yaniv Lerenthal,
Shazrul Fazry,
Gisela Taucher-Scholz,
Reinhard Kalb,
Detlev Schindler,
Regina Waltes,
Thilo Dörk,
Martin F. Lavin
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ABSTRACT: The Mre11/Rad50/NBN complex plays a central role in coordinating the cellular response to DNA double-strand breaks. The importance
of Rad50 in that response is evident from the recent description of a patient with Rad50 deficiency characterized by chromosomal
instability and defective ATM-dependent signaling. We report here that ATM (defective in ataxia-telangiectasia) phosphorylates
Rad50 at a single site (Ser-635) that plays an important adaptor role in signaling for cell cycle control and DNA repair.
Although a Rad50 phosphosite-specific mutant (S635G) supported normal activation of ATM in Rad50-deficient cells, it was defective
in correcting DNA damage-induced signaling through the ATM-dependent substrate SMC1. This mutant also failed to correct radiosensitivity,
DNA double-strand break repair, and an S-phase checkpoint defect in Rad50-deficient cells. This was not due to disruption
of the Mre11/Rad50/NBN complex revealing for the first time that phosphorylation of Rad50 plays a key regulatory role as an
adaptor for specific ATM-dependent downstream signaling through SMC1 for DNA repair and cell cycle checkpoint control in the
maintenance of genome integrity.
Journal of Biological Chemistry 09/2011; 286(36):31542-31556. · 4.77 Impact Factor
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Magtouf Gatei,
Burkhard Jakob,
Philip Chen,
Amanda W Kijas,
Olivier J Becherel,
Nuri Gueven,
Geoff Birrell, Ji-Hoon Lee,
Tanya T Paull,
Yaniv Lerenthal,
Shazrul Fazry,
Gisela Taucher-Scholz,
Reinhard Kalb,
Detlev Schindler,
Regina Waltes,
Thilo Dörk,
Martin F Lavin
[show abstract]
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ABSTRACT: The Mre11/Rad50/NBN complex plays a central role in coordinating the cellular response to DNA double-strand breaks. The importance of Rad50 in that response is evident from the recent description of a patient with Rad50 deficiency characterized by chromosomal instability and defective ATM-dependent signaling. We report here that ATM (defective in ataxia-telangiectasia) phosphorylates Rad50 at a single site (Ser-635) that plays an important adaptor role in signaling for cell cycle control and DNA repair. Although a Rad50 phosphosite-specific mutant (S635G) supported normal activation of ATM in Rad50-deficient cells, it was defective in correcting DNA damage-induced signaling through the ATM-dependent substrate SMC1. This mutant also failed to correct radiosensitivity, DNA double-strand break repair, and an S-phase checkpoint defect in Rad50-deficient cells. This was not due to disruption of the Mre11/Rad50/NBN complex revealing for the first time that phosphorylation of Rad50 plays a key regulatory role as an adaptor for specific ATM-dependent downstream signaling through SMC1 for DNA repair and cell cycle checkpoint control in the maintenance of genome integrity.
Journal of Biological Chemistry 07/2011; 286(36):31542-56. · 4.77 Impact Factor
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ABSTRACT: Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling.
PLoS ONE 01/2011; 6(3):e18259. · 4.09 Impact Factor
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ABSTRACT: In human cells, DNA double-strand breaks are repaired primarily by the non-homologous end joining (NHEJ) pathway. Given their critical nature, we expected NHEJ proteins to be evolutionarily conserved, with relatively little sequence change over time. Here, we report that while critical domains of these proteins are conserved as expected, the sequence of NHEJ proteins has also been shaped by recurrent positive selection, leading to rapid sequence evolution in other protein domains. In order to characterize the molecular evolution of the human NHEJ pathway, we generated large simian primate sequence datasets for NHEJ genes. Codon-based models of gene evolution yielded statistical support for the recurrent positive selection of five NHEJ genes during primate evolution: XRCC4, NBS1, Artemis, POLλ, and CtIP. Analysis of human polymorphism data using the composite of multiple signals (CMS) test revealed that XRCC4 has also been subjected to positive selection in modern humans. Crystal structures are available for XRCC4, Nbs1, and Polλ; and residues under positive selection fall exclusively on the surfaces of these proteins. Despite the positive selection of such residues, biochemical experiments with variants of one positively selected site in Nbs1 confirm that functions necessary for DNA repair and checkpoint signaling have been conserved. However, many viruses interact with the proteins of the NHEJ pathway as part of their infectious lifecycle. We propose that an ongoing evolutionary arms race between viruses and NHEJ genes may be driving the surprisingly rapid evolution of these critical genes.
PLoS Genetics 01/2010; 6(10):e1001169. · 8.69 Impact Factor
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ABSTRACT: The Mre11/Rad50/Nbs1 (MRN) complex has a central function in facilitating activation of the ATM protein kinase at sites of DNA double-strand breaks (DSBs). However, several other factors are also required in human cells for efficient signalling through MRN and ATM, including the tumour suppressor proteins p53-binding protein 1 (53BP1) and BRCA1. In this study, we investigate the functions of these mediator proteins in ATM activation and find that the presence of 53BP1 and BRCA1 can amplify the effects of MRN when interactions between MRN and ATM are compromised. This effect is dependent on a direct interaction between MRN and the tandem breast cancer carboxy-terminal (BRCT) repeats in 53BP1, and is accompanied by hyper-phosphorylation of both Nbs1 and 53BP1. We also find that the BRCT domains of 53BP1 affect the overall structure of 53BP1 multimers and that this structure is important for promoting ATM phosphorylation of substrates as well as for the repair of DNA DSBs in mammalian cells.
The EMBO Journal 12/2009; 29(3):574-85. · 9.20 Impact Factor
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ABSTRACT: Epsin and epsin-Related (epsinR) are multi-modular proteins that stimulate clathrin-coated vesicle formation. Epsin promotes endocytosis at the plasma membrane, and epsinR functions at the Golgi and early endosomes for trans-Golgi network/endosome vesicle trafficking. In Drosophila, endocytic epsin is known as Liquid facets, and it is essential specifically for Notch signaling. Here, by generating and analyzing loss-of-function mutants in the liquid facets-Related (lqfR) gene of Drosophila, we investigated the function of Golgi epsin in a multicellular context. We found that LqfR is indeed a Golgi protein, and that like liquid facets, lqfR is essential for Drosophila viability. In addition, primarily by analyzing mutant eye discs, we found that lqfR is required for cell proliferation, insulin-independent cell growth, and cell patterning, consistent with a role in one or several signaling pathways. Epsins in all organisms share an ENTH (epsin N-terminal homology) domain, which binds phosphoinositides enriched at the plasma membrane or the Golgi membrane. The epsinR ENTH domain is also the recognition element for particular cargos. By generating wild-type and mutant lqfR transgenes, we found that all apparent LqfR functions are independent of its ENTH domain. These results suggest that LqfR transports specific cargo critical to one or more signaling pathways, and lays the foundation for identifying those proteins.
Developmental Biology 05/2009; 331(1):1-13. · 4.07 Impact Factor
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Aude Dupré,
Louise Boyer-Chatenet,
Rose M Sattler,
Ami P Modi, Ji-Hoon Lee,
Matthew L Nicolette,
Levy Kopelovich,
Maria Jasin,
Richard Baer,
Tanya T Paull,
Jean Gautier
Nature Chemical Biology 04/2009; 5(3):191. · 14.69 Impact Factor
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ABSTRACT: Cellular responses to both physiological and pathological DNA double-strand breaks are initiated through activation of the evolutionarily conserved ataxia telangiectasia mutated (ATM) kinase. Upon DNA damage, an activation mechanism involving autophosphorylation has been reported to allow ATM to phosphorylate downstream targets important for cell cycle checkpoints and DNA repair. In humans, serine residues 367, 1893, and 1981 have been shown to be autophosphorylation sites that are individually required for ATM activation. To test the physiological importance of these sites, we generated a transgenic mouse model in which all three conserved ATM serine autophosphorylation sites (S367/1899/1987) have been replaced with alanine. In this study, we show that ATM-dependent responses at both cellular and organismal levels are functional in mice that express a triple serine mutant form of ATM as their sole ATM species. These results lend further support to the notion that ATM autophosphorylation correlates with the DNA damage-induced activation of the kinase but is not required for ATM function in vivo.
The Journal of Cell Biology 01/2009; 183(5):777-83. · 10.26 Impact Factor
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Aude Dupré,
Louise Boyer-Chatenet,
Rose M Sattler,
Ami P Modi, Ji-Hoon Lee,
Matthew L Nicolette,
Levy Kopelovich,
Maria Jasin,
Richard Baer,
Tanya T Paull,
Jean Gautier
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ABSTRACT: The MRN (Mre11-Rad50-Nbs1)-ATM (ataxia-telangiectasia mutated) pathway is essential for sensing and signaling from DNA double-strand breaks. The MRN complex acts as a DNA damage sensor, maintains genome stability during DNA replication, promotes homology-dependent DNA repair and activates ATM. MRN is essential for cell viability, which has limited functional studies of the complex. Small-molecule inhibitors of MRN could circumvent this experimental limitation and could also be used as cellular radio- and chemosensitization compounds. Using cell-free systems that recapitulate faithfully the MRN-ATM signaling pathway, we designed a forward chemical genetic screen to identify inhibitors of the pathway, and we isolated 6-(4-hydroxyphenyl)-2-thioxo-2,3-dihydro-4(1H)-pyrimidinone (mirin, 1) as an inhibitor of MRN. Mirin prevents MRN-dependent activation of ATM without affecting ATM protein kinase activity, and it inhibits Mre11-associated exonuclease activity. Consistent with its ability to target the MRN complex, mirin abolishes the G2/M checkpoint and homology-dependent repair in mammalian cells.
Nature Chemical Biology 03/2008; 4(2):119-25. · 14.69 Impact Factor
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Clayton R Hunt,
Raj K Pandita,
Andrei Laszlo,
Ryuji Higashikubo,
Manjula Agarwal,
Tetsuya Kitamura,
Arun Gupta,
Nicole Rief,
Nobuo Horikoshi,
Rajeskaran Baskaran, Ji-Hoon Lee,
Markus Löbrich,
Tanya T Paull,
Joseph L Roti Roti,
Tej K Pandita
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ABSTRACT: All cells have intricately coupled sensing and signaling mechanisms that regulate the cellular outcome following exposure to genotoxic agents such as ionizing radiation (IR). In the IR-induced signaling pathway, specific protein events, such as ataxia-telangiectasia mutated protein (ATM) activation and histone H2AX phosphorylation (gamma-H2AX), are mechanistically well characterized. How these mechanisms can be altered, especially by clinically relevant agents, is not clear. Here we show that hyperthermia, an effective radiosensitizer, can induce several steps associated with IR signaling in cells. Hyperthermia induces gamma-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced gamma-H2AX foci formation is dependent on ATM but independent of heat shock protein 70 expression. Hyperthermia also enhanced ATM kinase activity and increased cellular ATM autophosphorylation. The hyperthermia-induced increase in ATM phosphorylation was independent of Mre11 function. Similar to IR, hyperthermia also induced MDC1 foci formation; however, it did not induce all of the characteristic signals associated with irradiation because formation of 53BP1 and SMC1 foci was not observed in heated cells but occurred in irradiated cells. Additionally, induction of chromosomal DNA strand breaks was observed in IR-exposed but not in heated cells. These results indicate that hyperthermia activates signaling pathways that overlap with those activated by IR-induced DNA damage. Moreover, prior activation of ATM or other components of the IR-induced signaling pathway by heat may interfere with the normal IR-induced signaling required for chromosomal DNA double-strand break repair, thus resulting in increased cellular radiosensitivity.
Cancer Research 05/2007; 67(7):3010-7. · 7.86 Impact Factor
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ABSTRACT: We have performed mutagenesis screens of the Drosophila X chromosome and the autosomes for dominant enhancers of the rough eye resulting from overexpression of liquid facets. The liquid facets gene encodes the homolog of vertebrate endocytic Epsin, an endocytic adapter protein. In Drosophila, Liquid facets is a core component of the Notch signaling pathway required in the signaling cells for ligand endocytosis and signaling. Why ligand internalization by the signaling cells is essential for signaling is a mystery. The requirement for Liquid facets is a hint at the answer, and the genes identified in this screen provide further clues. Mutant alleles of clathrin heavy chain, Rala, split ends, and auxilin were identified as enhancers. We describe the mutant alleles and mutant phenotypes of Rala and aux. We discuss the relevance of all of these genetic interactions to the function of Liquid facets in Notch signaling.
Genetics 04/2007; 175(3):1163-74. · 4.01 Impact Factor
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ABSTRACT: Mre11 and Rad50 are the catalytic components of a highly conserved DNA repair complex that functions in many aspects of DNA metabolism involving double-strand breaks. The ATPase domains in Rad50 are related to the ABC transporter family of ATPases, previously shown to share structural similarities with adenylate kinases. Here we demonstrate that Mre11/Rad50 complexes from three organisms catalyze the reversible adenylate kinase reaction in vitro. Mutation of the conserved signature motif reduces the adenylate kinase activity of Rad50 but does not reduce ATP hydrolysis. This mutant resembles a rad50 null strain with respect to meiosis and telomere maintenance in S. cerevisiae, correlating adenylate kinase activity with in vivo functions. An adenylate kinase inhibitor blocks Mre11/Rad50-dependent DNA tethering in vitro and in cell-free extracts, indicating that adenylate kinase activity by Mre11/Rad50 promotes DNA-DNA associations. We propose a model for Rad50 that incorporates both ATPase and adenylate kinase reactions as critical activities that regulate Rad50 functions.
Molecular Cell 04/2007; 25(5):647-61. · 14.18 Impact Factor
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ABSTRACT: Ataxia-telangiectasia mutated (ATM) is a serine-threonine kinase that is activated by DNA double strand breaks to phosphorylate many cellular proteins involved in cell cycle regulation and DNA repair. We have shown previously that the activation of ATM can be reconstituted in an in vitro system using recombinant human ATM. In this system, ATM activity is dependent on the Mre11/Rad50/Nbs1 (MRN) complex and linear DNA, similar to requirements observed in human cells. This chapter describes methods used for the overexpression and purification of human ATM and MRN, as well as a protocol for in vitro kinase assays.
Methods in Enzymology 02/2006; 408:529-39. · 2.04 Impact Factor
