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Sample records for acvr1 mutation activates

  1. 进行性骨化性纤维增殖不良症临床及ACVR1基因c·774G>C突变分析%A novel mutation c.774G>C in the ACVR1 gene causes fibrodysplasia ossificans progressiva in one Chinese patient

    Institute of Scientific and Technical Information of China (English)

    张菀姣; 张伟; 张克勤

    2012-01-01

    ,radiological findings and biochemical tests. For mutation detection, the blood samples from the FOP patient, his parents and 60 normal controls were collected with informed consent. Genomic DNA was isolated from peripheral lymphocytes and all the exons of ACVR1 were amplified by PCR. The PCR products sequenced directly with the cycle sequencing methods. Then we generated the three-dimensional model of protein structure for the cytoplasmic domain of the R258S in ACVR1 to evaluate its receptor function. Results; The patient had congenital minimal malformations of the great toes and radiographic evidence of heterotopic ossification at the time of evaluation. The process of heterotopic bone formation was a mild course and did not follow the typical temporal and spatial patterns. Analysis of ACVR1 gene revealed that the patient had a heterozygous missense mutation,c.774 G>C(R258S), which is located in the kinase domain of AGVR1. We also find that all the people,including the patient,the parents and 60 normal controls,occurred nonsense mutation,c.690 G> A(E230E). In the protein modeling,ARG258 and SER194 can form the H-bonds. When the ARG258 is substituted by SER.the H-bonds are lost,so the αGS1 and the αC helix conformations do shift and make ACVR1 into an "open" conformation and constituently activated. Conclusion; Most patients showing typical FOP phenotypes have the heterozygous c.617G>A(R206H) mutation belonging to the GS domain of ACVR1. Our report describes a patient affected with FOP showing mild progressive symptoms,and these atypical FOP phenotypesmay associate with a novel mutation (c.774 G>C),affecting a conserved residue of the ACVR1 kinase domain.Our findings make therelations between phenotypes and genotypes of FOP better understood.

  2. Sporadic Fibrodysplasia Ossificans Progressiva in an Egyptian Infant with c.617G > A Mutation in ACVR1 Gene: A Case Report and Review of Literature

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    Mohammad Al-Haggar

    2013-01-01

    Full Text Available Fibrodysplasia ossificans progressiva (FOP is an autosomal dominant severe musculoskeletal disease characterized by extensive new bone formation within soft connective tissues and unique skeletal malformations of the big toes which represent a birth hallmark for the disease. Most of the isolated classic cases of FOP showed heterozygous mutation in the ACVR1 gene on chromosome 2q23 that encodes a bone morphogenetic protein BMP (ALK2. The most common mutation is (c.617G > A leading to the amino acid substitution of arginine by histidine (p.Arg206His. We currently report on an Egyptian infant with a sporadic classic FOP in whom c.617G > A mutation had been documented. The patient presented with the unique congenital malformation of big toe and radiological evidence of heterotopic ossification in the back muscles. The triggering trauma was related to the infant's head, however; neither neck region nor sites of routine intramuscular vaccination given during the first year showed any ossifications. Characterization of the big toe malformation is detailed to serve as an early diagnostic marker for this rare disabling disease.

  3. The tumor suppressor gene Trp53 protects the mouse lens against posterior subcapsular cataracts and the BMP receptor Acvr1 acts as a tumor suppressor in the lens

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    Luke A. Wiley

    2011-07-01

    We previously found that lenses lacking the Acvr1 gene, which encodes a bone morphogenetic protein (BMP receptor, had abnormal proliferation and cell death in epithelial and cortical fiber cells. We tested whether the tumor suppressor protein p53 (encoded by Trp53 affected this phenotype. Acvr1 conditional knockout (Acvr1CKO mouse fiber cells had increased numbers of nuclei that stained for p53 phosphorylated on serine 15, an indicator of p53 stabilization and activation. Deletion of Trp53 rescued the Acvr1CKO cell death phenotype in embryos and reduced Acvr1-dependent apoptosis in postnatal lenses. However, deletion of Trp53 alone increased the number of fiber cells that failed to withdraw from the cell cycle. Trp53CKO and Acvr1;Trp53DCKO (double conditional knockout, but not Acvr1CKO, lenses developed abnormal collections of cells at the posterior of the lens that resembled posterior subcapsular cataracts. Cells from human posterior subcapsular cataracts had morphological and molecular characteristics similar to the cells at the posterior of mouse lenses lacking Trp53. In Trp53CKO lenses, cells in the posterior plaques did not proliferate but, in Acvr1;Trp53DCKO lenses, many cells in the posterior plaques continued to proliferate, eventually forming vascularized tumor-like masses at the posterior of the lens. We conclude that p53 protects the lens against posterior subcapsular cataract formation by suppressing the proliferation of fiber cells and promoting the death of any fiber cells that enter the cell cycle. Acvr1 acts as a tumor suppressor in the lens. Enhancing p53 function in the lens could contribute to the prevention of steroid- and radiation-induced posterior subcapsular cataracts.

  4. Deficient signaling via Alk2 (Acvr1 leads to bicuspid aortic valve development.

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    Penny S Thomas

    Full Text Available Bicuspid aortic valve (BAV is the most common congenital cardiac anomaly in humans. Despite recent advances, the molecular basis of BAV development is poorly understood. Previously it has been shown that mutations in the Notch1 gene lead to BAV and valve calcification both in human and mice, and mice deficient in Gata5 or its downstream target Nos3 have been shown to display BAVs. Here we show that tissue-specific deletion of the gene encoding Activin Receptor Type I (Alk2 or Acvr1 in the cushion mesenchyme results in formation of aortic valve defects including BAV. These defects are largely due to a failure of normal development of the embryonic aortic valve leaflet precursor cushions in the outflow tract resulting in either a fused right- and non-coronary leaflet, or the presence of only a very small, rudimentary non-coronary leaflet. The surviving adult mutant mice display aortic stenosis with high frequency and occasional aortic valve insufficiency. The thickened aortic valve leaflets in such animals do not show changes in Bmp signaling activity, while Map kinase pathways are activated. Although dysfunction correlated with some pro-osteogenic differences in gene expression, neither calcification nor inflammation were detected in aortic valves of Alk2 mutants with stenosis. We conclude that signaling via Alk2 is required for appropriate aortic valve development in utero, and that defects in this process lead to indirect secondary complications later in life.

  5. Recurrent copy number gains of ACVR1 and corresponding transcript overexpression are associated with survival in head and neck squamous cell carcinomas

    DEFF Research Database (Denmark)

    Ambrosio, Eliane P; Drigo, Sandra A; Bérgamo, Nádia A;

    2011-01-01

    AIMS: This study aimed to evaluate the copy number alteration on 2q24, its association with ACVR1 transcript expression and the prognostic value of these data in head and neck squamous cell carcinomas. METHODS AND RESULTS: Twenty-eight samples of squamous cell carcinoma were evaluated by fluoresc...

  6. Mutation specific functions of EGFR result in a mutation-specific downstream pathway activation

    NARCIS (Netherlands)

    L. Eraslan-Erdem (Lale); Y. Gao; N.K. Kloosterhof (Nanne); Y. Atlasi (Yaser); J.A.A. Demmers (Jeroen); A. Sacchetti (Andrea); J.M. Kros (Johan); P.A.E. Sillevis Smitt (Peter); J.G.J.V. Aerts (Joachim); P.J. French (Pim)

    2015-01-01

    markdownabstractBackground: Epidermal growth factor receptor (EGFR) is frequently mutated in various types of cancer. Although all oncogenic mutations are considered activating, different tumour types have different mutation spectra. It is possible that functional differences underlie this tumour-ty

  7. Novel point mutations attenuate autotaxin activity

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    Stracke Mary L

    2009-02-01

    Full Text Available Abstract Background The secreted enzyme autotaxin (ATX stimulates tumor cell migration, tumorigenesis, angiogenesis, and metastasis. ATX hydrolyzes nucleotides, but its hydrolysis of lysophospholipids to produce lysophosphatidic acid (LPA accounts for its biological activities. ATX has been identified only as a constitutively active enzyme, and regulation of its activity is largely unexplored. In spite of its presence in plasma along with abundant putative substrate LPC, the product LPA is found in plasma at unexpectedly low concentrations. It is plausible that the LPA-producing activity of ATX is regulated by its expression and by access to substrate(s. For this reason studying the interaction of enzyme with substrate is paramount to understanding the regulation of LPA production. Results In this study we determine ATX hydrolytic activities toward several artificial and natural substrates. Two novel point mutations near the enzyme active site (H226Q and H434Q confer attenuated activity toward all substrates tested. The Vmax for LPC compounds depends upon chain length and saturation; but this order does not differ among wild type and mutants. However the mutant forms show disproportionately low activity toward two artificial substrates, pNpTMP and FS-3. The mutant forms did not significantly stimulate migration responses at concentrations that produced a maximum response for WT-ATX, but this defect could be rescued by inclusion of exogenous LPC. Conclusion H226Q-ATX and H434Q-ATX are the first point mutations of ATX/NPP2 demonstrated to differentially impair substrate hydrolysis, with hydrolysis of artificial substrates being disproportionately lower than that of LPC. This implies that H226 and H434 are important for substrate interaction. Assays that rely on hydrolyses of artificial substrates (FS-3 and pNpTMP, or that rely on hydrolysis of cell-derived substrate, might fail to detect certain mutated forms of ATX that are nonetheless capable of

  8. Comprehensive fine mapping of chr12q12-14 and follow-up replication identify activin receptor 1B (ACVR1B) as a muscle strength gene

    NARCIS (Netherlands)

    Windelinckx, An; De Mars, Gunther; Huygens, Wim; Peeters, Maarten W.; Vincent, Barbara; Wijmenga, Cisca; Lambrechts, Diether; Delecluse, Christophe; Roth, Stephen M.; Metter, E. Jeffrey; Ferrucci, Luigi; Aerssens, Jeroen; Vlietinck, Robert; Beunen, Gaston P.; Thomis, Martine A.

    2011-01-01

    Muscle strength is important in functional activities of daily living and the prevention of common pathologies. We describe the two-staged fine mapping of a previously identified linkage peak for knee strength on chr12q12-14. First, 209 tagSNPs in/around 74 prioritized genes were genotyped in 500 Ca

  9. Frequent PIK3CA-activating mutations in hidradenoma papilliferums.

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    Liau, Jau-Yu; Lan, Jui; Hong, Jin-Bon; Tsai, Jia-Huei; Kuo, Kuan-Tin; Chu, Chia-Yu; Sheen, Yi-Shuan; Huang, Wen-Chang

    2016-09-01

    Hidradenoma papilliferum (HP) is a benign epithelial tumor most commonly seen in the vulva. It is proposed to be derived from the anogenital mammary-like glands and is histologically very similar to the mammary intraductal papilloma (IP). Approximately 60% of mammary IPs have activating mutations in either PIK3CA or AKT1, with each gene accounting for 30% of cases. In this study, we screened the mutation statuses of PIK3CA, AKT1, RAS, and BRAF in 30 HPs. The results showed that activating mutations in either PIK3CA or AKT1 were identified in 20 tumors (67%); 19 tumors had PIK3CA mutations (63%; 13 in exon 20 and 6 in exon 9), and 1 had an AKT1 E17K mutation (3%). BRAF V600E mutation was found in an HP that also had a PIK3CA H1047R mutation. No RAS mutation was found. The mutation status was not correlated with the degree of epithelial cell hyperplasia. We conclude that although there might be site-related variations in the mutation frequencies of PIK3CA and AKT1 genes, HP is histologically and also genetically very similar to the mammary IP, suggesting that HP can be viewed as the extramammary counterpart of mammary IP. PMID:27184479

  10. Glucocerebrosidase enzyme activity in GBA mutation Parkinson's disease.

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    Ortega, Roberto A; Torres, Paola A; Swan, Matthew; Nichols, William; Boschung, Sarah; Raymond, Deborah; Barrett, Matthew J; Johannes, Brooke A; Severt, Lawrence; Shanker, Vicki; Hunt, Ann L; Bressman, Susan; Pastores, Gregory M; Saunders-Pullman, Rachel

    2016-06-01

    Mutations in the glucocerebrosidase (GBA1) gene, the most common genetic contributor to Parkinson's disease (PD), are associated with an increased risk of PD in heterozygous and homozygous carriers. While glucocerebrosidase enzyme (GCase) activity is consistently low in Gaucher disease, there is a range of leukocyte GCase activity in healthy heterozygous GBA1 mutation carriers. To determine whether GCase activity may be a marker for PD with heterozygous GBA1 mutations (GBA1 mutation PD, GBA PD), GBA PD patients (n=15) were compared to PD patients without heterozygous GBA1 mutations (idiopathic PD; n=8), heterozygous GBA1 carriers without PD (asymptomatic carriers; n=4), and biallelic mutation carriers with PD (Gaucher disease with PD, GD1 PD; n=3) in a pilot study. GCase activity (nmol/mg protein/hour) in GD1 PD (median [interquartile range]; minimum-maximum: 6.4 [5.7]; 5.3-11) was lower than that of GBA PD (16.0 [7.0]; 11-40) (p=0.01), while GCase activity in GBA PD was lower than idiopathic PD (28.5 [15.0]; 16-56) (p=0.01) and asymptomatic carriers (25.5 [2.5]; 23-27) (p=0.04). Therefore, GCase activity appears to be a possible marker of heterozygous GBA1 mutation PD, and larger studies are warranted. Prospective studies are also necessary to determine whether lower GCase activity precedes development of PD. PMID:26857292

  11. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

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    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

  12. Novel point mutations attenuate autotaxin activity

    OpenAIRE

    Stracke Mary L; Roberts David D; Bandle Russell W; Koh Eunjin; Clair Timothy

    2009-01-01

    Abstract Background The secreted enzyme autotaxin (ATX) stimulates tumor cell migration, tumorigenesis, angiogenesis, and metastasis. ATX hydrolyzes nucleotides, but its hydrolysis of lysophospholipids to produce lysophosphatidic acid (LPA) accounts for its biological activities. ATX has been identified only as a constitutively active enzyme, and regulation of its activity is largely unexplored. In spite of its presence in plasma along with abundant putative substrate LPC, the product LPA is ...

  13. An Activin Receptor IA/Activin-Like Kinase-2 (R206H Mutation in Fibrodysplasia Ossificans Progressiva

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    Rafael Herrera-Esparza

    2013-01-01

    Full Text Available Fibrodysplasia ossificans progressiva (FOP is an exceptionally rare genetic disease that is characterised by congenital malformations of the great toes and progressive heterotopic ossification (HO in specific anatomical areas. This disease is caused by a mutation in activin receptor IA/activin-like kinase-2 (ACVR1/ALK2. A Mexican family with one member affected by FOP was studied. The patient is a 19-year-old female who first presented with symptoms of FOP at 8 years old; she developed spontaneous and painful swelling of the right scapular area accompanied by functional limitation of movement. Mutation analysis was performed in which genomic DNA as PCR amplified using primers flanking exons 4 and 6, and PCR products were digested with Cac8I and HphI restriction enzymes. The most informative results were obtained with the exon 4 flanking primers and the Cac8I restriction enzyme, which generated a 253 bp product that carries the ACVR1 617G>A mutation, which causes an amino acid substitution of histidine for arginine at position 206 of the glycine-serine (GS domain, and its mutation results in the dysregulation of bone morphogenetic protein (BMP signalling that causes FOP.

  14. Mosaic Activating Mutations in FGFR1 Cause Encephalocraniocutaneous Lipomatosis

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    Bennett, James T.; Tan, Tiong Yang; Alcantara, Diana; Tétrault, Martine; Timms, Andrew E.; Jensen, Dana; Collins, Sarah; Nowaczyk, Malgorzata J.M.; Lindhurst, Marjorie J.; Christensen, Katherine M.; Braddock, Stephen R.; Brandling-Bennett, Heather; Hennekam, Raoul C.M.; Chung, Brian; Lehman, Anna; Su, John; Ng, SuYuen; Amor, David J.; Majewski, Jacek; Biesecker, Les G.; Boycott, Kym M.; Dobyns, William B.; O’Driscoll, Mark; Moog, Ute; McDonell, Laura M.

    2016-01-01

    Encephalocraniocutaneous lipomatosis (ECCL) is a sporadic condition characterized by ocular, cutaneous, and central nervous system anomalies. Key clinical features include a well-demarcated hairless fatty nevus on the scalp, benign ocular tumors, and central nervous system lipomas. Seizures, spasticity, and intellectual disability can be present, although affected individuals without seizures and with normal intellect have also been reported. Given the patchy and asymmetric nature of the malformations, ECCL has been hypothesized to be due to a post-zygotic, mosaic mutation. Despite phenotypic overlap with several other disorders associated with mutations in the RAS-MAPK and PI3K-AKT pathways, the molecular etiology of ECCL remains unknown. Using exome sequencing of DNA from multiple affected tissues from five unrelated individuals with ECCL, we identified two mosaic mutations, c.1638C>A (p.Asn546Lys) and c.1966A>G (p.Lys656Glu) within the tyrosine kinase domain of FGFR1, in two affected individuals each. These two residues are the most commonly mutated residues in FGFR1 in human cancers and are associated primarily with CNS tumors. Targeted resequencing of FGFR1 in multiple tissues from an independent cohort of individuals with ECCL identified one additional individual with a c.1638C>A (p.Asn546Lys) mutation in FGFR1. Functional studies of ECCL fibroblast cell lines show increased levels of phosphorylated FGFRs and phosphorylated FRS2, a direct substrate of FGFR1, as well as constitutive activation of RAS-MAPK signaling. In addition to identifying the molecular etiology of ECCL, our results support the emerging overlap between mosaic developmental disorders and tumorigenesis. PMID:26942290

  15. Base substitution mutations induced by metabolically activated aflatoxin B1.

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    Foster, P L; Eisenstadt, E; Miller, J H

    1983-05-01

    We have determined the base substitutions generated by metabolically activated aflatoxin B1 in the lacI gene of a uvrB- strain of Escherichia coli. By monitoring over 70 different nonsense mutation sites, we show that activated aflatoxin B1 specifically induced GxC leads to TxA transversions. One possible pathway leading to this base change involves depurination at guanine residues. We consider this mechanism of mutagenesis in the light of our other findings that the carcinogens benzo[a]pyrene diol epoxide and N-acetoxyacetylaminofluorene also specifically induce GxC leads to TxA transversions. PMID:6405385

  16. Base substitution mutations induced by metabolically activated aflatoxin B1.

    OpenAIRE

    Foster, P. L.; Eisenstadt, E; Miller, J H

    1983-01-01

    We have determined the base substitutions generated by metabolically activated aflatoxin B1 in the lacI gene of a uvrB- strain of Escherichia coli. By monitoring over 70 different nonsense mutation sites, we show that activated aflatoxin B1 specifically induced GxC leads to TxA transversions. One possible pathway leading to this base change involves depurination at guanine residues. We consider this mechanism of mutagenesis in the light of our other findings that the carcinogens benzo[a]pyren...

  17. Protein kinase C gamma mutations in spinocerebellar ataxia 14 increase kinase activity and alter membrane targeting

    NARCIS (Netherlands)

    Verbeek, D. S.; Knight, M. A.; Harmison, G. G.; Fischbeck, K. H.; Howell, B. W.

    2005-01-01

    The protein kinase C gamma (PKCgamma) gene is mutated in spinocerebellar ataxia type 14 (SCA14). In this study, we investigated the effects of two SCA14 missense mutations, G118D and C150F, on PKCgamma function. We found that these mutations increase the intrinsic activity of PKCgamma. Direct visual

  18. Real-Time Bidirectional Pyrophosphorolysis-Activated Polymerization for Quantitative Detection of Somatic Mutations

    OpenAIRE

    Song, Najie; Zhong, Xueting; Li, Qingge

    2014-01-01

    Detection of somatic mutations for targeted therapy is increasingly used in clinical settings. However, due to the difficulties of detecting rare mutations in excess of wild-type DNA, current methods often lack high sensitivity, require multiple procedural steps, or fail to be quantitative. We developed real-time bidirectional pyrophosphorolysis-activated polymerization (real-time Bi-PAP) that allows quantitative detection of somatic mutations. We applied the method to quantify seven mutation...

  19. The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer

    DEFF Research Database (Denmark)

    Hartung, Anne-Mette; Swensen, Jeff; Uriz, Inaki E;

    2016-01-01

    Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We...

  20. ARRHYTHMOGENIC CALMODULIN MUTATIONS AFFECT THE ACTIVATION AND TERMINATION OF CARDIAC RYANODINE RECEPTOR MEDIATED CA2+ RELEASE

    DEFF Research Database (Denmark)

    Søndergaard, Mads Toft; Chazin, Walter J.; Chen, Wayne S.R.;

    We recently identified the first two human missense mutations in a calmodulin (CaM) gene (CALM1) and linked these to catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden cardiac death in young individuals1. More CaM mutations have since been identified in CALM1 and also...... in the other two CaM genes (CALM2 and CALM3). All CaM mutations are associated with severe ventricular arrhythmias. CaM regulates several key proteins governing cardiac excitation-contraction coupling (ECC), including the cardiac ryanodine receptor (RyR2) Ca2+ release channel. RyR2 mutations also dominantly...... cause CPVT, where the mutations increase the channel sensitivity to activation and enhance the propensity for pro-arrhythmogenic spontaneous Ca2+ release. Here we investigated the effect of CPVT-linked CaM mutations (N53I and N97S) and two CaM mutations identified in individuals with early onset severe...

  1. c-src activating mutation analysis in Chinese patients with colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Ye-Xiong Tan; Han-Tao Wang; Peng Zhang; Zhong-Hua Yan; Guan-Long Dai; Meng-Chao Wu; Hong-Yang Wang

    2005-01-01

    AIM: To investigate the occurrence of cellular src (c-src)activating mutation at codon 531 in colorectal cancer patients from Chinese mainland.METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay followed by sequencing and single-strand conformation polymorphism analysis were carried out to screen 110 samples of primary colorectal cancer and 20 colorectal liver metastases.RESULTS: Only one sample showed PCR-RFLP-positive results and carried somatic codon 531 mutations. No additional mutation of c-src exon 12 was found.CONCLUSION: c-src codon 531 mutation in colorectal cancer is not the cause of c-src activation.

  2. Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects.

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    Yang, Gloria; Hong, Nansook; Baier, Florian; Jackson, Colin J; Tokuriki, Nobuhiko

    2016-08-16

    How remote mutations can lead to changes in enzyme function at a molecular level is a central question in evolutionary biochemistry and biophysics. Here, we combine laboratory evolution with biochemical, structural, genetic, and computational analysis to dissect the molecular basis for the functional optimization of phosphotriesterase activity in a bacterial lactonase (AiiA) from the metallo-β-lactamase (MBL) superfamily. We show that a 1000-fold increase in phosphotriesterase activity is caused by a more favorable catalytic binding position of the paraoxon substrate in the evolved enzyme that resulted from conformational tinkering of the active site through peripheral mutations. A nonmutated active site residue, Phe68, was displaced by ∼3 Å through the indirect effects of two second-shell trajectory mutations, allowing molecular interactions between the residue and paraoxon. Comparative mutational scanning, i.e., examining the effects of alanine mutagenesis on different genetic backgrounds, revealed significant changes in the functional roles of Phe68 and other nonmutated active site residues caused by the indirect effects of trajectory mutations. Our work provides a quantitative measurement of the impact of second-shell mutations on the catalytic contributions of nonmutated residues and unveils the underlying intramolecular network of strong epistatic mutational relationships between active site residues and more remote residues. Defining these long-range conformational and functional epistatic relationships has allowed us to better understand the subtle, but cumulatively significant, role of second- and third-shell mutations in evolution. PMID:27444875

  3. Suppressor Mutations for Presenilin 1 Familial Alzheimer Disease Mutants Modulate γ-Secretase Activities.

    Science.gov (United States)

    Futai, Eugene; Osawa, Satoko; Cai, Tetsuo; Fujisawa, Tomoya; Ishiura, Shoichi; Tomita, Taisuke

    2016-01-01

    γ-Secretase is a multisubunit membrane protein complex containing presenilin (PS1) as a catalytic subunit. Familial Alzheimer disease (FAD) mutations within PS1 were analyzed in yeast cells artificially expressing membrane-bound substrate, amyloid precursor protein, or Notch fused to Gal4 transcriptional activator. The FAD mutations, L166P and G384A (Leu-166 to Pro and Gly-384 to Ala substitution, respectively), were loss-of-function in yeast. We identified five amino acid substitutions that suppress the FAD mutations. The cleavage of amyloid precursor protein or Notch was recovered by the secondary mutations. We also found that secondary mutations alone activated the γ-secretase activity. FAD mutants with suppressor mutations, L432M or S438P within TMD9 together with a missense mutation in the second or sixth loops, regained γ-secretase activity when introduced into presenilin null mouse fibroblasts. Notably, the cells with suppressor mutants produced a decreased amount of Aβ42, which is responsible for Alzheimer disease. These results indicate that the yeast system is useful to screen for mutations and chemicals that modulate γ-secretase activity.

  4. Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Uhlin, Ulla; Munch-Petersen, Birgitte

    2012-01-01

    ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket...

  5. Characterization of a tumor-associated activating mutation of the p110β PI 3-kinase.

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    Hashem A Dbouk

    Full Text Available The PI3-kinase pathway is commonly activated in tumors, most often by loss of PTEN lipid phosphatase activity or the amplification or mutation of p110α. Oncogenic mutants have commonly been found in p110α, but rarely in any of the other catalytic subunits of class I PI3-kinases. We here characterize a p110β helical domain mutation, E633K, first identified in a Her2-positive breast cancer. The mutation increases basal p110β activity, but does not affect activation of p85/p110β dimers by phosphopeptides or Gβγ. Expression of the mutant causes increases in Akt and S6K1 activation, transformation, chemotaxis, proliferation and survival in low serum. E633 is conserved among class I PI3 Ks, and its mutation in p110β is also activating. Interestingly, the E633K mutant occurs near a region that interacts with membranes in activated PI 3-kinases, and its mutation abrogates the requirement for an intact Ras-binding domain in p110β-mediated transformation. We propose that the E633K mutant activates p110β by enhancing its basal association with membranes. This study presents the first analysis of an activating oncogenic mutation of p110β.

  6. GNA14 Somatic Mutation Causes Congenital and Sporadic Vascular Tumors by MAPK Activation.

    Science.gov (United States)

    Lim, Young H; Bacchiocchi, Antonella; Qiu, Jingyao; Straub, Robert; Bruckner, Anna; Bercovitch, Lionel; Narayan, Deepak; McNiff, Jennifer; Ko, Christine; Robinson-Bostom, Leslie; Antaya, Richard; Halaban, Ruth; Choate, Keith A

    2016-08-01

    Vascular tumors are among the most common neoplasms in infants and children; 5%-10% of newborns present with or develop lesions within the first 3 months of life. Most are benign infantile hemangiomas that typically regress by 5 years of age; other vascular tumors include congenital tufted angiomas (TAs), kaposiform hemangioendotheliomas (KHEs), and childhood lobular capillary hemangiomas (LCHs). Some of these lesions can become locally invasive and unresponsive to pharmacologic intervention, leading to significant complications. Recent investigation has revealed that activating mutations in HRAS, KRAS, NRAS, GNAQ, and GNA11 can cause certain types of rare childhood vascular tumors, and we have now identified causal recurrent somatic activating mutations in GNA14 by whole-exome and targeted sequencing. We found somatic activating GNA14 c.614A>T (p.Gln205Leu) mutations in one KHE, one TA, and one LCH and a GNA11 c.547C>T (p.Arg183Cys) mutation in two LCH lesions. We examined mutation pathobiology via expression of mutant GNA14 or GNA11 in primary human endothelial cells and melanocytes. GNA14 and GNA11 mutations induced changes in cellular morphology and rendered cells growth-factor independent by upregulating the MAPK pathway. Our findings identify GNA14 mutations as a cause of childhood vascular tumors, offer insight into mechanisms of oncogenic transformation by mutations affecting Gaq family members, and identify potential targets for therapeutic intervention. PMID:27476652

  7. Mutation Analysis of the LH Receptor Gene in Leydig Cell Adenoma and Hyperplasia and Functional and Biochemical Studies of Activating Mutations of the LH Receptor Gene

    NARCIS (Netherlands)

    Boot, Annemieke M.; Lumbroso, Serge; Verhoef-Post, Miriam; Richter-Unruh, Annette; Looijenga, Leendert H. J.; Funaro, Ada; Beishuizen, Auke; van Marle, Andre; Drop, Stenvert L. S.; Themmen, Axel P. N.

    2011-01-01

    Context: Germline and somatic activating mutations in the LH receptor (LHR) gene have been reported. Objective: Our objective was to perform mutation analysis of the LHR gene of patients with Leydig cell adenoma or hyperplasia. Functional studies were conducted to compare the D578H-LHR mutant with t

  8. ISOLATION OF ENDOPHYTIC ACTINOMYCETES FROM MEDICINAL PLANTS AND ITS MUTATIONAL EFFECT IN BIOCONTROL ACTIVITY

    Directory of Open Access Journals (Sweden)

    Hema Shenpagam N.*, D. Kanchana Devi ** and Sinduja G.

    2012-11-01

    Full Text Available In the present study, the endophytic actinomycetes were collected from three medicinal plants Azadiracta indica, Ocimum sanctum and Phyllanthus amarus. Endophytic actinomycetes were isolated using different media like Starch casein agar, Starch casein nitrate agar, Actinomycetes isolation agar and Soyabean agar, while it showed more colonies in Starch casein agar. The endophytic actinomycetes were stained and biochemical tests were performed. Antimicrobial compound was purified from the filtrate by ethanol extraction method. Antagonistic activities of endophytic actinomycetes isolates were tested against bacterial pathogens (Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa and the fungi Rhizopus. For the selected isolates antibiotic resistance was checked using various antibiotic discs like Amoxycillin, Penicillin, Rifampicin and Ampicillin. The strains which showed efficient antibacterial activity were selected to study the effect of mutation by physical and chemical method. In this study, UV mutated endophytic actinomycetes increase antibiotic production than non-mutated endophytic Actinomycetes, whereas in chemical mutation it does not increase the antibiotic production.

  9. Oncogenic JAK1 and JAK2-activating mutations resistant to ATP-competitive inhibitors

    OpenAIRE

    Hornakova, Tekla; Springuel, Lorraine; Devreux, Julien; Dusa, Alexandra; Constantinescu, Stefan,; Knoops, Laurent; Renauld, Jean-Christophe

    2011-01-01

    BACKGROUND: Activating mutations in JAK1 and JAK2 have been described in patients with various hematologic malignancies including acute lymphoblastic leukemia and myeloproliferative neoplasms, leading to clinical trials with JAK inhibitors. While there has been a tremendous effort towards the development of specific JAK inhibitors, mutations conferring resistance to such drugs have not yet been observed. DESIGN AND METHODS: Taking advantage of a model of spontaneous cellular transformation...

  10. PROKR2 missense mutations associated with Kallmann syndrome impair receptor signalling activity.

    Science.gov (United States)

    Monnier, Carine; Dodé, Catherine; Fabre, Ludovic; Teixeira, Luis; Labesse, Gilles; Pin, Jean-Philippe; Hardelin, Jean-Pierre; Rondard, Philippe

    2009-01-01

    Kallmann syndrome (KS) combines hypogonadism due to gonadotropin-releasing hormone deficiency, and anosmia or hyposmia, related to defective olfactory bulb morphogenesis. In a large series of KS patients, ten different missense mutations (p.R85C, p.R85H, p.R164Q, p.L173R, p.W178S, p.Q210R, p.R268C, p.P290S, p.M323I, p.V331M) have been identified in the gene encoding the G protein-coupled receptor prokineticin receptor-2 (PROKR2), most often in the heterozygous state. Many of these mutations were, however, also found in clinically unaffected individuals, thus raising the question of their actual implication in the KS phenotype. We reproduced each of the ten mutations in a recombinant murine Prokr2, and tested their effects on the signalling activity in transfected HEK-293 cells, by measuring intracellular calcium release upon ligand-activation of the receptor. We found that all mutated receptors except one (M323I) had decreased signalling activities. These could be explained by different defective mechanisms. Three mutations (L173R, W178S, P290S) impaired cell surface-targeting of the receptor. One mutation (Q210R) abolished ligand-binding. Finally, five mutations (R85C, R85H, R164Q, R268C, V331M) presumably impaired G protein-coupling of the receptor. In addition, when wild-type and mutant receptors were coexpressed in HEK-293 cells, none of the mutant receptors that were retained within the cells did affect cell surface-targeting of the wild-type receptor, and none of the mutant receptors properly addressed at the plasma membrane did affect wild-type receptor signalling activity. This argues against a dominant negative effect of the mutations in vivo. PMID:18826963

  11. Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer.

    Directory of Open Access Journals (Sweden)

    Paolo Cossu-Rocca

    Full Text Available Triple Negative Breast Cancer (TNBC accounts for 12-24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20-40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data.PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components.PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC.Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.

  12. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    Science.gov (United States)

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS.

  13. Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERT expression in multiple cancers.

    Science.gov (United States)

    Stern, Josh Lewis; Theodorescu, Dan; Vogelstein, Bert; Papadopoulos, Nickolas; Cech, Thomas R

    2015-11-01

    Somatic mutations in the promoter of the gene for telomerase reverse transcriptase (TERT) are the most common noncoding mutations in cancer. They are thought to activate telomerase, contributing to proliferative immortality, but the molecular events driving TERT activation are largely unknown. We observed in multiple cancer cell lines that mutant TERT promoters exhibit the H3K4me2/3 mark of active chromatin and recruit the GABPA/B1 transcription factor, while the wild-type allele retains the H3K27me3 mark of epigenetic silencing; only the mutant promoters are transcriptionally active. These results suggest how a single-base-pair mutation can cause a dramatic epigenetic switch and monoallelic expression. PMID:26515115

  14. Frequencies, Laboratory Features, and Granulocyte Activation in Chinese Patients with CALR-Mutated Myeloproliferative Neoplasms.

    Directory of Open Access Journals (Sweden)

    Haixiu Guo

    Full Text Available Somatic mutations in the CALR gene have been recently identified as acquired alterations in myeloproliferative neoplasms (MPNs. In this study, we evaluated mutation frequencies, laboratory features, and granulocyte activation in Chinese patients with MPNs. A combination of qualitative allele-specific polymerase chain reaction and Sanger sequencing was used to detect three driver mutations (i.e., CALR, JAK2V617F, and MPL. CALR mutations were identified in 8.4% of cases with essential thrombocythemia (ET and 5.3% of cases with primary myelofibrosis (PMF. Moreover, 25% of polycythemia vera, 29.5% of ET, and 48.1% of PMF were negative for all three mutations (JAK2V617F, MPL, and CALR. Compared with those patients with JAK2V617F mutation, CALR-mutated ET patients displayed unique hematological phenotypes, including higher platelet counts, and lower leukocyte counts and hemoglobin levels. Significant differences were not found between Chinese PMF patients with mutants CALR and JAK2V617F in terms of laboratory features. Interestingly, patients with CALR mutations showed markedly decreased levels of leukocyte alkaline phosphatase (LAP expression, whereas those with JAK2V617F mutation presented with elevated levels. Overall, a lower mutant rate of CALR gene and a higher triple-negative rate were identified in the cohort of Chinese patients with MPNs. This result indicates that an undiscovered mutant gene may have a significant role in these patients. Moreover, these pathological features further imply that the disease biology varies considerably between mutants CALR and JAK2V617F.

  15. Activation-Induced Cytidine Deaminase Contributes to Pancreatic Tumorigenesis by Inducing Tumor-Related Gene Mutations.

    Science.gov (United States)

    Sawai, Yugo; Kodama, Yuzo; Shimizu, Takahiro; Ota, Yuji; Maruno, Takahisa; Eso, Yuji; Kurita, Akira; Shiokawa, Masahiro; Tsuji, Yoshihisa; Uza, Norimitsu; Matsumoto, Yuko; Masui, Toshihiko; Uemoto, Shinji; Marusawa, Hiroyuki; Chiba, Tsutomu

    2015-08-15

    Pancreatic ductal adenocarcinoma (PDAC) develops via an accumulation of various gene mutations. The mechanism underlying the mutations in PDAC development, however, is not fully understood. Recent insight into the close association between the mutation pattern of various cancers and specific mutagens led us to investigate the possible involvement of activation-induced cytidine deaminase (AID), a DNA editing enzyme, in pancreatic tumorigenesis. Our immunohistochemical findings revealed AID protein expression in human acinar ductal metaplasia, pancreatic intraepithelial neoplasia, and PDAC. Both the amount and intensity of the AID protein expression increased with the progression from precancerous to cancerous lesions in human PDAC tissues. To further assess the significance of ectopic epithelial AID expression in pancreatic tumorigenesis, we analyzed the phenotype of AID transgenic (AID Tg) mice. Consistent with our hypothesis that AID is involved in the mechanism of the mutations underlying pancreatic tumorigenesis, we found precancerous lesions developing in the pancreas of AID Tg mice. Using deep sequencing, we also detected Kras and c-Myc mutations in our analysis of the whole pancreas of AID Tg mice. In addition, Sanger sequencing confirmed the presence of Kras, c-Myc, and Smad4 mutations, with the typical mutational footprint of AID in precancerous lesions in AID Tg mice separated by laser capture microdissection. Taken together, our findings suggest that AID contributes to the development of pancreatic precancerous lesions by inducing tumor-related gene mutations. Our new mouse model without intentional manipulation of specific tumor-related genes provides a powerful system for analyzing the mutations involved in PDAC.

  16. Segmental basal cell naevus syndrome caused by an activating mutation in smoothened.

    Science.gov (United States)

    Khamaysi, Z; Bochner, R; Indelman, M; Magal, L; Avitan-Hersh, E; Sarig, O; Sprecher, E; Bergman, R

    2016-07-01

    Aberrant sonic hedgehog signalling, mostly due to PTCH1 mutations, has been shown to play a central role in the pathogenesis of basal cell carcinoma (BCC), as well as in basal cell naevus syndrome (BCNS). Mutations in smoothened (SMO) encoding a receptor for sonic hedgehog have been reported in sporadic BCCs but not in BCNS. We report a case with multiple BCCs, pits and comedones in a segmental distribution over the upper part of the body, along with other findings compatible with BCNS. Histopathologically, there were different types of BCC. A heterozygous mutation (c.1234C>T, p.L412F) in SMO was detected in three BCCs but not in peripheral blood lymphocytes or the uninvolved skin. These were compatible with the type 1 mosaic form of BCNS. The p.L412F mutation was found experimentally to result in increased SMO transactivating activity, and the patient responded to vismodegib therapy. Activating mutations in SMO may cause BCNS. The identification of a gain-of-function mutation in SMO causing a type 1 mosaic form of BCNS further expands our understanding of the pathogenesis of BCC, with implications for the treatment of these tumours, whether sporadic or inherited. PMID:26822128

  17. Mutations in the catalytic loop HRD motif alter the activity and function of Drosophila Src64.

    Directory of Open Access Journals (Sweden)

    Taylor C Strong

    Full Text Available The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele.

  18. Epidermal Growth Factor Receptor Activating Mutations in Squamous Histology of Lung Cancer Patients of Southern Bulgaria

    Directory of Open Access Journals (Sweden)

    Genova Silvia N.

    2015-12-01

    Full Text Available There is only limited data on the prevalence of epidermal growth factor receptor (EGFR activating mutations in squamous cell carcinomas and adenosquamous carcinomas of the lung in patients of the Southern Bulgarian region and the efficacy of EGFR tyrosine kinase inhibitors. AIM: Previous reports for Bulgarian population showed high incidence of EGFR mutations in the squamous cell carcinomas, so we set the goal to investigate their frequency in Southern Bulgaria, after precise immunohistochemical verification of lung cancers. MATERIALS AND METHODS: Two hundred and thirty-six lung carcinomas were included in this prospective study. All biopsies were initially analysed with p63, TTF1, Napsin A, CK7, CK34βE12, synaptophysin, CK20 and CDX2. Two hundred and twenty-five non-small cell lung carcinomas were studied with real-time PCR technology to assess the status of the EGFR gene. RESULTS: We detected 132 adenocarcinomas (58.7%, 89 squamous cell carcinomas (39.2%, 4 adenosquamous carcinomas (1.8%, 9 large cell neuroendocrine carcinomas (3.8% and 2 metastatic colorectal adenocarcinomas (0.8%. Activating mutations in the EGF receptor had 3 out of 89 squamous cell carcinomas (3.37%. We have established mutations in L858R, deletion in exon 19 and rare mutation in S7681. One out of four adenosquamous carcinomas had a point mutation in the L858R (25%. CONCLUSIONS: The frequency of EGFR mutations we found in lung squamous cell carcinomas in a Southern Bulgarian region is lower than that in European countries. Ethnic diversity in the region does not play role of an independent predictive factor in terms of mutation frequency.

  19. Antitumor effects and molecular mechanisms of ponatinib on endometrial cancer cells harboring activating FGFR2 mutations.

    Science.gov (United States)

    Kim, Do-Hee; Kwak, Yeonui; Kim, Nam Doo; Sim, Taebo

    2016-01-01

    Aberrant mutational activation of FGFR2 is associated with endometrial cancers (ECs). AP24534 (ponatinib) currently undergoing clinical trials has been known to be an orally available multi-targeted tyrosine kinase inhibitor. Our biochemical kinase assay showed that AP24534 is potent against wild-type FGFR1-4 and 5 mutant FGFRs (V561M-FGFR1, N549H-FGFR2, K650E-FGFR3, G697C-FGFR3, N535K-FGFR4) and possesses the strongest kinase-inhibitory activity on N549H-FGFR2 (IC50 of 0.5 nM) among all FGFRs tested. We therefore investigated the effects of AP24534 on endometrial cancer cells harboring activating FGFR2 mutations and explored the underlying molecular mechanisms. AP24534 significantly inhibited the proliferation of endometrial cancer cells bearing activating FGFR2 mutations (N549K, K310R/N549K, S252W) and mainly induced G1/S cell cycle arrest leading to apoptosis. AP24534 also diminished the kinase activity of immunoprecipitated FGFR2 derived from MFE-296 and MFE-280 cells and reduced the phosphorylation of FGFR2 and FRS2 on MFE-296 and AN3CA cells. AP24534 caused substantial reductions in ERK phosphorylation, PLCγ signaling and STAT5 signal transduction on ECs bearing FGFR2 activating mutations. Akt signaling pathway was also deactivated by AP24534. AP24534 causes the chemotherapeutic effect through mainly the blockade of ERK, PLCγ and STAT5 signal transduction on ECs. Moreover, AP24534 inhibited migration and invasion of endometrial cancer cells with FGFR2 mutations. In addition, AP24534 significantly blocked anchorage-independent growth of endometrial cancer cells. We, for the first time, report the molecular mechanisms by which AP24534 exerts antitumor effects on ECs with FGFR2 activating mutations, which would provide mechanistic insight into ongoing clinical investigations of AP24534 for ECs.

  20. SCN10A Mutation in a Patient with Erythromelalgia Enhances C-Fiber Activity Dependent Slowing.

    Science.gov (United States)

    Kist, Andreas M; Sagafos, Dagrun; Rush, Anthony M; Neacsu, Cristian; Eberhardt, Esther; Schmidt, Roland; Lunden, Lars Kristian; Ørstavik, Kristin; Kaluza, Luisa; Meents, Jannis; Zhang, Zhiping; Carr, Thomas Hedley; Salter, Hugh; Malinowsky, David; Wollberg, Patrik; Krupp, Johannes; Kleggetveit, Inge Petter; Schmelz, Martin; Jørum, Ellen; Lampert, Angelika; Namer, Barbara

    2016-01-01

    Gain-of-function mutations in the tetrodotoxin (TTX) sensitive voltage-gated sodium channel (Nav) Nav1.7 have been identified as a key mechanism underlying chronic pain in inherited erythromelalgia. Mutations in TTX resistant channels, such as Nav1.8 or Nav1.9, were recently connected with inherited chronic pain syndromes. Here, we investigated the effects of the p.M650K mutation in Nav1.8 in a 53 year old patient with erythromelalgia by microneurography and patch-clamp techniques. Recordings of the patient's peripheral nerve fibers showed increased activity dependent slowing (ADS) in CMi and less spontaneous firing compared to a control group of erythromelalgia patients without Nav mutations. To evaluate the impact of the p.M650K mutation on neuronal firing and channel gating, we performed current and voltage-clamp recordings on transfected sensory neurons (DRGs) and neuroblastoma cells. The p.M650K mutation shifted steady-state fast inactivation of Nav1.8 to more hyperpolarized potentials and did not significantly alter any other tested gating behaviors. The AP half-width was significantly broader and the stimulated action potential firing rate was reduced for M650K transfected DRGs compared to WT. We discuss the potential link between enhanced steady state fast inactivation, broader action potential width and the potential physiological consequences.

  1. Transforming activity of the c-Ha-ras oncogene having two point mutations in codons 12 and 61.

    Science.gov (United States)

    Sekiya, T; Prassolov, V S; Fushimi, M; Nishimura, S

    1985-09-01

    A recombinant plasmid carrying the human c-Ha-ras gene with two point mutations in codons 12 and 61 was constructed and its transforming activity on mouse NIH 3T3 cells was compared with those of genes with a single mutation in either codon 12 or 61. Quantitative analyses revealed that the gene with two mutations had essentially the same transforming activity as the genes with single mutations. These results indicate that a single mutation of the c-Ha-ras gene in either codon 12 or 61 is sufficient to activate the gene and that neither of the two mutation sites involved in activation of the gene needs to be intact for transforming activity.

  2. The second activating glucokinase mutation (A456V)

    DEFF Research Database (Denmark)

    Christesen, Henrik B T; Jacobsen, Bendt B; Odili, Stella;

    2002-01-01

    concentration of glucose needed to achieve the half-maximal rate of phosphorylation) from 8.04 (wild-type) to 2.53 mmol/l. The mutant's Hill coefficient was decreased, and its maximal specific activity k(cat) was increased. Mathematical modeling predicted a markedly lowered GSIR threshold of 1.5 mmol...

  3. XPD Helicase Structures And Activities: Insights Into the Cancer And Aging Phenotypes From XPD Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Fan, L.; Fuss, J.O.; Cheng, Q.J.; Arvai, A.S.; Hammel, M.; Roberts, V.A.; Cooper, P.K.; Tainer, J.A.

    2009-05-18

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  4. XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Tainer, John; Fan, Li; Fuss, Jill O.; Cheng, Quen J.; Arvai, Andrew S.; Hammel, Michal; Roberts, Victoria A.; Cooper, Priscilla K.; Tainer, John A.

    2008-06-02

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  5. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses.

    Science.gov (United States)

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F X; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A; Roffler, Stefan

    2016-01-01

    DNA (class 2) transposons are mobile genetic elements which move within their 'host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind. PMID:27599761

  6. Analysis of phenotype, enzyme activity and genotype of Chinese patients with POMT1 mutation.

    Science.gov (United States)

    Yang, Haipo; Manya, Hiroshi; Kobayashi, Kazuhiro; Jiao, Hui; Fu, Xiaona; Xiao, Jiangxi; Li, Xiaoqing; Wang, Jingmin; Jiang, Yuwu; Toda, Tatsushi; Endo, Tamao; Wu, Xiru; Xiong, Hui

    2016-08-01

    Protein O-mannosyltransferase 1 (POMT1) is a glycosyltransferase involved in α-dystroglycan glycosylation. POMT1 mutations cause a wide spectrum of clinical conditions from Walker-Warburg syndrome (WWS), which involves muscle, eye and brain abnormalities, to mild forms of limb-girdle muscular dystrophy with mental retardation. We aimed to elucidate the impact of different POMT1 mutations on the clinical phenotype. We report five Chinese patients with POMT1 mutations: one had a typical clinical manifestation of WWS, and the other four were diagnosed with congenital muscular dystrophy with mental retardation of varying severity. We analyzed the influence of the POMT1 mutations on POMT activity by assaying the patients' muscles and cultured skin fibroblasts. We demonstrated different levels of decreased POMT activity that correlated highly with decreased α-dystroglycan glycosylation. Our results suggest that POMT activity is inversely proportional to clinical severity, and demonstrate that skin fibroblasts can be used for differential diagnosis of patients with α-dystroglycanopathies. We have provided clinical, histological, enzymatic and genetic evidence of POMT1 involvement in five unrelated Chinese patients.

  7. Increased sleep spindle activity in patients with Costello syndrome (HRAS gene mutation).

    Science.gov (United States)

    Della Marca, Giacomo; Leoni, Chiara; Dittoni, Serena; Battaglia, Domenica; Losurdo, Anna; Testani, Elisa; Colicchio, Salvatore; Gnoni, Valentina; Gambardella, Maria L; Mariotti, Paolo; Alfieri, Paolo; Tartaglia, Marco; Zampino, Giuseppe

    2011-06-01

    Costello syndrome is a congenital disorder because of HRAS gene mutation, frequently associated with neurologic impairment and sleep disorders. The aims of the study were to evaluate the sleep EEG, and particularly the sleep spindles, in a population of patients with Costello syndrome and to compare them with those characterizing unaffected subjects. Eleven subjects (5 men and 6 women) with Costello syndrome were included in the study; age ranged between 18 months and 31 years (mean, 9.6 ± 9.4 years). The diagnosis was posed on the basis of established clinical criteria and confirmed molecularly. Sleep EEG was studied by means of full-night, laboratory-based video-polysomnography, performed overnight, during hospitalization. Sleep activity was quantified by means of power spectral analysis. Patients heterozygous for an HRAS mutation exhibited increased EEG power in 12- to 15-Hz activity band compared with age-matched control subjects. In conclusion, the authors observed a consistent increase in the amplitude of cortical sleep spindles in all our subjects with an HRAS mutation. These "giant" spindles were not associated with any evidence of structural damage of the cortex or the thalami and should be considered as phenotypic feature of sleep EEG activity in Costello syndrome because of HRAS mutation.

  8. Effect of point mutations on Herbaspirillum seropedicae NifA activity

    Directory of Open Access Journals (Sweden)

    B. Aquino

    2015-08-01

    Full Text Available NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

  9. Effect of point mutations on Herbaspirillum seropedicae NifA activity

    Energy Technology Data Exchange (ETDEWEB)

    Aquino, B.; Stefanello, A.A.; Oliveira, M.A.S.; Pedrosa, F.O.; Souza, E.M.; Monteiro, R.A.; Chubatsu, L.S. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

    2015-07-10

    NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

  10. Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation.

    Science.gov (United States)

    Ohka, Fumiharu; Ito, Maki; Ranjit, Melissa; Senga, Takeshi; Motomura, Ayako; Motomura, Kazuya; Saito, Kaori; Kato, Keiko; Kato, Yukinari; Wakabayashi, Toshihiko; Soga, Tomoyoshi; Natsume, Atsushi

    2014-06-01

    Isocitrate dehydrogenase 1 (IDH1), which localizes to the cytosol and peroxisomes, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and in parallel converts NADP(+) to NADPH. IDH1 mutations are frequently detected in grades 2-4 gliomas and in acute myeloid leukemias (AML). Mutations of IDH1 have been identified at codon 132, with arginine being replaced with histidine in most cases. Mutant IDH1 gains novel enzyme activity converting α-KG to D-2-hydroxyglutarate (2-HG) which acts as a competitive inhibitor of α-KG. As a result, the activity of α-KG-dependent enzyme is reduced. Based on these findings, 2-HG has been proposed to be an oncometabolite. In this study, we established HEK293 and U87 cells that stably expressed IDH1-WT and IDH1-R132H and investigated the effect of glutaminase inhibition on cell proliferation with 6-diazo-5-oxo-L-norleucine (DON). We found that cell proliferation was suppressed in IDH1-R132H cells. The addition of α-KG restored cell proliferation. The metabolic features of 33 gliomas with wild type IDH1 (IDH1-WT) and with IDH1-R132H mutation were examined by global metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the 2-HG levels were highly elevated in gliomas with IDH1-R132H mutation. Intriguingly, in gliomas with IDH1-R132H, glutamine and glutamate levels were significantly reduced which implies replenishment of α-KG by glutaminolysis. Based on these results, we concluded that glutaminolysis is activated in gliomas with IDH1-R132H mutation and that development of novel therapeutic approaches targeting activated glutaminolysis is warranted.

  11. Mutations in MAP3K7 that Alter the Activity of the TAK1 Signaling Complex Cause Frontometaphyseal Dysplasia.

    Science.gov (United States)

    Wade, Emma M; Daniel, Philip B; Jenkins, Zandra A; McInerney-Leo, Aideen; Leo, Paul; Morgan, Tim; Addor, Marie Claude; Adès, Lesley C; Bertola, Debora; Bohring, Axel; Carter, Erin; Cho, Tae-Joon; Duba, Hans-Christoph; Fletcher, Elaine; Kim, Chong A; Krakow, Deborah; Morava, Eva; Neuhann, Teresa; Superti-Furga, Andrea; Veenstra-Knol, Irma; Wieczorek, Dagmar; Wilson, Louise C; Hennekam, Raoul C M; Sutherland-Smith, Andrew J; Strom, Tim M; Wilkie, Andrew O M; Brown, Matthew A; Duncan, Emma L; Markie, David M; Robertson, Stephen P

    2016-08-01

    Frontometaphyseal dysplasia (FMD) is a progressive sclerosing skeletal dysplasia affecting the long bones and skull. The cause of FMD in some individuals is gain-of-function mutations in FLNA, although how these mutations result in a hyperostotic phenotype remains unknown. Approximately one half of individuals with FMD have no identified mutation in FLNA and are phenotypically very similar to individuals with FLNA mutations, except for an increased tendency to form keloid scars. Using whole-exome sequencing and targeted Sanger sequencing in 19 FMD-affected individuals with no identifiable FLNA mutation, we identified mutations in two genes-MAP3K7, encoding transforming growth factor β (TGF-β)-activated kinase (TAK1), and TAB2, encoding TAK1-associated binding protein 2 (TAB2). Four mutations were found in MAP3K7, including one highly recurrent (n = 15) de novo mutation (c.1454C>T [ p.Pro485Leu]) proximal to the coiled-coil domain of TAK1 and three missense mutations affecting the kinase domain (c.208G>C [p.Glu70Gln], c.299T>A [p.Val100Glu], and c.502G>C [p.Gly168Arg]). Notably, the subjects with the latter three mutations had a milder FMD phenotype. An additional de novo mutation was found in TAB2 (c.1705G>A, p.Glu569Lys). The recurrent mutation does not destabilize TAK1, or impair its ability to homodimerize or bind TAB2, but it does increase TAK1 autophosphorylation and alter the activity of more than one signaling pathway regulated by the TAK1 kinase complex. These findings show that dysregulation of the TAK1 complex produces a close phenocopy of FMD caused by FLNA mutations. Furthermore, they suggest that the pathogenesis of some of the filaminopathies caused by FLNA mutations might be mediated by misregulation of signaling coordinated through the TAK1 signaling complex. PMID:27426733

  12. TERT promoter mutations lead to high transcriptional activity under hypoxia and temozolomide treatment and predict poor prognosis in gliomas.

    Directory of Open Access Journals (Sweden)

    Chen Chen

    Full Text Available This study explored the effects of telomerase reverse transcriptase (TERT promoter mutations on transcriptional activity of the TERT gene under hypoxic and temozolomide (TMZ treatment conditions, and investigated the status and prognostic value of these mutations in gliomas.The effect of TERT promoter mutations on the transcriptional activity of the TERT gene under hypoxic and TMZ treatment conditions was investigated in glioma cells using the luciferase assay. TERT promoter mutations were detected in 101 glioma samples (grades I-IV and 49 other brain tumors by sequencing. TERT mRNA expression in gliomas was examined by real-time PCR. Hazard ratios from survival analysis of glioma patients were determined relative to the presence of TERT promoter mutations.Mutations in the TERT promoter enhanced gene transcription even under hypoxic and TMZ treatment conditions, inducing upregulation of TERT mRNA expression. Mutations were detected in gliomas, but not in meningiomas, pituitary adenomas, cavernomas, intracranial metastases, normal brain tissues, or peripheral blood of glioma patients. Patients with TERT promoter mutations had lower survival rates, even after adjusting for other known or potential risk factors, and the incidence of mutation was correlated with patient age.TERT promoter mutations were specific to gliomas. TERT promoter mutations maintained its ability of inducing high transcriptional activity even under hypoxic and TMZ treatment conditions, and the presence of mutations was associated with poor prognosis in glioma patients. These findings demonstrate that TERT promoter mutations are novel prognostic markers for gliomas that can inform prospective therapeutic strategies.

  13. Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

    KAUST Repository

    Lissanu Deribe, Yonathan

    2016-03-01

    PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

  14. The protist Trichomonas vaginalis harbors multiple lineages of transcriptionally active Mutator-like elements

    Directory of Open Access Journals (Sweden)

    Pereira Gonçalo AG

    2009-07-01

    Full Text Available Abstract Background For three decades the Mutator system was thought to be exclusive of plants, until the first homolog representatives were characterized in fungi and in early-diverging amoebas earlier in this decade. Results Here, we describe and characterize four families of Mutator-like elements in a new eukaryotic group, the Parabasalids. These Trichomonas vaginalis Mutator- like elements, or TvMULEs, are active in T. vaginalis and patchily distributed among 12 trichomonad species and isolates. Despite their relatively distinctive amino acid composition, the inclusion of the repeats TvMULE1, TvMULE2, TvMULE3 and TvMULE4 into the Mutator superfamily is justified by sequence, structural and phylogenetic analyses. In addition, we identified three new TvMULE-related sequences in the genome sequence of Candida albicans. While TvMULE1 is a member of the MuDR clade, predominantly from plants, the other three TvMULEs, together with the C. albicans elements, represent a new and quite distinct Mutator lineage, which we named TvCaMULEs. The finding of TvMULE1 sequence inserted into other putative repeat suggests the occurrence a novel TE family not yet described. Conclusion These findings expand the taxonomic distribution and the range of functional motif of MULEs among eukaryotes. The characterization of the dynamics of TvMULEs and other transposons in this organism is of particular interest because it is atypical for an asexual species to have such an extreme level of TE activity; this genetic landscape makes an interesting case study for causes and consequences of such activity. Finally, the extreme repetitiveness of the T. vaginalis genome and the remarkable degree of sequence identity within its repeat families highlights this species as an ideal system to characterize new transposable elements.

  15. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    Science.gov (United States)

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase. PMID:17850513

  16. Disease Mutations in Rab7 Result in Unregulated Nucleotide Exchange and Inappropriate Activation

    Energy Technology Data Exchange (ETDEWEB)

    B McCray; E Skordalakes; J Taylor

    2011-12-31

    Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 A crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.

  17. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    Science.gov (United States)

    Ledoux, Sarah; Guthrie, Christine

    2016-06-01

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. PMID:27072132

  18. The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer.

    Directory of Open Access Journals (Sweden)

    Anne-Mette Hartung

    2016-05-01

    Full Text Available Costello syndrome (CS may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE and creation of an Exonic Splicing Silencer (ESS. We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

  19. Convergent mutations and kinase fusions lead to oncogenic STAT3 activation in anaplastic large cell lymphoma.

    Science.gov (United States)

    Crescenzo, Ramona; Abate, Francesco; Lasorsa, Elena; Tabbo', Fabrizio; Gaudiano, Marcello; Chiesa, Nicoletta; Di Giacomo, Filomena; Spaccarotella, Elisa; Barbarossa, Luigi; Ercole, Elisabetta; Todaro, Maria; Boi, Michela; Acquaviva, Andrea; Ficarra, Elisa; Novero, Domenico; Rinaldi, Andrea; Tousseyn, Thomas; Rosenwald, Andreas; Kenner, Lukas; Cerroni, Lorenzo; Tzankov, Alexander; Ponzoni, Maurilio; Paulli, Marco; Weisenburger, Dennis; Chan, Wing C; Iqbal, Javeed; Piris, Miguel A; Zamo', Alberto; Ciardullo, Carmela; Rossi, Davide; Gaidano, Gianluca; Pileri, Stefano; Tiacci, Enrico; Falini, Brunangelo; Shultz, Leonard D; Mevellec, Laurence; Vialard, Jorge E; Piva, Roberto; Bertoni, Francesco; Rabadan, Raul; Inghirami, Giorgio

    2015-04-13

    A systematic characterization of the genetic alterations driving ALCLs has not been performed. By integrating massive sequencing strategies, we provide a comprehensive characterization of driver genetic alterations (somatic point mutations, copy number alterations, and gene fusions) in ALK(-) ALCLs. We identified activating mutations of JAK1 and/or STAT3 genes in ∼20% of 88 [corrected] ALK(-) ALCLs and demonstrated that 38% of systemic ALK(-) ALCLs displayed double lesions. Recurrent chimeras combining a transcription factor (NFkB2 or NCOR2) with a tyrosine kinase (ROS1 or TYK2) were also discovered in WT JAK1/STAT3 ALK(-) ALCL. All these aberrations lead to the constitutive activation of the JAK/STAT3 pathway, which was proved oncogenic. Consistently, JAK/STAT3 pathway inhibition impaired cell growth in vitro and in vivo. PMID:25873174

  20. Elastase Activity in Aspergillus fumigatus Can Arise by Random, Spontaneous Mutations

    Science.gov (United States)

    Álvarez-Pérez, Sergio; Blanco, Jose L.; López-Rodas, Victoria; Flores-Moya, Antonio; Costas, Eduardo; García, Marta E.

    2010-01-01

    Aspergillus fumigatus Fresenius has the capacity to degrade elastin (the principal protein of the lungs) and it is considered that elastase activity (EA) is among the most important pathogenicity factors of this mold. In particular, there is a strong correlation between EA in A. fumigatus and invasive aspergillosis. However, EA is not universal in this mold, and it is unknown whether the capacity to degrade elastin is the consequence of physiological mechanisms and/or genetic changes (putative adaptive mutations) induced after the exposure to this substrate or, on the contrary, it is due to random spontaneous mutations that occur under nonselective conditions. In order to discriminate between these possibilities, a Luria-Delbrück fluctuation analysis was carried out on an elastase-negative (EA−) A. fumigatus strain, using as selective factor a culture medium containing elastin as the sole source of nitrogen. Here we show that the EA− → EA+ transformation in A. fumigatus appears by rare, random mutations before the exposure of the strain to selective conditions. This work represents the first experimental evidence of pathogenicity factor acquisition in mycelial fungi by preselective mutation. PMID:21350652

  1. Elastase Activity in Aspergillus fumigatus Can Arise by Random, Spontaneous Mutations

    Directory of Open Access Journals (Sweden)

    Sergio Álvarez-Pérez

    2010-01-01

    Full Text Available Aspergillus fumigatus Fresenius has the capacity to degrade elastin (the principal protein of the lungs and it is considered that elastase activity (EA is among the most important pathogenicity factors of this mold. In particular, there is a strong correlation between EA in A. fumigatus and invasive aspergillosis. However, EA is not universal in this mold, and it is unknown whether the capacity to degrade elastin is the consequence of physiological mechanisms and/or genetic changes (putative adaptive mutations induced after the exposure to this substrate or, on the contrary, it is due to random spontaneous mutations that occur under nonselective conditions. In order to discriminate between these possibilities, a Luria-Delbrück fluctuation analysis was carried out on an elastase-negative (EA− A. fumigatus strain, using as selective factor a culture medium containing elastin as the sole source of nitrogen. Here we show that the EA−→EA+ transformation in A. fumigatus appears by rare, random mutations before the exposure of the strain to selective conditions. This work represents the first experimental evidence of pathogenicity factor acquisition in mycelial fungi by preselective mutation.

  2. Progranulin Mutations Affects Brain Oscillatory Activity in Fronto-Temporal Dementia

    Science.gov (United States)

    Moretti, Davide V.; Benussi, Luisa; Fostinelli, Silvia; Ciani, Miriam; Binetti, Giuliano; Ghidoni, Roberta

    2016-01-01

    Background: Mild cognitive impairment (MCI) is a clinical stage indicating a prodromal phase of dementia. This practical concept could be used also for fronto-temporal dementia (FTD). Progranulin (PGRN) has been recently recognized as a useful diagnostic biomarker for fronto-temporal lobe degeneration (FTLD) due to GRN null mutations. Electroencephalography (EEG) is a reliable tool in detecting brain networks changes. The working hypothesis of the present study is that EEG oscillations could detect different modifications among FTLD stages (FTD-MCI versus overt FTD) as well as differences between GRN mutation carriers versus non-carriers in patients with overt FTD. Materials and Methods: EEG in all patients and PGRN dosage in patients with a clear FTD were detected. The cognitive state has been investigated through mini mental state examination (MMSE). Results: MCI-FTD showed a significant lower spectral power in both alpha and theta oscillations as compared to overt FTD. GRN mutations carriers affected by FTLD show an increase in high alpha and decrease in theta oscillations as compared to non-carriers. Conclusion: EEG frequency rhythms are sensible to different stage of FTD and could detect changes in brain oscillatory activity affected by GRN mutations. PMID:26973510

  3. Novel Mutations in the Transcriptional Activator Domain of the Human TBX20 in Patients with Atrial Septal Defect

    Directory of Open Access Journals (Sweden)

    Irma Eloisa Monroy-Muñoz

    2015-01-01

    Full Text Available Background. The relevance of TBX20 gene in heart development has been demonstrated in many animal models, but there are few works that try to elucidate the effect of TBX20 mutations in human congenital heart diseases. In these studies, all missense mutations associated with atrial septal defect (ASD were found in the DNA-binding T-box domain, none in the transcriptional activator domain. Methods. We search for TBX20 mutations in a group of patients with ASD or ventricular septal defect (VSD using the High Resolution Melting (HRM method and DNA sequencing. Results. We report three missense mutations (Y309D, T370O, and M395R within the transcriptional activator domain of human TBX20 that were associated with ASD. Conclusions. This is the first association of TBX20 transcriptional activator domain missense mutations with ASD. These findings could have implications for diagnosis, genetic screening, and patient follow-up.

  4. Effects of antiandrogens on transformation and transcription activation of wild-type and mutated (LNCaP) androgen receptors

    NARCIS (Netherlands)

    C.A. Berrevoets (Cor); J. Veldscholte (Jos); E. Mulder (Eppo)

    1993-01-01

    textabstractLNCaP cells contain androgen receptors with a mutation in the steroid binding domain (Thr 868 changed to Ala) resulting in a changed hormone specificity. Both the wild-type and mutated androgen receptors were transfected into COS cells. Transcription activation was studied in cells co-tr

  5. Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

    Science.gov (United States)

    Zhong, Franklin L; Mamaï, Ons; Sborgi, Lorenzo; Boussofara, Lobna; Hopkins, Richard; Robinson, Kim; Szeverényi, Ildikó; Takeichi, Takuya; Balaji, Reshmaa; Lau, Aristotle; Tye, Hazel; Roy, Keya; Bonnard, Carine; Ahl, Patricia J; Jones, Leigh Ann; Baker, Paul; Lacina, Lukas; Otsuka, Atsushi; Fournie, Pierre R; Malecaze, François; Lane, E Birgitte; Akiyama, Masashi; Kabashima, Kenji; Connolly, John E; Masters, Seth L; Soler, Vincent J; Omar, Salma Samir; McGrath, John A; Nedelcu, Roxana; Gribaa, Moez; Denguezli, Mohamed; Saad, Ali; Hiller, Sebastian; Reversade, Bruno

    2016-09-22

    Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition. PMID:27662089

  6. Target DNA sequence directly regulates the frequency of activation-induced deaminase-dependent mutations.

    Science.gov (United States)

    Chen, Zhangguo; Viboolsittiseri, Sawanee S; O'Connor, Brian P; Wang, Jing H

    2012-10-15

    Activation-induced deaminase (AID) catalyses class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes to enhance Ab diversity. CSR involves breaking and rejoining highly repetitive switch (S) regions in the IgH (Igh) locus. S regions appear to be preferential targets of AID. To determine whether S region sequence per se, independent of Igh cis regulatory elements, can influence AID targeting efficiency and mutation frequency, we established a knock-in mouse model by inserting a core Sγ1 region into the first intron of proto-oncogene Bcl6, which is a non-Ig target of SHM. We found that the mutation frequency of the inserted Sγ1 region was dramatically higher than that of the adjacent Bcl6 endogenous sequence. Mechanistically, S region-enhanced SHM was associated with increased recruitment of AID and RNA polymerase II, together with Spt5, albeit to a lesser extent. Our studies demonstrate that target DNA sequences influence mutation frequency via regulating AID recruitment. We propose that the nucleotide sequence preference may serve as an additional layer of AID regulation by restricting its mutagenic activity to specific sequences despite the observation that AID has the potential to access the genome widely.

  7. The Use of EGFR Exon 19 and 21 Unlabeled DNA Probes to Screen for Activating Mutations in Non–Small Cell Lung Cancer

    OpenAIRE

    Willmore-Payne, Carlynn; Holden, Joseph A.; Wittwer, Carl T.; Layfield, Lester J.

    2008-01-01

    Activating mutations in epidermal growth factor receptor-1 (EGFR) are found in 10–15% of Caucasian patients with non–small cell lung carcinoma (NSCLC). Approximately 90% of the mutations are deletions of several amino acids in exon 19 or point mutations in exon 21. Some studies suggest that these mutations identify patients that might benefit from targeted EGFR inhibitor therapy. DNA melting analysis of polymerase chain reaction products can screen for these mutations to identify this patient...

  8. Disruption of dopamine neuron activity pattern regulation through selective expression of a human KCNN3 mutation.

    Science.gov (United States)

    Soden, Marta E; Jones, Graham L; Sanford, Christina A; Chung, Amanda S; Güler, Ali D; Chavkin, Charles; Luján, Rafael; Zweifel, Larry S

    2013-11-20

    The calcium-activated small conductance potassium channel SK3 plays an essential role in the regulation of dopamine neuron activity patterns. Here we demonstrate that expression of a human disease-related SK3 mutation (hSK3Δ) in dopamine neurons of mice disrupts the balance between tonic and phasic dopamine neuron activity. Expression of hSK3Δ suppressed endogenous SK currents, reducing coupling between SK channels and NMDA receptors (NMDARs) and increasing permissiveness for burst firing. Consistent with enhanced excitability of dopamine neurons, hSK3Δ increased evoked calcium signals in dopamine neurons in vivo and potentiated evoked dopamine release. Specific expression of hSK3Δ led to deficits in attention and sensory gating and heightened sensitivity to a psychomimetic drug. Sensory-motor alterations and psychomimetic sensitivity were recapitulated in a mouse model of transient, reversible dopamine neuron activation. These results demonstrate the cell-autonomous effects of a human ion channel mutation on dopamine neuron physiology and the impact of activity pattern disruption on behavior.

  9. Identification of a mutation affecting an alanine-alpha-ketoisovalerate transaminase activity in Escherichia coli K-12.

    Science.gov (United States)

    Falkinham, J O

    1979-10-01

    A mutation affecting alanine-alpha-ketoisovalerate transaminase activity has been shown to be cotransducible with ilv gene cluster. The transaminase deficiency results in conditional isoleucine auxotrophy in the presence of alanine. PMID:396446

  10. Mutations in the fourth EGF-like domain affect thrombomodulin-induced changes in the active site of thrombin

    OpenAIRE

    Koeppe, Julia R.; Beach, Muneera A.; Baerga-Ortiz, Abel; Jordan Kerns, S.; Komives, Elizabeth A.

    2008-01-01

    A number of alanine and more conservative mutants of residues in the fourth domain of thrombomodulin (TM) were prepared and assayed for protein C activation and for thrombin binding. Several of the alanine mutations appeared to cause misfolding or structural defects as assessed by poor expression and/or NMR HSQC experiments, while more conservative mutations at the same site appeared to fold correctly and retain activity. Several of the conservative mutants bound more weakly to thrombin despi...

  11. De novo RRAGC mutation activates mTORC1 signaling in syndromic fetal dilated cardiomyopathy.

    Science.gov (United States)

    Long, Pamela A; Zimmermann, Michael T; Kim, Maengjo; Evans, Jared M; Xu, Xiaolei; Olson, Timothy M

    2016-08-01

    Idiopathic dilated cardiomyopathy (DCM) is a heritable, genetically heterogeneous disorder with variable age-dependent penetrance. We sought to identify the genetic underpinnings of syndromic, sporadic DCM in a newborn female diagnosed in utero. Postnatal evaluation revealed ventricular dilation and systolic dysfunction, bilateral cataracts, and mild facial dysmorphisms. Comprehensive metabolic and genetic testing, including chromosomal microarray, mitochondrial DNA and targeted RASopathy gene sequencing, and clinical whole exome sequencing for known cardiomyopathy genes was non-diagnostic. Following exclusion of asymptomatic DCM in the parents, trio-based whole exome sequencing was carried out on a research basis, filtering for rare, predicted deleterious de novo and recessive variants. An unreported de novo S75Y mutation was discovered in RRAGC, encoding Ras-related GTP binding C, an essential GTPase in nutrient-activated mechanistic target of rapamycin complex 1 (mTORC1) signaling. In silico protein modeling and molecular dynamics simulation predicted the mutation to disrupt ligand interactions and increase the GDP-bound state. Overexpression of RagC(S75Y) rendered AD293 cells partially insensitive to amino acid deprivation, resulting in increased mTORC1 signaling compared to wild-type RagC. These findings implicate mTORC1 dysregulation through a gain-of-function mutation in RagC as a novel molecular basis for syndromic forms of pediatric heart failure, and expand genotype-phenotype correlation in RASopathy-related syndromes. PMID:27234373

  12. VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations.

    Science.gov (United States)

    Hervé, Virginie; Rabbe, Nathalie; Guilleminault, Laurent; Paul, Flora; Schlick, Laurène; Azzopardi, Nicolas; Duruisseaux, Michael; Fouquenet, Delphine; Montharu, Jérôme; Redini, Françoise; Paintaud, Gilles; Lemarié, Etienne; Cadranel, Jacques; Wislez, Marie; Heuzé-Vourc'h, Nathalie

    2014-01-01

    K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras(LA1) model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations. PMID:25484066

  13. Activating mutations in ERBB2 and their impact on diagnostics and treatment

    Directory of Open Access Journals (Sweden)

    Grit Sophie Herter-Sprie

    2013-04-01

    Full Text Available Despite the ongoing ‘war on cancer’, cancer remains one of the major causes of human morbidity and mortality. A new paradigm of targeted therapies holds the most promise for the future, making identification of tumor-specific therapeutic targets of prime importance. ERBB2, best known for its role in breast cancer tumorigenesis, can be targeted by two types of pharmacological manipulation: antibody therapy against the extracellular receptor domain and small molecule compounds against the intracellular tyrosine kinase domain. Aberrant activation of ERBB2 by gene amplification has been shown to participate in the pathophysiology of breast, ovarian, gastric, colorectal, lung, brain and head and neck tumors. However, the advent of next-generation sequencing technologies has enabled efficient identification of activating molecular alterations of ERBB2. In this review, we will focus on the functional role of these somatic mutations that cause ERBB2 receptor activation. We will additionally discuss the current preclinical and clinical therapeutic strategies for targeting mutationally activated ERBB2.

  14. Characterization of two MODY2 mutations with different susceptibility to activation

    Energy Technology Data Exchange (ETDEWEB)

    Langer, Sara; Platz, Christian; Waterstradt, Rica; Baltrusch, Simone, E-mail: simone.baltrusch@med.uni-rostock.de

    2015-09-04

    Glucokinase plays a key role in glucose sensing in pancreatic beta cells and in liver metabolism. Heterozygous inactivating glucokinase mutations cause the autosomal dominantly inherited MODY2 subtype of maturity-onset diabetes of the young. The goal of this study was to elucidate the pathogenicity of the recently described glucokinase mutants L304P and L315H, located in an alpha-helix and connecting region, respectively, at the outer region of the large domain of glucokinase. Both mutants showed wild-type-like cytosolic localization, but faster protein degradation in insulin-secreting MIN6 cells. However, strongly reduced nuclear/cytoplasmic localization of the mutants was observed in primary hepatocytes suggesting reduced interaction with the liver specific glucokinase regulatory protein. Both mutants displayed a significantly lowered glucokinase activity compared to the wild-type protein. Even though the L315H protein showed the lowest enzymatic activity, this mutant was very sensitive to allosteric activation. The endogenous activator fructose-2,6-bisphosphatase evoked an increase in glucokinase activity for both mutants, but much stronger for L315H compared to L304P. The synthetic activator RO281675 was ineffective against the L304P mutant. Expression of the mutant proteins evoked loss of glucose-induced insulin secretion in MIN6 cells. Administration of RO281675 increased insulin secretion, however, only for the L315H mutant. Thus, a glucokinase activator drug therapy may help MODY2 patients not in general, but seems to be a useful strategy for carriers of the L315H glucokinase mutation. - Highlights: • The GK mutants L304P and L315H display a highly reduced enzymatic activity. • In hepatocytes both mutations lower the nuclear/cytoplasmic localization ratio of GK. • Both mutants inhibit stimulus-secretion coupling in insulin-producing cells. • Activation by fructose-2,6-bisphosphatase and by RO281675 is stronger for L315H. • RO281675 stimulates

  15. Mutations, kataegis, and translocations in B lymphocytes: towards a mechanistic understanding of AID promiscuous activity

    Science.gov (United States)

    Casellas, Rafael; Basu, Uttiya; Yewdell, William T.; Chaudhuri, Jayanta; Robbiani, Davide F.; Di Noia, Javier M.

    2016-01-01

    As B cells engage in the immune response they express the deaminase AID to initiate the hypermutation and recombination of immunoglobulin genes, which are crucial processes for the efficient recognition and disposal of pathogens, However, AID must be tightly controlled in B cells to minimize off-targeting mutations, which can drive chromosomal translocations and the development of B cell malignancies, such as lymphomas. Recent genomic and biochemical analyses have begun to unravel the crucial question of how AID-mediated deamination is targeted outside immunoglobulin genes. Here, we discuss the transcriptional and topological features that are emerging as key drivers of AID promiscuous activity. PMID:26898111

  16. Implications of compound heterozygous insulin receptor mutations in congenital muscle fibre type disproportion myopathy for the receptor kinase activation

    DEFF Research Database (Denmark)

    Klein, H H; Müller, R; Vestergaard, H;

    1999-01-01

    We studied insulin receptor kinase activation in two brothers with congenital muscle fibre type disproportion myopathy and compound heterozygous mutations of the insulin receptor gene, their parents, and their unaffected brother. In the father who has a heterozygote Arg1174-->Gln mutation, in situ......% of the receptors to become insulin-dependently activated. The mother carries a point mutation at the last base pair in exon 17 which, due to abnormal alternative splicing, could lead to normally transcribed receptor or truncated receptor lacking the kinase region. Kinase activation was normal in the mother...... activation of the receptor kinase in skeletal muscle was reduced about 70%. Selection of only those receptors that bound to anti-phosphotyrosine antibody showed that these receptors had normal kinase activity and that the reduction in overall kinase activity was due to the inability of about 70...

  17. M-CSF receptor mutations in hereditary diffuse leukoencephalopathy with spheroids impair not only kinase activity but also surface expression

    Energy Technology Data Exchange (ETDEWEB)

    Hiyoshi, Masateru; Hashimoto, Michihiro; Yukihara, Mamiko; Bhuyan, Farzana; Suzu, Shinya, E-mail: ssuzu06@kumamoto-u.ac.jp

    2013-11-01

    Highlights: •Many mutations were identified in Fms as a putative genetic cause of HDLS. •All of the mutations tested severely impair the kinase activity. •Most of the mutations also impair the trafficking to the cell surface. •These defects further suggest that HDLS is caused by a loss of Fms function. -- Abstract: The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important role of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.

  18. A Crouzon syndrome synonymous mutation activates a 5{prime} splice site within the IIIC exon of the FGFR2 gene

    Energy Technology Data Exchange (ETDEWEB)

    Gatto, F.D.; Breathnach, R. [INSERM, Nantes (France)

    1995-06-10

    Crouzon syndrome, an autosomal dominant condition causing premature fusion of cranial structures, appears to be caused by mutations in the FGFR2 gene. Several mutations have been identified in the IIIc or bek exon that alter the amino acid sequence of the receptor in a zone known to be involved in ligand binding. In addition, a synonymous G to A transition has been described in three familial Crouzon syndrome cases (mutation at the third position of the alanine 344 codon). It has been suggested that this mutation may activate a cryptic 5{prime} or 3{prime} splice site. The significance of this latter mutation in Crouzon syndrome will be established only when it is known whether it does in fact affect splicing. If it does, prediction of the structure of the mutated receptor requires us to know whether a cryptic 5{prime} or a cryptic 3{prime} splice site has been activated. Ideally, splicing of the pre-mRNA would be studied in the cell type in which the mutated receptor is supposed to exert its effect. However, in our case this information is not available. An alternative strategy is to study splicing in cultured cells using cloned genes. The validity of this approach has been established in other disease systems, for example, thalassemias. 9 refs., 1 fig.

  19. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Monika B Dolinska

    Full Text Available BACKGROUND: Tyrosinase (TYR catalyzes the rate-limiting, first step in melanin production and its gene (TYR is mutated in many cases of oculocutaneous albinism (OCA1, an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. METHODOLOGY/PRINCIPAL FINDINGS: The intra-melanosomal domain of human tyrosinase (residues 19-469 and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. CONCLUSIONS/SIGNIFICANCE: The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

  20. Molecular spectrum of BRAF, NRAS and KRAS gene mutations in plasma cell dyscrasias: implication for MEK-ERK pathway activation.

    Science.gov (United States)

    Lionetti, Marta; Barbieri, Marzia; Todoerti, Katia; Agnelli, Luca; Marzorati, Simona; Fabris, Sonia; Ciceri, Gabriella; Galletti, Serena; Milesi, Giulia; Manzoni, Martina; Mazzoni, Mara; Greco, Angela; Tonon, Giovanni; Musto, Pellegrino; Baldini, Luca; Neri, Antonino

    2015-09-15

    Multiple myeloma (MM) is a clinically and genetically heterogeneous plasma cell (PC) malignancy. Whole-exome sequencing has identified therapeutically targetable mutations such as those in the mitogen-activated protein kinase (MAPK) pathway, which are the most prevalent MM mutations. We used deep sequencing to screen 167 representative patients with PC dyscrasias [132 with MM, 24 with primary PC leukemia (pPCL) and 11 with secondary PC leukemia (sPCL)] for mutations in BRAF, NRAS and KRAS, which were respectively found in 12%, 23.9% and 29.3% of cases. Overall, the MAPK pathway was affected in 57.5% of the patients (63.6% of those with sPCL, 59.8% of those with MM, and 41.7% of those with pPCL). The majority of BRAF variants were comparably expressed at transcript level. Additionally, gene expression profiling indicated the MAPK pathway is activated in mutated patients. Finally, we found that vemurafenib inhibition of BRAF activation in mutated U266 cells affected the expression of genes known to be associated with MM. Our data confirm and extend previous published evidence that MAPK pathway activation is recurrent in myeloma; the finding that it is mediated by BRAF mutations in a significant fraction of patients has potentially immediate clinical implications. PMID:26090869

  1. Targeting Mitogen-Activated Protein Kinase Signaling in Mouse Models of Cardiomyopathy Caused by Lamin A/C Gene Mutations.

    Science.gov (United States)

    Muchir, Antoine; Worman, Howard J

    2016-01-01

    The most frequently occurring mutations in the gene encoding nuclear lamin A and nuclear lamin C cause striated muscle diseases virtually always involving the heart. In this review, we describe the approaches and methods used to discover that cardiomyopathy-causing lamin A/C gene mutations increase MAP kinase signaling in the heart and that this plays a role in disease pathogenesis. We review different mouse models of cardiomyopathy caused by lamin A/C gene mutations and how transcriptomic analysis of one model identified increased cardiac activity of the ERK1/2, JNK, and p38α MAP kinases. We describe methods used to measure the activity of these MAP kinases in mouse hearts and then discuss preclinical treatment protocols using pharmacological inhibitors to demonstrate their role in pathogenesis. Several of these kinase inhibitors are in clinical development and could potentially be used to treat human subjects with cardiomyopathy caused by lamin A/C gene mutations.

  2. Lack of SOD1 gene mutations and activity alterations in two Italian families with amyotrophic lateral sclerosis.

    Science.gov (United States)

    Gestri, D; Cecchi, C; Tedde, A; Latorraca, S; Orlacchio, A; Grassi, E; Massaro, A M; Liguri, G; St George-Hyslop, P H; Sorbi, S

    2000-08-11

    Amyotrophic lateral sclerosis (ALS) is a progressive fatal disorder, which results from the degeneration of motor neurons in the brain and spinal cord. Approximately 20% of the inherited autosomal dominant cases are due to mutations within the gene coding for Cu/Zn superoxide dismutase 1 (SOD1), a cytosolic homodimeric enzyme that catalyzes the dismutation of toxic superoxide anion. We investigated the presence of SOD1 gene mutations and activity alterations in two unrelated families of ALS patients from Elba, an island of central Italy. No mutation in SOD1 exon 1 to 5 and no activity alteration were observed in all members of the two analyzed ALS families (FALS). These data show an apparent heterogeneous distribution of ALS patients with SOD1 gene mutations among different populations and suggest that another genetic locus could be involved in the disease. PMID:10961653

  3. Site-directed mutation of a laccase from Thermus thermophilus: Effect on the activity profile

    Directory of Open Access Journals (Sweden)

    Liu Xin

    2012-01-01

    Full Text Available A site-directed mutant R453T of a laccase from Thermus thermophilus HB27 (Tth-laccase was constructed in order to investigate the effect on laccase catalytic properties. The mutated gene was cloned and overexpressed in Escherichia coli. Nickel-affinity purification was achieved and followed by copper ion incorporation. The mature mutated enzyme was quantitatively equal to the wild type. A photometric assay based on the oxidation of the substrate 2,2-azino-bis-(3- ethylbenzthiazoline-6-sulfonate (ABTS was employed in comparison with the wild-type Tth-laccase on catalytic properties. The R453T mutant exhibited improvement in substrate affinity and specific activity at room temperature, whereas those parameters were not significantly influenced when the temperature increased up to 65°C or higher. The mutant had better catalytic activity than that of the wild type at acidic pH. Investigated by circular dichroism spectroscopy, the mutant Tth-laccase displayed similar profiles at low and high temperatures.

  4. Multiple-site mutations of phage Bp7 endolysin improves its activities against target bacteria

    Institute of Scientific and Technical Information of China (English)

    Can; Zhang; Yuanchao; Wang; Huzhi; Sun; Huiying; Ren

    2015-01-01

    The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7(Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a p ET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated(L99A, M102E) to construct p ET28a-Bp7Δe, with p ET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.

  5. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    Science.gov (United States)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  6. Detection of K-ras point mutation and telomerase activity during endoscopic retrograde cholangiopancreatography in diagnosis of pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Guo-Xiong Zhou; Jie-Fei Huang; Zhao-Shen Li; Guo-Ming Xu; Feng Liu; Hong Zhang

    2004-01-01

    AIM: To study the value of monitoring K-ras point mutation at codon 12 and telomerase activity in exfoliated cells obtained from pancreatic duct brushings during endoscopic retrograde cholangiopancreatography (ERCP) in the diagnosis of pancreatic cancer.METHODS: Exfoliated cells obtained from pancreatic duct brushings during ERCP were examined in 27 patients: 23with pancreatic cancers, 4 with chronic pancreatitis. K-fas point mutation was detected with the polymerase chain reaction and restriction fragment-length polymorphism (PCR-RFLP). Telomerase activity was detected by PCR and telomeric repeat amplification protocol assay (PCR-TRAPELISA).RESULTS: The telomerase activities in 27 patients were measured in 21 exfoliated cell samples obtained from pancreatic duct brushings. D450 value of telomerase activities in pancreatic cancer and chronic pancreatitis were 0.446±0.27and 0.041±0.0111, respectively. Seventy-seven point eight percent (14/18) of patients with pancreatic cancer and none of the patients with chronic pancreatitis showed telomerase activity in cells collected from pancreatic duct brushings when cutoff value of telomerase activity was set at 2.0. The K-ras gene mutation rate (72.2%) in pancreatic cancer was higher than that in chronic pancreatitis (33.3%)(P<0.05). In considering of both telomerase activities and K-ras point mutation, the total positive rate was 83.3%(15/18), and the specificity was 100%.CONCLUSION: Changes of telomerase activities and K-ras point mutation at codon 12 may be an early event of malignant progression in pancreatic cancer. Detection of telomerase activity and K-ras point mutation at codon 12may be complementary to each other, and is useful in diagnosis of pancreatic cancer.

  7. Acquisition of p53 mutations in response to the non-genotoxic p53 activator Nutlin-3

    OpenAIRE

    Aziz, Moammir H.; Shen, Hong; Carl G Maki

    2011-01-01

    Wild-type p53 is a stress-responsive tumor suppressor and potent growth inhibitor. Genotoxic stresses (e.g. ionizing and UV radiation or chemotherapeutic drug treatment) can activate p53, but also induce mutations in the P53 gene and thus select for p53-mutated cells. Nutlin-3a (Nutlin) is pre-clinical drug that activates p53 in a non-genotoxic fashion. Nutlin occupies the p53-binding pocket of MDM2, activating p53 by blocking the p53-MDM2 interaction. Because Nutlin neither binds p53 directl...

  8. Constitutive activation of the thyroid-stimulating hormone receptor (TSHR by mutating Ile691 in the cytoplasmic tail segment.

    Directory of Open Access Journals (Sweden)

    Zheng Liu

    Full Text Available BACKGROUND: Autosomal dominant non-autoimmune hyperthyroidism (ADNAH is a rare genetic disorder of the endocrine system. Molecular genetic studies in ADNAH have revealed heterozygous germline mutations in the TSHR. To data, mutations leading to an increase in the constitutive activation of the TSHR have been described in the transmembrane segments, exoloops and cytoplasmic loop of TSHR. These mutations result in constitutive activation of the G(αs/cAMP or G(αq/11/inositol phosphate (IP pathways, which stimulate thyroid hormone production and thyroid proliferation. METHODOLOGY/PRINCIPAL FINDINGS: In a previous study, we reported a new TSHR mutation located in the C-terminal domain of TSHR, which results in a substitution of the conserved Ile(691 for Phe. In this study, to address the question of whether the I691F mutated receptor could be responsible for G(αs/cAMP or G(αq/11/IP constitutive activity, wild-type and TSHR mutants were expressed in COS-7 cells to determine cAMP constitutive activity and IP formation. Compared to the cell surface with expression of the A623V mutated receptor as positive control, the I691F mutated receptor showed a slight increase of cAMP accumulation. Furthermore, I691F resulted in constitutive activation of the G(αq/11/IP signaling pathway. CONCLUSIONS/SIGNIFICANCE: Our results indicate that Ile(691 not only contributes to keeping TSHR inactive in the G(αs/cAMP pathways but also in the G(αq/11/IP cascade.

  9. The use of EGFR exon 19 and 21 unlabeled DNA probes to screen for activating mutations in non-small cell lung cancer.

    Science.gov (United States)

    Willmore-Payne, Carlynn; Holden, Joseph A; Wittwer, Carl T; Layfield, Lester J

    2008-07-01

    Activating mutations in epidermal growth factor receptor-1 (EGFR) are found in 10-15% of Caucasian patients with non-small cell lung carcinoma (NSCLC). Approximately 90% of the mutations are deletions of several amino acids in exon 19 or point mutations in exon 21. Some studies suggest that these mutations identify patients that might benefit from targeted EGFR inhibitor therapy. DNA melting analysis of polymerase chain reaction products can screen for these mutations to identify this patient population. However, amplicon DNA melting analysis, although easily capable of detecting heterozygous mutations by heterodimer formation, becomes more difficult if mutations are homozygous or if the mutant allele is selectively amplified over wild type. Amplification of EGFR is common in NSCLC and this could compromise mutation detection by amplicon melting analysis. To overcome this potential limitation, we developed unlabeled, single-stranded DNA probes, complimentary to EGFR exon 19 and exon 21 where the common activating mutations occur. The unlabeled probes are incorporated into a standard polymerase chain reaction during the amplification of EGFR exons 19 and 21. The probe melting peak is easily distinguished from the amplicon melting peak, and probe melting is altered if mutations are present. This allows for easy identification of activating mutations even in homozygous or amplified states and is useful in the screening of NSCLC for the common EGFR activating mutations. PMID:19137110

  10. New hyperekplexia mutations provide insight into glycine receptor assembly, trafficking, and activation mechanisms

    DEFF Research Database (Denmark)

    Bode, Anna; Wood, Sian-Elin; Mullins, Jonathan G L;

    2013-01-01

    to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated...

  11. Mutation in E1, the ubiquitin activating enzyme, reduces Drosophila lifespan and results in motor impairment.

    Science.gov (United States)

    Liu, Hsiu-Yu; Pfleger, Cathie M

    2013-01-01

    Neurodegenerative diseases cause tremendous suffering for those afflicted and their families. Many of these diseases involve accumulation of mis-folded or aggregated proteins thought to play a causal role in disease pathology. Ubiquitinated proteins are often found in these protein aggregates, and the aggregates themselves have been shown to inhibit the activity of the proteasome. These and other alterations in the Ubiquitin Pathway observed in neurodegenerative diseases have led to the question of whether impairment of the Ubiquitin Pathway on its own can increase mortality or if ongoing neurodegeneration alters Ubiquitin Pathway function as a side-effect. To address the role of the Ubiquitin Pathway in vivo, we studied loss-of-function mutations in the Drosophila Ubiquitin Activating Enzyme, Uba1 or E1, the most upstream enzyme in the Ubiquitin Pathway. Loss of only one functional copy of E1 caused a significant reduction in adult lifespan. Rare homozygous hypomorphic E1 mutants reached adulthood. These mutants exhibited further reduced lifespan and showed inappropriate Ras activation in the brain. Removing just one functional copy of Ras restored the lifespan of heterozygous E1 mutants to that of wild-type flies and increased the survival of homozygous E1 mutants. E1 homozygous mutants also showed severe motor impairment. Our findings suggest that processes that impair the Ubiquitin Pathway are sufficient to cause early mortality. Reduced lifespan and motor impairment are seen in the human disease X-linked Infantile Spinal Muscular Atrophy, which is associated with mutation in human E1 warranting further analysis of these mutants as a potential animal model for study of this disease.

  12. A mutation in a new gene bglJ, activates the bgl operon in Escherichia coli K-12

    Energy Technology Data Exchange (ETDEWEB)

    Giel, M.; Desnoyer, M.; Lopilato, J. [Simmons College, Boston, MA (United States)

    1996-06-01

    A new mutation , bglJ4, has been characterized that results in the expression of the silent bgl operon. The bgl operon encodes proteins necessary for the transport and utilization of the aromatic {beta}-glucosides arbutin and salicin. A variety of mutations activate the operon and result in a Bgl{sup +} phenotype. Activating mutations are located upstream of the bgl promoter and in genes located elsewhere on the chromosome. Mutations outside of the bgl operon occur in the genes encoding DNA gyrase and in the gene encoding the nucleoid associated protein H-NS. The mutation described here, bglJ4, has been mapped to a new locus at min 99 on the Escherichia coli K-12 genetic map. The putative protein encoded by the bglJ gene has homology to a family of transcriptional activators. Evidence is presented that increased expression of the bglJ product is needed for activation of the bgl operon. 56 refs., 3 figs., 3 tabs.

  13. Rhabdomyolysis-Associated Mutations in Human LPIN1 Lead to Loss of Phosphatidic Acid Phosphohydrolase Activity.

    Science.gov (United States)

    Schweitzer, George G; Collier, Sara L; Chen, Zhouji; Eaton, James M; Connolly, Anne M; Bucelli, Robert C; Pestronk, Alan; Harris, Thurl E; Finck, Brian N

    2015-01-01

    Rhabdomyolysis is an acute syndrome due to extensive injury of skeletal muscle. Recurrent rhabdomyolysis is often caused by inborn errors in intermediary metabolism, and recent work has suggested that mutations in the human gene encoding lipin 1 (LPIN1) may be a common cause of recurrent rhabdomyolysis in children. Lipin 1 dephosphorylates phosphatidic acid to form diacylglycerol (phosphatidic acid phosphohydrolase; PAP) and acts as a transcriptional regulatory protein to control metabolic gene expression. Herein, a 3-year-old boy with severe recurrent rhabdomyolysis was determined to be a compound heterozygote for a novel c.1904T>C (p.Leu635Pro) substitution and a previously reported genomic deletion of exons 18-19 (E766-S838_del) in LPIN1. Western blotting with patient muscle biopsy lysates demonstrated a marked reduction in lipin 1 protein, while immunohistochemical staining for lipin 1 showed abnormal subcellular localization. We cloned cDNAs to express recombinant lipin 1 proteins harboring pathogenic mutations and showed that the E766-S838_del allele was not expressed at the RNA or protein level. Lipin 1 p.Leu635Pro was expressed, but the protein was less stable, was aggregated in the cytosol, and was targeted for proteosomal degradation. Another pathogenic single amino acid substitution, lipin 1 p.Arg725His, was well expressed and retained its transcriptional regulatory function. However, both p.Leu635Pro and p.Arg725His proteins were found to be deficient in PAP activity. Kinetic analyses demonstrated a loss of catalysis rather than diminished substrate binding. These data suggest that loss of lipin 1-mediated PAP activity may be involved in the pathogenesis of rhabdomyolysis in lipin 1 deficiency. PMID:25967228

  14. Rhabdomyolysis-Associated Mutations in Human LPIN1 Lead to Loss of Phosphatidic Acid Phosphohydrolase Activity.

    Science.gov (United States)

    Schweitzer, George G; Collier, Sara L; Chen, Zhouji; Eaton, James M; Connolly, Anne M; Bucelli, Robert C; Pestronk, Alan; Harris, Thurl E; Finck, Brian N

    2015-01-01

    Rhabdomyolysis is an acute syndrome due to extensive injury of skeletal muscle. Recurrent rhabdomyolysis is often caused by inborn errors in intermediary metabolism, and recent work has suggested that mutations in the human gene encoding lipin 1 (LPIN1) may be a common cause of recurrent rhabdomyolysis in children. Lipin 1 dephosphorylates phosphatidic acid to form diacylglycerol (phosphatidic acid phosphohydrolase; PAP) and acts as a transcriptional regulatory protein to control metabolic gene expression. Herein, a 3-year-old boy with severe recurrent rhabdomyolysis was determined to be a compound heterozygote for a novel c.1904T>C (p.Leu635Pro) substitution and a previously reported genomic deletion of exons 18-19 (E766-S838_del) in LPIN1. Western blotting with patient muscle biopsy lysates demonstrated a marked reduction in lipin 1 protein, while immunohistochemical staining for lipin 1 showed abnormal subcellular localization. We cloned cDNAs to express recombinant lipin 1 proteins harboring pathogenic mutations and showed that the E766-S838_del allele was not expressed at the RNA or protein level. Lipin 1 p.Leu635Pro was expressed, but the protein was less stable, was aggregated in the cytosol, and was targeted for proteosomal degradation. Another pathogenic single amino acid substitution, lipin 1 p.Arg725His, was well expressed and retained its transcriptional regulatory function. However, both p.Leu635Pro and p.Arg725His proteins were found to be deficient in PAP activity. Kinetic analyses demonstrated a loss of catalysis rather than diminished substrate binding. These data suggest that loss of lipin 1-mediated PAP activity may be involved in the pathogenesis of rhabdomyolysis in lipin 1 deficiency.

  15. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.

    Directory of Open Access Journals (Sweden)

    Yana Pikman

    2006-07-01

    Full Text Available BACKGROUND: The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV, essential thrombocytosis (ET, and myelofibrosis with myeloid metaplasia (MF. Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR, the thrombopoietin receptor (MPL, or the granulocyte-colony stimulating factor receptor (GCSFR. METHODS AND FINDINGS: DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L in 9% (4/45 of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9-4.0 x 10(12/L, marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis. CONCLUSIONS: Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L

  16. Substrate activation of brewers' yeast pyruvate decarboxylase is abolished by mutation of cysteine 221 to serine.

    Science.gov (United States)

    Baburina, I; Gao, Y; Hu, Z; Jordan, F; Hohmann, S; Furey, W

    1994-05-10

    Brewers' yeast pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate and Mg(II)-dependent enzyme, isolated from Saccharomyces cerevisiae possesses four cysteines/subunit at positions 69, 152, 221, and 222. Earlier studies conducted on a variant of the enzyme with a single Cys at position 221 (derived from a gene that was the product of spontaneous fusion) showed that this enzyme is still subject to substrate activation [Zeng, X., Farrenkopf, B., Hohmann, S., Jordan, F., Dyda, F., & Furey, W. (1993) Biochemistry 32, 2704-2709], indicating that if Cys was responsible for this activation, it had to be C221. To further test the hypothesis, the C221S and C222S single and the C221S-C222S double mutants were constructed. It is clearly shown that the mutation at C221, but not at C222, leads to abolished substrate activation according to a number of kinetic criteria, both steady state and pre steady state. On the basis of the three-dimensional structure of the enzyme [Dyda, F., Furey, W., Swaminathan, S., Sax, M., Farrenkopf, B., Jordan, F. (1993) Biochemistry 32, 6165-6170], it is obvious that while C221 is located on the beta domain, whereas thiamin diphosphate is wedged at the interface of the alpha and gamma domains, addition of pyruvate or pyruvamide as a hemiketal adduct to the sulfur of C221 can easily bridge the gap between the beta and alpha domains. In fact, residues in one or both domains must be dislocated by this adduct formation. It is very likely that regulation as expressed in substrate activation is transmitted via this direct contact made between the two domains in the presence of the activator.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Hierarchical modeling of activation mechanisms in the ABL and EGFR kinase domains: thermodynamic and mechanistic catalysts of kinase activation by cancer mutations.

    Directory of Open Access Journals (Sweden)

    Anshuman Dixit

    2009-08-01

    Full Text Available Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition

  18. The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia

    OpenAIRE

    Levine, Ross L; Loriaux, Marc; Huntly, Brian J.P.; Loh, Mignon L.; Beran, Miroslav; Stoffregen, Eric; Berger, Roland; Clark, Jennifer J; Willis, Stephanie G; Kim T. Nguyen; Flores, Nikki J.; Estey, Elihu; Gattermann, Norbert; Armstrong, Scott; Look, A. Thomas

    2005-01-01

    Activating mutations in tyrosine kinases have been identified in hematopoietic and nonhematopoietic malignancies. Recently, we and others identified a single recurrent somatic activating mutation (JAK2V617F) in the Janus kinase 2 (JAK2) tyrosine kinase in the myeloproliferative disorders (MPDs) polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. We used direct sequence analysis to determine if the JAK2V617F mutation was present in acute myeloid leukemia (A...

  19. Analysis of Somatic Mutations in Cancer: Molecular Mechanisms of Activation in the ErbB Family of Receptor Tyrosine Kinases

    International Nuclear Information System (INIS)

    The ErbB/EGFR/HER family of kinases consists of four homologous receptor tyrosine kinases which are important regulatory elements in many cellular processes, including cell proliferation, differentiation, and migration. Somatic mutations in, or over-expression of, the ErbB family is found in many cancers and is correlated with a poor prognosis; particularly, clinically identified mutations found in non-small-cell lung cancer (NSCLC) of ErbB1 have been shown to increase its basal kinase activity and patients carrying these mutations respond remarkably to the small tyrosine kinase inhibitor gefitinib. Here, we analyze the potential effects of the currently catalogued clinically identified mutations in the ErbB family kinase domains on the molecular mechanisms of kinase activation. Recently, we identified conserved networks of hydrophilic and hydrophobic interactions characteristic to the active and inactive conformation, respectively. Here, we show that the clinically identified mutants influence the kinase activity in distinctive fashion by affecting the characteristic interaction networks

  20. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    Science.gov (United States)

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. PMID:26980729

  1. Gain-of-function STAT1 mutations impair STAT3 activity in patients with chronic mucocutaneous candidiasis (CMC)

    NARCIS (Netherlands)

    Zheng, J.; Veerdonk, F.L. van de; Crossland, K.L.; Smeekens, S.P.; Chan, C.M.; Shehri, T. Al; Abinun, M.; Gennery, A.R.; Mann, J.; Lendrem, D.W.; Netea, M.G.; Rowan, A.D.; Lilic, D.

    2015-01-01

    Signal transducer and activator of transcription 3 (STAT3) triggered production of Th-17 cytokines mediates protective immunity against fungi. Mutations affecting the STAT3/interleukin 17 (IL-17) pathway cause selective susceptibility to fungal (Candida) infections, a hallmark of chronic mucocutaneo

  2. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    Science.gov (United States)

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

  3. Fine tuning of the catalytic activity of colicin e7 nuclease domain by systematic n-terminal mutations

    DEFF Research Database (Denmark)

    Németh, Eszter; Körtvélyesi, Tamás; Thulstrup, Peter W.;

    2014-01-01

    The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446–449NColE75KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuc...

  4. KDR activating mutations in human angiosarcomas are sensitive to specific kinase inhibitors.

    Science.gov (United States)

    Antonescu, Cristina R; Yoshida, Akihiko; Guo, Tianhuo; Chang, Ning-En; Zhang, Lei; Agaram, Narasimhan P; Qin, Li-Xuan; Brennan, Murray F; Singer, Samuel; Maki, Robert G

    2009-09-15

    Angiosarcomas (AS) represent a heterogeneous group of malignant vascular tumors occurring not only in different anatomic locations but also in distinct clinical settings, such as radiation or associated chronic lymphedema. Although representing only 1% to 2% of soft tissue sarcomas, vascular sarcomas provide unique insight into the general process of tumor angiogenesis. However, no molecular candidates have been identified to guide a specific therapeutic intervention. By expression profiling, AS show distinct up-regulation of vascular-specific receptor tyrosine kinases, including TIE1, KDR, SNRK, TEK, and FLT1. Full sequencing of these five candidate genes identified 10% of patients harboring KDR mutations. A KDR-positive genotype was associated with strong KDR protein expression and was restricted to the breast anatomic site with or without prior exposure to radiation. Transient transfection of KDR mutants into COS-7 cells showed ligand-independent activation of the kinase, which was inhibited by specific KDR inhibitors. These data provide a basis for the activity of vascular endothelial growth factor receptor-directed therapy in the treatment of primary and radiation-induced AS. PMID:19723655

  5. Splice mutations preserve myophosphorylase activity that ameliorates the phenotype in McArdle disease

    DEFF Research Database (Denmark)

    Vissing, John; Duno, Morten; Schwartz, Marianne;

    2009-01-01

    Over 100 mutations in the myophosphorylase gene, which cause McArdle disease, are known. All these mutations have resulted in a complete block of muscle glycogenolysis, and accordingly, no genotype-phenotype correlation has been identified in this condition. We evaluated physiologic and genetic...

  6. Targeting EZH2 methyltransferase activity in ARID1A mutated cancer cells is synthetic lethal

    Science.gov (United States)

    Biter, Benjamin G.; Aird, Katherine M.; Garipov, Azat; Li, Hua; Amatangelo, Michael; Kossenkov, Andrew V.; Schultz, David C.; Liu, Qin; Shih, Ie-Ming; Conejo-Garcia, Jose R.; Speicher, David W.; Zhang, Rugang

    2015-01-01

    ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently has no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A mutated ovarian cancer cells. ARID1A mutational status correlates with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct ARID1A/EZH2 target, which is upregulated by EZH2 inhibition and contributes to the observed synthetic lethality by inhibiting PI3K/AKT signaling. Significantly, EZH2 inhibition causes regression of ARID1A mutated ovarian tumors in vivo. Together, these data demonstrate for the first time a synthetic lethality between ARID1A mutation and EZH2 inhibition. They indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for ARID1A mutated cancers. PMID:25686104

  7. Achievement of Cure with Gefitinib in Advanced Lung Adenocarcinoma Harboring an Activating EGFR Mutation: A Case Report

    Directory of Open Access Journals (Sweden)

    Taiji Kuwata

    2016-10-01

    Full Text Available Tyrosine kinase inhibitors (TKIs of the epidermal growth factor receptor (EGFR may achieve long-term survival in selected cases with advanced non-small cell lung cancer harboring activating mutations in the EGFR gene, but a cured case has not been reported yet. Here, we present the first case of EGFR-mutated lung adenocarcinoma cured with an EGFR-TKI, as the 75-year-old Japanese man has achieved complete response with gefitinib treatment and has survived without tumor 10 years after termination of gefitinib treatment.

  8. Activating mutations of STAT5B and STAT3 in lymphomas derived from ??-T or NK cells.

    OpenAIRE

    Lack, Nathan A.; Şen, Emel; Kucuk, Can; Jiang, Bei; Hu, Xiaozhou; Zhang, Wenyan; Chan, John K. C.; Xiao, Wenming; Alkan, Can; Williams, John C.; Avery, Kendra N.; Kavak, Pinar; Scuto, Anna; Gaulard, Philippe; Staudt, Lou; Iqbal, Javeed; Zhang, Weiwei; Cornish, Adam; Gong, Qiang; Yang, Qunpei; Sun, Hong; d'Amore, Francesco; Leppa, Sirpa; Liu, Weiping; Fu, Kai; de Leval, Laurence; McKeithan, Timothy; Chan, Wing C.

    2015-01-01

    Lymphomas arising from NK or gamma delta-T cells are very aggressive diseases and little is known regarding their pathogenesis. Here we report frequent activating mutations of STAT3 and STAT5B in NK/T-cell lymphomas (n - 51), gamma delta-T-cell lymphomas (n - 43) and their cell lines (n = 9) through next generation and/or Sanger sequencing. STAT5B N642H is particularly frequent in all forms of gamma delta-T-cell lymphomas. STAT3 and STAT5B mutations are associated with increased phosphorylate...

  9. Localization of active, dually phosphorylated extracellular signal-regulated kinase 1 and 2 in colorectal cancer with or without activating BRAF and KRAS mutations

    DEFF Research Database (Denmark)

    Holck, Susanne; Bonde, Jesper; Pedersen, Helle;

    2016-01-01

    . The same applied to 4 of the 14 KRAS(m) tumors. A phophorylation-insensitive antibody demonstrated that lack of pERK staining did not reflect defect expression of ERK1/2 protein. Thus, increased staining for pERK does not correlate to BRAF or KRAS mutations even with a highly optimized procedure. Further......Colorectal cancers (CRC) often show activating mutations of the KRAS or BRAF genes, which stimulate the extracellular signal-regulated kinase (ERK) pathway, thus increasing cell proliferation and inhibiting apoptosis. However, immunohistochemical results on ERK activation in such tumors differ...... greatly. Recently, using a highly optimized immunohistochemical method, we obtained evidence that high levels of ERK activation in rectal adenocarcinomas were associated with resistance to radiochemotherapy. In order to determine whether KRAS and/or BRAF mutations correlate to immunohistochemically...

  10. In vitro mutation breeding for seed protein, alpha amylase activity and herbicide resistance in bulrush millet (Peenisetum nigritarum)

    International Nuclear Information System (INIS)

    In vitro mutation breeding in bulrush millet (Penissetum nigritarum) was investigated using chemical mutagenesis followed by selection. Mutations were induced by soaking dry viable seeds at room temperature in 8 mM ethylmethanesulphonate (EMS) for 15 hours or in 64 mM EMS for 3, 6, 9, 12, 15 and 24 hours. After treatment and rinsing in running water, the seeds were germinated in Petri dishes and later transplanted to loamy sand oil and grown to maturity in the greenhouse. Screening and analysis of 2500 M2 plants yielded a broad spectrum of mutations, including leaf variation and agronomically useful attributes such as improved and early germinability (+15% and +10 hours, respectively), and a higher seed protein content, alpha amylase activity and herbicide resistance. EMS did not reduce cell viability, but it produced a high frequency of mutations, accompanied by a relatively low frequency of chromosomal aberrations. The appropriate dosage and duration for mutation breeding was 8 mM for 15 hours or 64 mM for 3 hours. (author). 9 refs, 2 figs, 4 tabs

  11. Impact of nonsynonymous mutations of factor X on the functions of factor X and anticoagulant activity of edoxaban.

    Science.gov (United States)

    Noguchi, Kengo; Morishima, Yoshiyuki; Takahashi, Shinichi; Ishihara, Hiroaki; Shibano, Toshiro; Murata, Mitsuru

    2015-03-01

    Edoxaban is an oral direct factor Xa (FXa) inhibitor and its efficacy as an oral anticoagulant is less subject to drug-food and drug-drug interaction than existing vitamin K antagonists. Although this profile of edoxaban suggests it is well suited for clinical use, it is not clear whether genetic variations of factor X influence the activity of edoxaban. Our aim was to investigate a possible impact of single-nucleotide polymorphisms (SNPs) in the factor X gene on the functions of factor X and the activity of edoxaban. Two nonsynonymous SNPs within mature factor X, Ala152Thr and Gly192Arg, were selected as possible candidates that might affect the functions of FXa and the activity of edoxaban. We measured catalytic activities of wild type and mutant FXas in a chromogenic assay using S-2222 and coagulation times including prothrombin time (PT) and activated partial thrombin time (aPTT) of plasma-containing recombinant FXs in the presence and absence of edoxaban. Michaelis-Menten kinetic parameters of FXas, Km and Vmax values, PT and aPTT were not influenced by either mutation indicating these mutations do not affect the FXa catalytic and coagulation activities. The Ki values of edoxaban for the FXas and the concentrations of edoxaban required to double PT and aPTT were not different between wild type and mutated FXas indicating that both mutations have little impact on the activity of edoxaban. In conclusion, these data suggest that edoxaban has little interpatient variability stemming from SNPs in the factor X gene. PMID:24911450

  12. Cellular hyper-excitability caused by mutations that alter the activation process of voltage-gated sodium channels

    Directory of Open Access Journals (Sweden)

    Mohamed-Yassine eAMAROUCH

    2015-02-01

    Full Text Available Voltage-gated sodium channels (Nav are widely expressed as macro-molecular complexes in both excitable and non-excitable tissues. In excitable tissues, the upstroke of the action potential is the result of the passage of a large and rapid influx of sodium ions through these channels. NaV dysfunction has been associated with an increasingly wide range of neurological, muscular and cardiac disorders. The purpose of this review is to summarize the recently identified sodium channel mutations that are linked to hyper-excitability phenotypes and associated with the alteration of the activation process of voltage gated sodium channels. Indeed, several clinical manifestations that demonstrate an alteration of tissue excitability were recently shown to be strongly associated with the presence of mutations that affect the activation process of the voltage-gated sodium channels. These emerging genotype-phenotype correlations have expanded the clinical spectrum of sodium channelopathies to include disorders which feature a hyper-excitability phenotype that may or may not be associated with a cardiomyopathy. The p.I141V mutation in SCN4A and SCN5A, as well as its homologous p.I136V mutation in SCN9A, are interesting examples of mutations that have been linked to inherited hyperexcitability myotonia, exercise-induced polymorphic ventricular arrhythmias and erythromelalgia, respectively. Regardless of which sodium channel isoform is investigated, the substitution of the isoleucine to valine in the locus 141 induces similar modifications in the biophysical properties of the voltage-gated sodium channels by shifting the voltage-dependence of steady state activation towards more negative potentials.

  13. The m.13051G>A mitochondrial DNA mutation results in variable neurology and activated mitophagy

    OpenAIRE

    Dombi, E.; Diot, A.; Morten, K.; Carver, J; Lodge, T.; Fratter, C.; Ng, Y.S.; Liao, C.; Muir, R; Blakely, E.L.; Hargreaves, I; Al-Dosary, M.; Sarkar, G; Hickman, S. J.; Downes, S M

    2016-01-01

    Maternally inherited mitochondrial DNA (mtDNA) mutations cause symptoms of Leber hereditary optic neuropathy (LHON) in -1 in 30,000 individuals. Most of the affected individuals lack respiratory chain defects1 and there is no proven prophylactic treatment.

  14. Moxifloxacin Retains Antimycobacterial Activity in the Presence of gyrA Mutations

    OpenAIRE

    McGrath, Marieta; Gey van Pittius, Nico C.; Sirgel, Frederick A.; van Helden, Paul D.; Warren, Robin M

    2014-01-01

    Moxifloxacin-resistant Mycobacterium tuberculosis mutants were selected in vitro using different concentrations of moxifloxacin. gyrA mutations at codons 88 and 94 were associated with resistance (defined as an MIC of ≥2 μg/ml) (P < 0.0001 and P = 0.0053, respectively). Despite the presence of gyrA mutations, moxifloxacin significantly impedes bacterial growth, supporting its use for the treatment of ofloxacin-resistant M. tuberculosis.

  15. VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations

    OpenAIRE

    Hervé, Virginie; Rabbe, Nathalie; Guilleminault, Laurent; Paul, Flora; Schlick, Laurène; Azzopardi, Nicolas; Duruisseaux, Michael; Fouquenet, Delphine; Montharu, Jérôme; Redini, Françoise; Paintaud, Gilles; Lemarié, Etienne; Cadranel, Jacques; Wislez, Marie; Heuzé-Vourc’h, Nathalie

    2014-01-01

    K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the del...

  16. Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations

    Directory of Open Access Journals (Sweden)

    Pavlov Youri I

    2004-05-01

    Full Text Available Abstract Background DNA polymerase ε (Pol ε is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol ε active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect. Results Purified four-subunit Y831A Pol ε turns over more deoxynucleoside triphosphates to deoxynucleoside monophosphates than does wild-type Pol ε, suggesting altered coordination between the polymerase and exonuclease active sites. The pol2-Y831A mutation suppresses the mutator effect of the pol2-4 mutation in the exonuclease active site that abolishes proofreading by Pol ε, as measured in haploid strain with the pol2-Y831A,4 double mutation. Analysis of mutation rates in diploid strains reveals that the pol2-Y831A allele is recessive to pol2-4. In addition, the mutation rates of strains with the pol2-4 mutation in combination with active site mutator mutations in Pol δ and Pol α suggest that Pol ε may proofread certain errors made by Pol α and Pol δ during replication in vivo. Conclusions Our data suggest that Y831A replacement in Pol ε reduces replication fidelity and its participation in chromosomal replication, but without eliminating an additional function that is essential for viability. This suggests that other polymerases can substitute for certain functions of polymerase ε.

  17. Mutator activity induced by microRNA-155 (miR-155) links inflammation and cancer.

    Science.gov (United States)

    Tili, Esmerina; Michaille, Jean-Jacques; Wernicke, Dorothee; Alder, Hansjuerg; Costinean, Stefan; Volinia, Stefano; Croce, Carlo M

    2011-03-22

    Infection-driven inflammation has been implicated in the pathogenesis of ~15-20% of human tumors. Expression of microRNA-155 (miR-155) is elevated during innate immune response and autoimmune disorders as well as in various malignancies. However, the molecular mechanisms providing miR-155 with its oncogenic properties remain unclear. We examined the effects of miR-155 overexpression and proinflammatory environment on the frequency of spontaneous hypoxanthine phosphoribosyltransferase (HPRT) mutations that can be detected based on the resistance to 6-thioguanine. Both miR-155 overexpression and inflammatory environment increased the frequency of HPRT mutations and down-regulated WEE1 (WEE1 homolog-S. pombe), a kinase that blocks cell-cycle progression. The increased frequency of HPRT mutation was only modestly attributable to defects in mismatch repair machinery. This result suggests that miR-155 enhances the mutation rate by simultaneously targeting different genes that suppress mutations and decreasing the efficiency of DNA safeguard mechanisms by targeting of cell-cycle regulators such as WEE1. By simultaneously targeting tumor suppressor genes and inducing a mutator phenotype, miR-155 may allow the selection of gene alterations required for tumor development and progression. Hence, we anticipate that the development of drugs reducing endogenous miR-155 levels might be key in the treatment of inflammation-related cancers. PMID:21383199

  18. Up-regulation effect of hepatitis B virus genome A1846T mutation on viral replication and core promoter activity

    Directory of Open Access Journals (Sweden)

    Ling JIANG

    2013-01-01

    Full Text Available Objective  To evaluate the influence of hepatitis B virus (HBV genome nucleotide A1846T mutation on the viral replication capacity and the transcription activity of HBV core promoter (CP in vitro. Methods  A total of 385 patients with hepatitis B admitted to the 302 Hospital of PLA were enrolled in the study, including 116 with moderate chronic hepatitis B (CHB-M, 123 with severe chronic hepatitis B (CHB-S, and 146 with acute-on-chronic liver failure (ACLF. Serum HBV DNA was isolated and full-length HBV genome was amplified. The incidence of A1846T was analyzed. Full-length HBV genomes containing 1846T mutation were cloned into pGEM-T easy vector, and the counterpart wild-type 1846A plasmids were obtained by site-directed mutagenesis. The full-length HBV genome was released from recombinant plasmid by BspQ Ⅰ/Sca Ⅰ digestion, and then transfected into HepG2 cells. Secreted HBsAg level and intracellular HBV core particles were measured 72 hours post-transfection to analyze the replication capacity (a 1.0-fold HBV genome model. 1846 mutant and wild-type full-length HBV genomes were extracted to amplify the fragment of HBV CP region, and the dual luciferase reporter of the pGL3-CP was constructed. The luciferase activity was detected 48 hours post-transfection. Results  The incidence of A1846T mutation gradually increased with the severity of hepatitis B, reaching 31.03%, 42.27%, and 55.48% in CHB-M, CHB-S and ACLF patients respectively (P<0.01. The replication capacity of 1846T mutants, level of secreted HBsAg, and transcriptional activity of CP promoter were increased by 320%, 28% and 85% respectively, compared with 1846A wild-type strains. While the more common double mutation A1762T/G1764A in CP region was increased by 67%, 9% and 72% respectively, compared with its counterpart wild-type strains. A1846T had a greater influence on viral replication capacity in vitro. Conclusions A1846T mutation could significantly increase the

  19. Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity.

    Directory of Open Access Journals (Sweden)

    Wenyan Xie

    Full Text Available Human parainfluenza virus type 3 (HPIV3 can cause severe respiratory tract diseases in infants and young children, but no licensed vaccines or antiviral agents are currently available for treatment. Fusing the viral and target cell membranes is a prerequisite for its entry into host cells and is directly mediated by the fusion (F protein. Although several domains of F are known to have important effects on regulating the membrane fusion activity, the roles of the DI-DII linker (residues 369-374 of the HPIV3 F protein in the fusogenicity still remains ill-defined. To facilitate our understanding of the role of this domain might play in F-induced cell-cell fusion, nine single mutations were engineered into this domain by site-directed mutagenesis. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated F proteins. These mutants were analyzed for membrane fusion activity, cell surface expression, and interaction between F and HN protein. Each of the mutated F proteins in this domain has a cell surface expression level similar to that of wild-type F. All of them resulted in a significant reduction in fusogenic activity in all steps of membrane fusion. Furthermore, all these fusion-deficient mutants reduced the amount of the HN-F complexes at the cell surface. Together, the results of our work suggest that this region has an important effect on the fusogenic activity of F.

  20. LMNA E82K mutation activates FAS and mitochondrial pathways of apoptosis in heart tissue specific transgenic mice.

    Directory of Open Access Journals (Sweden)

    Dan Lu

    Full Text Available The lamin A/C (LMNA, nuclear intermediate filament proteins, is a basic component of the nuclear lamina. Mutations in LMNA are associated with a broad range of laminopathies, congenital diseases affecting tissue regeneration and homeostasis. Heart tissue specific transgenic mice of human LMNA E82K, a mutation causing dilated cardiomyopathy, were generated. Lmna(E82K transgenic mouse lines exhibited thin-walled, dilated left and right ventricles, a progressive decrease of contractile function assessed by echocardiography. Abnormalities of the conduction system, myocytes disarray, collagen accumulation and increased levels of B-type natriuretic peptide (BNP, procollagen type III α1 (Col3α1 and skeletal muscle actin α1 (Actα1 were detected in the hearts of Lmna(E82K transgenic mice. The LMNA E82K mutation caused mislocation of LMNA in the nucleus and swollen mitochondria with loss of critae, together with the loss of nuclear envelope integrity. Most interestingly, we found that the level of apoptosis was 8.5-fold higher in the Lmna(E82K transgenic mice than that of non-transgenic (NTG mice. In the presence of the LMNA E82K, both of FAS and mitochondrial pathways of apoptosis were activated consistent with the increase of FAS expression, the release of cytochrome c from mitochondria to cytosol and activation of caspase-8, -9 and -3. Our results suggested that the apoptosis, at least for the LMNA E82K or the mutations in the rod region of Lamin A/C, might be an important mechanism causing continuous loss of myocytes and lead to myocardial dysfunction. It could be a potential therapeutic means to suppress and/or prevent inappropriate cardiac cell death in patients carrying LMNA mutation.

  1. Mutational analysis of PI3K/AKT and RAS/RAF pathway activation in malignant salivary gland tumours with a new mutation of PIK3CA.

    Science.gov (United States)

    Shalmon, B; Drendel, M; Wolf, M; Hirshberg, A; Cohen, Y

    2016-06-01

    The phosphoinositide 3-kinase (PIK3)/v-akt murine thymoma (AKT) oncogene pathway and the RAS/RAF pathway are involved in regulating the signalling of multiple biological processes, including apoptosis, metabolism, cell proliferation, and cell growth. Mutations in the genes within these pathways are frequently found in several tumours. The aim of this study was to investigate the frequency of mutations in the PIK3CA, BRAF, and KRAS genes in cases of malignant salivary gland tumours. Mutational analysis of the PIK3CA, KRAS, and BRAF genes was performed by direct sequencing of material from 21 patients with malignant salivary gland tumours who underwent surgery between 1992 and 2001. No mutations were found in the KRAS exon 2, BRAF exon 15, or PIK3CA exon 9 genes. However, an unpublished mutation of the PIK3CA gene in exon 20 (W1051 stop mutation) was found in one case of adenocarcinoma NOS. The impact of this mutation on the biological behaviour of the tumour has yet to be explored, however the patient with adenocarcinoma NOS harbouring this mutation has survived for over 20 years following surgery despite a high stage at presentation. Further studies with more homogeneous patient cohorts are needed to address whether this mutation reflects a different clinical presentation and may benefit from targeted treatment strategies. PMID:26811072

  2. New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities.

    Science.gov (United States)

    Moroz, Yurii S; Dunston, Tiffany T; Makhlynets, Olga V; Moroz, Olesia V; Wu, Yibing; Yoon, Jennifer H; Olsen, Alissa B; McLaughlin, Jaclyn M; Mack, Korrie L; Gosavi, Pallavi M; van Nuland, Nico A J; Korendovych, Ivan V

    2015-12-01

    Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

  3. Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene

    Science.gov (United States)

    Känsäkoski, Johanna; Jääskeläinen, Jarmo; Jääskeläinen, Tiina; Tommiska, Johanna; Saarinen, Lilli; Lehtonen, Rainer; Hautaniemi, Sampsa; Frilander, Mikko J.; Palvimo, Jorma J.; Toppari, Jorma; Raivio, Taneli

    2016-01-01

    Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation. PMID:27609317

  4. Changing the treatment of permanent neonatal diabetes mellitus from insulin to glibenclamide in a 4-month-old infant with kcnj11 activating mutation

    Directory of Open Access Journals (Sweden)

    Homa Ilkhanipoor

    2013-01-01

    Full Text Available Permanent neonatal diabetes mellitus (PNDM is a rare type of diabetes and KCNJ11 gene activating mutation is one of its prevalent causes. We introduced a 4-month-old male infant with poor feeding, restlessness, tachypnea, hyperglycemia, metabolic acidosis, and ketonemia. He was discharged with insulin and after 2 months, KCNJ11 gene mutation was found and treatment was switched from subcutaneous insulin to oral glibenclamide. Now, he is 1 year old with desirable glycemic control; therefore, genetic study is recommended for KCNJ11 gene mutation in such patients because if the mutation is found, treatment can be switched from insulin to sulfonylurea.

  5. Mutations in Tau Gene Exon 10 Associated with FTDP-17 Alter the Activity of an Exonic Splicing Enhancer to Interact with Tra2β*

    OpenAIRE

    Jiang, Zhihong; Tang, Hao; Havlioglu, Necat; Zhang, Xiaochun; Stamm, Stefan; Yan, Riqiang; Jane Y Wu

    2003-01-01

    Mutations in the human tau gene leading to aberrant splicing have been identified in FTDP-17, an autosomal dominant hereditary neurodegenerative disorder. Molecular mechanisms by which such mutations cause tau aberrant splicing were not understood. We characterized two mutations in exon 10 of the tau gene, N279K and Del280K. Our results revealed an exonic splicing enhancer element located in exon 10. The activity of this AG-rich splicing enhancer was altered by N279K and Del280K mutations. Th...

  6. Structural insights into chaperone-activity enhancement by a K354E mutation in tomato acidic leucine aminopeptidase.

    Science.gov (United States)

    DuPrez, Kevin T; Scranton, Melissa A; Walling, Linda L; Fan, Li

    2016-05-01

    Tomato plants express acidic leucine aminopeptidase (LAP-A) in response to various environmental stressors. LAP-A not only functions as a peptidase for diverse peptide substrates, but also displays chaperone activity. A K354E mutation has been shown to abolish the peptidase activity but to enhance the chaperone activity of LAP-A. To better understand this moonlighting function of LAP-A, the crystal structure of the K354E mutant was determined at 2.15 Å resolution. The structure reveals that the K354E mutation destabilizes an active-site loop and causes significant rearrangement of active-site residues, leading to loss of the catalytic metal-ion coordination required for the peptidase activity. Although the mutant was crystallized in the same hexameric form as wild-type LAP-A, gel-filtration chromatography revealed an apparent shift from the hexamer to lower-order oligomers for the K354E mutant, showing a mixture of monomers to trimers in solution. In addition, surface-probing assays indicated that the K354E mutant has more accessible hydrophobic areas than wild-type LAP-A. Consistently, computational thermodynamic estimations of the interfaces between LAP-A monomers suggest that increased exposure of hydrophobic surfaces occurs upon hexamer breakdown. These results suggest that the K354E mutation disrupts the active-site loop, which also contributes to the hexameric assembly, and destabilizes the hexamers, resulting in much greater hydrophobic areas accessible for efficient chaperone activity than in the wild-type LAP-A. PMID:27139632

  7. Genetic and proteomic characterization of rpoB mutations and their effect on nematicidal activity in Photorhabdus luminescens LN2.

    Directory of Open Access Journals (Sweden)

    Xuehong Qiu

    Full Text Available Rifampin resistant (Rif(R mutants of the insect pathogenic bacterium Photorhabdus luminescens LN2 from entomopathogenic nematode Heterorhabditis indica LN2 were genetically and proteomically characterized. The Rif(R mutants showed typical phase one characters of Photorhabdus bacteria, and insecticidal activity against Galleria mellonella larvae, but surprisingly influenced their nematicidal activity against axenic infective juveniles (IJs of H. bacteriophora H06, an incompatible nematode host. 13 out of 34 Rif(R mutants lost their nematicidal activity against H06 IJs but supported the reproduction of H06 nematodes. 7 nematicidal-producing and 7 non-nematicidal-producing Rif(R mutants were respectively selected for rpoB sequence analysis. rpoB mutations were found in all 14 Rif(R mutants. The rpoB (P564L mutation was found in all 7 mutants which produced nematicidal activity against H06 nematodes, but not in the mutants which supported H06 nematode production. Allelic exchange assays confirmed that the Rif-resistance and the impact on nematicidal activity of LN2 bacteria were conferred by rpoB mutation(s. The non-nematicidal-producing Rif(R mutant was unable to colonize in the intestines of H06 IJs, but able to colonize in the intestines of its indigenous LN2 IJs. Proteomic analysis revealed different protein expression between wild-type strain and Rif(R mutants, or between nematicidal-producing and non nematicidal-producing mutants. At least 7 putative proteins including DsbA, HlpA, RhlE, RplC, NamB (a protein from T3SS, and 2 hypothetical proteins (similar to unknown protein YgdH and YggE of Escherichia coli respectively were probably involved in the nematicidal activity of LN2 bacteria against H06 nematodes. This hypothesis was further confirmed by creating insertion-deletion mutants of three selected corresponding genes (the downregulated rhlE and namB, and upregulated dsbA. These results indicate that the rpoB mutations greatly influence the

  8. Study on structure-activity relationship of mutation-dependent herbicide resistance acetohydroxyacid synthase through 3D-QSAR and mutation

    Institute of Scientific and Technical Information of China (English)

    YU ZhiHong; NIU CongWei; BAN ShuRong; WEN Xin; XI Zhen

    2007-01-01

    Seventy-four sulfonylureas were synthesized and tested for their inhibitory activity against the whole enzyme of E. Coli acetohydroxyacid synthase (AHAS, EC 2.2.1.6) isoenzyme Ⅱ, and 3D-QSAR analyses were performed based on these inhibitory activities. The binding conformation of chlorimuron-ethyl, a commercial herbicide of AHAS, in the crystal structure of AHAS complex was extracted and used as template to build the initial three-dimensional structure of other sulfonylureas, and then all structures were fully geometry optimized. After systematic optimization of the alignment rule, molecular orientation, grid space and attenuation factor, two satisfactory models with excellent performances (CoMFA: q2 = 0.735, r2 = 0.954, n = 7, r 2pred = 0.832; CoMSIA: q2 = 0.721, r2 = 0.913, n = 8, r 2pred = 0.844) were established. By mapping the 3D contour maps of CoMFA and CoMSIA models into the possible inhibitory active site in the crystal structure of catalytic subunit of yeast AHAS, a plausible binding model for AHAS, with best fit QSAR in the literature so far, was proposed. Moreover, the results of 3D-QSAR were further utilized to interpret resistance of site-directed mutants. A relative activity index (RAI) for AHAS enzyme mutant was defined for the first time to relate the 3D-QSAR and resistance of mutants. This study, for the first time, demonstrated that combination of 3D-QSAR and enzyme mutation can be used to decipher the molecular basis of ligand-receptor interaction mechanism. This study refined our understanding of the ligand-receptor interaction and resistance mechanism in AHAS-sulfonylurea system, and provided basis for designing new potent herbicides to combat the herbicide resistance.

  9. Lack of in vitro constitutive activity for four previously reported TSH receptor mutations identified in patients with nonautoimmune hyperthyroidism and hot thyroid carcinomas

    OpenAIRE

    Paschke, Ralf; Jaeschke, Holger; Mueller, Sandra; Eszlinger, Markus

    2010-01-01

    Abstract Objective: Constitutively activating mutations of the TSHR are the major cause for nonautoimmune hyperthyroidism. Re-examination of constitutive activity previously determined in CHO cell lines recently demonstrated the caveats for the in vitro determination of constitutive TSHR activity, which leads to false positive conclusions regarding the molecular origin of hyperthyroidism or hot thyroid carcinomas. Design: Mutations L677V and T620I identified in hot thyroid carc...

  10. Somatic Mutations in PI3Kalpha: Structural Basis for Enzyme Activation and Drug Design

    Energy Technology Data Exchange (ETDEWEB)

    S Gabelli; D Mandelker; O Schmidt-Kittler; B Vogelstein; L Amzel

    2011-12-31

    The PI3K pathway is a communication hub coordinating critical cell functions including cell survival, cell growth, proliferation, motility and metabolism. Because PI3K{alpha} harbors recurrent somatic mutations resulting in gains of function in human cancers, it has emerged as an important drug target for many types of solid tumors. Various PI3K isoforms are also being evaluated as potential therapeutic targets for inflammation, heart disease, and hematological malignancies. Structural biology is providing insights into the flexibility of the PI3Ks, and providing basis for understanding the effects of mutations, drug resistance and specificity.

  11. Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction.

    Science.gov (United States)

    Lozano, María Luisa; Cook, Aaron; Bastida, José María; Paul, David S; Iruin, Gemma; Cid, Ana Rosa; Adan-Pedroso, Rosa; Ramón González-Porras, José; Hernández-Rivas, Jesús María; Fletcher, Sarah J; Johnson, Ben; Morgan, Neil; Ferrer-Marin, Francisca; Vicente, Vicente; Sondek, John; Watson, Steve P; Bergmeier, Wolfgang; Rivera, José

    2016-09-01

    In addition to mutations in ITG2B or ITGB3 genes that cause defective αIIbβ3 expression and/or function in Glanzmann's thrombasthenia patients, platelet dysfunction can be a result of genetic variability in proteins that mediate inside-out activation of αIIbβ3 The RASGRP2 gene is strongly expressed in platelets and neutrophils, where its encoded protein CalDAG-GEFI facilitates the activation of Rap1 and subsequent activation of integrins. We used next-generation sequencing (NGS) and whole-exome sequencing (WES) to identify 2 novel function-disrupting mutations in RASGRP2 that account for bleeding diathesis and platelet dysfunction in 2 unrelated families. By using a panel of 71 genes, we identified a homozygous change (c.1142C>T) in exon 10 of RASGRP2 in a 9-year-old child of Chinese origin (family 1). This variant led to a p.Ser381Phe substitution in the CDC25 catalytic domain of CalDAG-GEFI. In 2 Spanish siblings from family 2, WES identified a nonsense homozygous variation (c.337C>T) (p.Arg113X) in exon 5 of RASGRP2 CalDAG-GEFI expression was markedly reduced in platelets from all patients, and by using a novel in vitro assay, we found that the nucleotide exchange activity was dramatically reduced in CalDAG-GEFI p.Ser381Phe. Platelets from homozygous patients exhibited agonist-specific defects in αIIbβ3 integrin activation and aggregation. In contrast, α- and δ-granule secretion, platelet spreading, and clot retraction were not markedly affected. Integrin activation in the patients' neutrophils was also impaired. These patients are the first cases of a CalDAG-GEFI deficiency due to homozygous RASGRP2 mutations that are linked to defects in both leukocyte and platelet integrin activation. PMID:27235135

  12. Presence of activating KRAS mutations correlates significantly with expression of tumour suppressor genes DCN and TPM1 in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Rems Miran

    2009-08-01

    Full Text Available Abstract Background Despite identification of the major genes and pathways involved in the development of colorectal cancer (CRC, it has become obvious that several steps in these pathways might be bypassed by other as yet unknown genetic events that lead towards CRC. Therefore we wanted to improve our understanding of the genetic mechanisms of CRC development. Methods We used microarrays to identify novel genes involved in the development of CRC. Real time PCR was used for mRNA expression as well as to search for chromosomal abnormalities within candidate genes. The correlation between the expression obtained by real time PCR and the presence of the KRAS mutation was investigated. Results We detected significant previously undescribed underexpression in CRC for genes SLC26A3, TPM1 and DCN, with a suggested tumour suppressor role. We also describe the correlation between TPM1 and DCN expression and the presence of KRAS mutations in CRC. When searching for chromosomal abnormalities, we found deletion of the TPM1 gene in one case of CRC, but no deletions of DCN and SLC26A3 were found. Conclusion Our study provides further evidence of decreased mRNA expression of three important tumour suppressor genes in cases of CRC, thus implicating them in the development of this type of cancer. Moreover, we found underexpression of the TPM1 gene in a case of CRCs without KRAS mutations, showing that TPM1 might serve as an alternative path of development of CRC. This downregulation could in some cases be mediated by deletion of the TPM1 gene. On the other hand, the correlation of DCN underexpression with the presence of KRAS mutations suggests that DCN expression is affected by the presence of activating KRAS mutations, lowering the amount of the important tumour suppressor protein decorin.

  13. Docetaxel for non small cell lung cancer harboring the activated EGFR mutation with T790M at initial presentation

    Directory of Open Access Journals (Sweden)

    Yamane H

    2013-03-01

    Full Text Available Hiromichi Yamane,1 Nobuaki Ochi,1 Masayuki Yasugi,2 Takayuki Tabayashi,1 Tomoko Yamagishi,1 Yasumasa Monobe,3 Akiko Hisamoto,4 Katsuyuki Kiura,4 Nagio Takigawa1 1Department of General Internal Medicine 4, Kawasaki Medical School, Okayama, Japan; 2Department of Respiratory Medicine, National Hospital Organization Fukuyama Medical Center, Fukuyama, Japan; 3Department of Pathology, Kawasaki Medical School Kawasaki Hospital, Okayama, Japan; 4Department of Hematology, Oncology, and Respiratory Medicine, Okayama University Graduate School of Medicine, Okayama, Japan Abstract: A 72-year-old woman was referred to our hospital with Stage IV non-small-cell lung cancer (NSCLC. Chest computed tomography revealed a mass in the upper lobe of the right lung, with pleural effusion. Cytologic examination identified adenocarcinoma cells in the right pleural effusion. Furthermore, both a deletion mutation in exon 19 and a threonine–methionine substitution mutation at position 790 in exon 20 (T790M were detected in the epidermal growth factor receptors (EGFR in the malignant cells. As systemic chemotherapy consisting of carboplatin and pemetrexed or erlotinib proved ineffective, docetaxel monotherapy was initiated as a third-line treatment. Following salvage chemotherapy, her Eastern Cooperative Oncology Group performance status improved from 3 to 1, with tumor regression over 5 months. To the best of our knowledge, this is the first report of successful docetaxel treatment for a patient with NSCLC harboring the T790M EGFR-activating mutation identified before treatment with EGFR tyrosine kinase inhibitors. Keywords: non-small-cell lung cancer, EGFR mutation, pretreatment mutation, T790M, docetaxel

  14. HMG CoA Lyase (HL): Mutation detection and development of a bacterial expression system for screening the activity of mutant alleles from HL-deficient patients

    Energy Technology Data Exchange (ETDEWEB)

    Robert, M.F.; Ashmarina, L.; Poitier, E. [Hospital Ste-Justine, Montreal (Canada)] [and others

    1994-09-01

    HL catalyzes the last step of ketogenesis, and autosomal recessive HL deficiency in humans can cause episodes of hypoglycemia and coma. Structurally, HL is a dimer of identical 325-residue peptides which requires a reducing environment to maintain activity. We cloned the human and mouse HL cDNAs and genes and have performed mutation analysis on cells from 30 HL-deficient probands. Using SSCP and also genomic Southern analysis we have identified putative mutations on 53/60 alleles of these patients (88%). To date, we have found 20 mutations: 3 large deletions, 4 termination mutations, 5 frameshift mutations, and 8 missense mutations which we suspect to be pathogenic based on evolutionary conservation and/or our previous studies on purified HL protein. We have also identified 3 polymorphic variants. In order to directly test the activity of the missense mutations, we established a pGEX-based system, using a glutathione S transferase (GST)-HL fusion protein. Expressed wild-type GST-HL was insoluble. We previously located a reactive Cys at the C-terminus of chicken HL which is conserved in human HL. We produced a mutant HL peptide, C323S, which replaced Cys323 with Ser. Purified C323S is soluble and has similar kinetics to wild-type HL. C323S-containing GST-HL is soluble and enzymatically active. We are cloning and expressing the 8 missense mutations.

  15. Accumulation of wildtype and ALS-linked mutated VAPB impairs activity of the proteasome.

    Directory of Open Access Journals (Sweden)

    Anice Moumen

    Full Text Available Cellular homeostasis relies on a tight control of protein synthesis, folding and degradation, in which the endoplasmic reticulum (ER quality control and the ubiquitin proteasome system (UPS have an instrumental function. ER stress and aberrant accumulation of misfolded proteins represent a pathological signature of amyotrophic lateral sclerosis (ALS, a fatal paralytic disorder caused by the selective degeneration of motoneurons in the brain and spinal cord. Mutations in the ER-resident protein VAPB have been associated with familial forms of the disease. ALS-linked mutations cause VAPB to form cytoplasmic aggregates. We previously demonstrated that viral-mediated expression of both wildtype and mutant human VAPB (hVAPB leads to an ER stress response that contributes to the selective death of motoneurons. However, the mechanisms behind ER stress, defective UPS and hVAPB-associated motoneuron degeneration remain elusive. Here, we show that the overexpression of wildtype and mutated hVAPB, which is found to be less stable than the wildtype protein, leads to the abnormal accumulation of ubiquitin and ubiquitin-like protein conjugates in non-human primate cells. We observed that overexpression of both forms of hVAPB elicited an ER stress response. Treatment of wildtype and mutated hVAPB expressing cells with the ER stress inhibitor salubrinal diminished the burden of ubiquitinated proteins, suggesting that ER stress contributes to the impairment of proteasome function. We also found that both wildtype and mutated hVAPB can associate with the 20S proteasome, which was found to accumulate at the ER with wildtype hVAPB or in mutant hVAPB aggregates. Our results suggest that ER stress and corruption of the proteasome function might contribute to the aberrant protein homeostasis associated with hVAPB.

  16. ALK kinase domain mutations in primary anaplastic large cell lymphoma: consequences on NPM-ALK activity and sensitivity to tyrosine kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Federica Lovisa

    Full Text Available ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.

  17. ALK kinase domain mutations in primary anaplastic large cell lymphoma: consequences on NPM-ALK activity and sensitivity to tyrosine kinase inhibitors.

    Science.gov (United States)

    Lovisa, Federica; Cozza, Giorgio; Cristiani, Andrea; Cuzzolin, Alberto; Albiero, Alessandro; Mussolin, Lara; Pillon, Marta; Moro, Stefano; Basso, Giuseppe; Rosolen, Angelo; Bonvini, Paolo

    2015-01-01

    ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.

  18. Altered promoter recycling rates contribute to dominant-negative activity of human peroxisome proliferator-activated receptor-gamma mutations associated with diabetes.

    Science.gov (United States)

    Li, Gang; Leff, Todd

    2007-04-01

    The transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma) plays an important role in regulating lipid and glucose metabolism and improves insulin sensitivity in diabetic patients when activated by thiazolidinedione drugs. Several loss-of-function mutations in PPARgamma have been identified that cause lipodystrophy and diabetes in humans. Because affected individuals are heterozygotes and have one normal PPARgamma allele, it is of interest to know whether these mutations act in a dominant-negative fashion to inhibit the activity of the wild-type (WT) receptor. Here we compare the molecular phenotypes of two previously identified PPARgamma mutations: P467L, reported to be dominant negative; and F388L, reported to be devoid of dominant-negative activity. We developed a competitive chromatin immunoprecipitation assay to measure the relative ability of mutant PPARgamma to compete with WT receptor for binding to a PPAR regulatory element (PPRE)-containing promoter. By determining the ratio of mutant and WT receptors bound to a PPRE over time, we estimated the relative promoter turnover rate of each receptor. This assay demonstrated that PPARgamma bearing the P467L had a reduced promoter turnover rate compared with the F388L receptor, and over time out-competed the WT receptor for promoter binding sites. We propose that the P467L receptor is dominant negative because in a cell containing both WT and mutant receptors, the majority of the PPAR-regulated promoters will be occupied by the transcriptionally defective mutant receptor. In contrast, the F388L mutation lacks dominant-negative activity because its more rapid promoter turnover rate prevented it from out-competing the WT receptor for promoter binding sites.

  19. The yeast ROAM mutation--identification of the sequences mediating host gene activation and cell-type control in the yeast retrotransposon, Ty.

    OpenAIRE

    Rathjen, P D; Kingsman, A J; Kingsman, S M

    1987-01-01

    When the yeast retrotransposon, Ty, integrates into the 5' flanking region of a gene it can activate the expression of that gene. At the same time the activated gene is brought under cell-type specific control such that expression is high in haploid a or alpha cells but low in a/alpha diploids. These Ty mediated mutations are known as ROAM mutations. In this study we have used a ROAM mutation created in vitro to identify the sequences within Ty that mediate this phenomenon. We show that a sin...

  20. Activating Ras mutations fail to ensure efficient replication of adenovirus mutants lacking VA-RNA

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    Adenoviruses lacking their PKR-antagonizing VA RNAs replicate poorly in primary cells. It has been suggested that these virus recombinants still replicate efficiently in tumor cells with Ras mutations and might therefore be useful in tumor therapy. The ability of interferon-sensitive viruses...... to grow in Ras-mutant tumor cells is generally ascribed to a postulated inhibitory effect of mutant Ras on PKR. We have constructed a set of isogenic adenoviruses that lack either or both VA RNA species, and tested virus replication in a variety of cell species with different Ras status. In tendency, VA......-less viruses replicated with higher efficiency in Ras-mutant cells, as compared to cell lines without Ras mutation. However, several exceptions to this rule were observed, arguing against a direct inhibition of PKR by mutant Ras. Phosphorylation of the PKR-substrate eIF2alpha was observed regardless of the Ras...

  1. Rhabdomyolysis-Associated Mutations in Human LPIN1 Lead to Loss of Phosphatidic Acid Phosphohydrolase Activity

    OpenAIRE

    Schweitzer, George G.; Collier, Sara L.; Chen, Zhouji; Eaton, James M.; Connolly, Anne M.; Bucelli, Robert C.; Pestronk, Alan; Harris, Thurl E.; Finck, Brian N.

    2015-01-01

    Rhabdomyolysis is an acute syndrome due to extensive injury of skeletal muscle. Recurrent rhabdomyolysis is often caused by inborn errors in intermediary metabolism, and recent work has suggested that mutations in the human gene encoding lipin 1 (LPIN1) may be a common cause of recurrent rhabdomyolysis in children. Lipin 1 dephosphorylates phosphatidic acid to form diacylglycerol (phosphatidic acid phosphohydrolase; PAP) and acts as a transcriptional regulatory protein to control metabolic ge...

  2. Deep Intronic Mutation and Pseudo Exon Activation as a Novel Muscular Hypertrophy Modifier in Cattle

    OpenAIRE

    Claire Bouyer; Lionel Forestier; Gilles Renand; Ahmad Oulmouden

    2014-01-01

    Myostatin is essential for proper regulation of myogenesis, and inactivation of Myostatin results in muscle hypertrophy. Here, we identified an unexpected mutation in the myostatin gene which is almost fixed in Blonde d'Aquitaine cattle. In skeletal muscle, the mutant allele was highly expressed leading to an abnormal transcript consisting of a 41-bp inclusion and premature termination codons and to residual levels of a correctly spliced transcript. This expression pattern, caused by a leaky ...

  3. Preventing AID, a physiological mutator, from deleterious activation: regulation of the genomic instability that is associated with antibody diversity.

    Science.gov (United States)

    Nagaoka, Hitoshi; Tran, Thinh Huy; Kobayashi, Maki; Aida, Masatoshi; Honjo, Tasuku

    2010-04-01

    Activation-induced cytidine deaminase (AID) is essential and sufficient to accomplish class-switch recombination and somatic hypermutation, which are two genetic events required for the generation of antibody-mediated memory responses. However, AID can also introduce genomic instability, giving rise to chromosomal translocation and/or mutations in proto-oncogenes. It is therefore important for cells to suppress AID expression unless B lymphocytes are stimulated by pathogens. The mechanisms for avoiding the accidental activation of AID and thereby avoiding genomic instability can be classified into three types: (i) transcriptional regulation, (ii) post-transcriptional regulation and (iii) target specificity. This review summarizes the recently elucidated comprehensive transcriptional regulation mechanisms of the AID gene and the post-transcriptional regulation that may be critical for preventing excess AID activity. Finally, we discuss why AID targets not only Igs but also other proto-oncogenes. AID targets many genes but it is not totally promiscuous and the criteria that specify its targets are unclear. A recent finding that a non-B DNA structure forms upon a decrease in topoisomerase 1 expression may explain this paradoxical target specificity determination. Evolution has chosen AID as a mutator of Ig genes because of its efficient DNA cleavage activity, even though its presence increases the risk of genomic instability. This is probably because immediate protection against pathogens is more critical for species survival than complete protection from the slower acting consequences of genomic instability, such as tumor formation.

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

    Directory of Open Access Journals (Sweden)

    Catherine E Smith

    2013-10-01

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

  5. TLR4 mutation reduces microglial activation, increases Aβ deposits and exacerbates cognitive deficits in a mouse model of Alzheimer's disease

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    Song Min

    2011-08-01

    Full Text Available Abstract Background Amyloid plaques, a pathological hallmark of Alzheimer's disease (AD, are accompanied by activated microglia. The role of activated microglia in the pathogenesis of AD remains controversial: either clearing Aβ deposits by phagocytosis or releasing proinflammatory cytokines and cytotoxic substances. Microglia can be activated via toll-like receptors (TLRs, a class of pattern-recognition receptors in the innate immune system. We previously demonstrated that an AD mouse model homozygous for a loss-of-function mutation of TLR4 had increases in Aβ deposits and buffer-soluble Aβ in the brain as compared with a TLR4 wild-type AD mouse model at 14-16 months of age. However, it is unknown if TLR4 signaling is involved in initiation of Aβ deposition as well as activation and recruitment of microglia at the early stage of AD. Here, we investigated the role of TLR4 signaling and microglial activation in early stages using 5-month-old AD mouse models when Aβ deposits start. Methods Microglial activation and amyloid deposition in the brain were determined by immunohistochemistry in the AD models. Levels of cerebral soluble Aβ were determined by ELISA. mRNA levels of cytokines and chemokines in the brain and Aβ-stimulated monocytes were quantified by real-time PCR. Cognitive functions were assessed by the Morris water maze. Results While no difference was found in cerebral Aβ load between AD mouse models at 5 months with and without TLR4 mutation, microglial activation in a TLR4 mutant AD model (TLR4M Tg was less than that in a TLR4 wild-type AD model (TLR4W Tg. At 9 months, TLR4M Tg mice had increased Aβ deposition and soluble Aβ42 in the brain, which were associated with decrements in cognitive functions and expression levels of IL-1β, CCL3, and CCL4 in the hippocampus compared to TLR4W Tg mice. TLR4 mutation diminished Aβ-induced IL-1β, CCL3, and CCL4 expression in monocytes. Conclusion This is the first demonstration of TLR4

  6. Gain-of-function STAT1 mutations impair STAT3 activity in patients with chronic mucocutaneous candidiasis (CMC).

    Science.gov (United States)

    Zheng, Jie; van de Veerdonk, Frank L; Crossland, Katherine L; Smeekens, Sanne P; Chan, Chun M; Al Shehri, Tariq; Abinun, Mario; Gennery, Andrew R; Mann, Jelena; Lendrem, Dennis W; Netea, Mihai G; Rowan, Andrew D; Lilic, Desa

    2015-10-01

    Signal transducer and activator of transcription 3 (STAT3) triggered production of Th-17 cytokines mediates protective immunity against fungi. Mutations affecting the STAT3/interleukin 17 (IL-17) pathway cause selective susceptibility to fungal (Candida) infections, a hallmark of chronic mucocutaneous candidiasis (CMC). In patients with autosomal dominant CMC, we and others previously reported defective Th17 responses and underlying gain-of-function (GOF) STAT1 mutations, but how this affects STAT3 function leading to decreased IL-17 is unclear. We also assessed how GOF-STAT1 mutations affect STAT3 activation, DNA binding, gene expression, cytokine production, and epigenetic modifications. We excluded impaired STAT3 phosphorylation, nuclear translocation, and sequestration of STAT3 into STAT1/STAT3 heterodimers and confirm significantly reduced transcription of STAT3-inducible genes (RORC/IL-17/IL-22/IL-10/c-Fos/SOCS3/c-Myc) as likely underlying mechanism. STAT binding to the high affinity sis-inducible element was intact but binding to an endogenous STAT3 DNA target was impaired. Reduced STAT3-dependent gene transcription was reversed by inhibiting STAT1 activation with fludarabine or enhancing histone, but not STAT1 or STAT3 acetylation with histone deacetylase (HDAC) inhibitors trichostatin A or ITF2357. Silencing HDAC1, HDAC2, and HDAC3 indicated a role for HDAC1 and 2. Reduced STAT3-dependent gene transcription underlies low Th-17 responses in GOF-STAT1 CMC, which can be reversed by inhibiting acetylation, offering novel targets for future therapies.

  7. A mutation in the dam gene of Vibrio cholerae: 2-aminopurine sensitivity with intact GATC methylase activity

    International Nuclear Information System (INIS)

    Vibrio cholerae mutants sensitive to 2-aminopurine (2AP) but with DNA adenine methylase activity similar to parental cells have been isolated. The mutant strains were sensitive to ultraviolet light (UV), methyl methanesulfonate (MMS) and 9-aminoacridine. The spontaneous mutation frequency of the mutants were not significantly affected. Attempts to isolate dam V. cholerae cells by screening 2AP sensitive cells have not been successful. All the mutant phenotypes could be suppressed by introducing the plasmid pRB103 carrying the dam gene of Escherichia coli into the mutant cells

  8. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.

    Science.gov (United States)

    Williams, Alison A; Mehler, Vera J; Mueller, Christina; Vonhoff, Fernando; White, Robin; Duch, Carsten

    2016-01-01

    Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X), a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80) and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2. PMID:27442528

  9. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.

    Directory of Open Access Journals (Sweden)

    Alison A Williams

    Full Text Available Methyl-CpG binding protein 2 (MeCP2 is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X, a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80 and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.

  10. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.

    Science.gov (United States)

    Williams, Alison A; Mehler, Vera J; Mueller, Christina; Vonhoff, Fernando; White, Robin; Duch, Carsten

    2016-01-01

    Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X), a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80) and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.

  11. The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation

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    Zarbock Ralf

    2012-03-01

    Full Text Available Abstract Background Surfactant protein C (SP-C is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects. Methods SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide. Results Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space. Conclusions We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy

  12. Recessive Inactivating Mutations in TBCK, Encoding a Rab GTPase-Activating Protein, Cause Severe Infantile Syndromic Encephalopathy.

    Science.gov (United States)

    Chong, Jessica X; Caputo, Viviana; Phelps, Ian G; Stella, Lorenzo; Worgan, Lisa; Dempsey, Jennifer C; Nguyen, Alina; Leuzzi, Vincenzo; Webster, Richard; Pizzuti, Antonio; Marvin, Colby T; Ishak, Gisele E; Ardern-Holmes, Simone; Richmond, Zara; Bamshad, Michael J; Ortiz-Gonzalez, Xilma R; Tartaglia, Marco; Chopra, Maya; Doherty, Dan

    2016-04-01

    Infantile encephalopathies are a group of clinically and biologically heterogeneous disorders for which the genetic basis remains largely unknown. Here, we report a syndromic neonatal encephalopathy characterized by profound developmental disability, severe hypotonia, seizures, diminished respiratory drive requiring mechanical ventilation, brain atrophy, dysgenesis of the corpus callosum, cerebellar vermis hypoplasia, and facial dysmorphism. Biallelic inactivating mutations in TBCK (TBC1-domain-containing kinase) were independently identified by whole-exome sequencing as the cause of this condition in four unrelated families. Matching these families was facilitated by the sharing of phenotypic profiles and WES data in a recently released web-based tool (Geno2MP) that links phenotypic information to rare variants in families with Mendelian traits. TBCK is a putative GTPase-activating protein (GAP) for small GTPases of the Rab family and has been shown to control cell growth and proliferation, actin-cytoskeleton dynamics, and mTOR signaling. Two of the three mutations (c.376C>T [p.Arg126(∗)] and c.1363A>T [p.Lys455(∗)]) are predicted to truncate the protein, and loss of the major TBCK isoform was confirmed in primary fibroblasts from one affected individual. The third mutation, c.1532G>A (p.Arg511His), alters a conserved residue within the TBC1 domain. Structural analysis implicated Arg511 as a required residue for Rab-GAP function, and in silico homology modeling predicted impaired GAP function in the corresponding mutant. These results suggest that loss of Rab-GAP activity is the underlying mechanism of disease. In contrast to other disorders caused by dysregulated mTOR signaling associated with focal or global brain overgrowth, impaired TBCK function results in progressive loss of brain volume. PMID:27040692

  13. Recessive Inactivating Mutations in TBCK, Encoding a Rab GTPase-Activating Protein, Cause Severe Infantile Syndromic Encephalopathy

    Science.gov (United States)

    Chong, Jessica X.; Caputo, Viviana; Phelps, Ian G.; Stella, Lorenzo; Worgan, Lisa; Dempsey, Jennifer C.; Nguyen, Alina; Leuzzi, Vincenzo; Webster, Richard; Pizzuti, Antonio; Marvin, Colby T.; Ishak, Gisele E.; Ardern-Holmes, Simone; Richmond, Zara; Bamshad, Michael J.; Ortiz-Gonzalez, Xilma R.; Tartaglia, Marco; Chopra, Maya; Doherty, Dan

    2016-01-01

    Infantile encephalopathies are a group of clinically and biologically heterogeneous disorders for which the genetic basis remains largely unknown. Here, we report a syndromic neonatal encephalopathy characterized by profound developmental disability, severe hypotonia, seizures, diminished respiratory drive requiring mechanical ventilation, brain atrophy, dysgenesis of the corpus callosum, cerebellar vermis hypoplasia, and facial dysmorphism. Biallelic inactivating mutations in TBCK (TBC1-domain-containing kinase) were independently identified by whole-exome sequencing as the cause of this condition in four unrelated families. Matching these families was facilitated by the sharing of phenotypic profiles and WES data in a recently released web-based tool (Geno2MP) that links phenotypic information to rare variants in families with Mendelian traits. TBCK is a putative GTPase-activating protein (GAP) for small GTPases of the Rab family and has been shown to control cell growth and proliferation, actin-cytoskeleton dynamics, and mTOR signaling. Two of the three mutations (c.376C>T [p.Arg126∗] and c.1363A>T [p.Lys455∗]) are predicted to truncate the protein, and loss of the major TBCK isoform was confirmed in primary fibroblasts from one affected individual. The third mutation, c.1532G>A (p.Arg511His), alters a conserved residue within the TBC1 domain. Structural analysis implicated Arg511 as a required residue for Rab-GAP function, and in silico homology modeling predicted impaired GAP function in the corresponding mutant. These results suggest that loss of Rab-GAP activity is the underlying mechanism of disease. In contrast to other disorders caused by dysregulated mTOR signaling associated with focal or global brain overgrowth, impaired TBCK function results in progressive loss of brain volume. PMID:27040692

  14. Gain of function AMP-activated protein kinase γ3 mutation (AMPKγ3R200Q) in pig muscle increases glycogen storage regardless of AMPK activation.

    Science.gov (United States)

    Scheffler, Tracy L; Park, Sungkwon; Roach, Peter J; Gerrard, David E

    2016-06-01

    Chronic activation of AMP-activated protein kinase (AMPK) increases glycogen content in skeletal muscle. Previously, we demonstrated that a mutation in the ryanodine receptor (RyR1(R615C)) blunts AMPK phosphorylation in longissimus muscle of pigs with a gain of function mutation in the AMPKγ3 subunit (AMPKγ3(R200Q)); this may decrease the glycogen storage capacity of AMPKγ3(R200Q) + RyR1(R615C) muscle. Therefore, our aim in this study was to utilize our pig model to understand how AMPKγ3(R200Q) and AMPK activation contribute to glycogen storage and metabolism in muscle. We selected and bred pigs in order to generate offspring with naturally occurring AMPKγ3(R200Q), RyR1(R615C), and AMPKγ3(R200Q) + RyR1(R615C) mutations, and also retained wild-type littermates (control). We assessed glycogen content and parameters of glycogen metabolism in longissimus muscle. Regardless of RyR1(R615C), AMPKγ3(R200Q) increased the glycogen content by approximately 70%. Activity of glycogen synthase (GS) without the allosteric activator glucose 6-phosphate (G6P) was decreased in AMPKγ3(R200Q) relative to all other genotypes, whereas both AMPKγ3(R200Q) and AMPKγ3(R200Q) + RyR1(R615C) muscle exhibited increased GS activity with G6P. Increased activity of GS with G6P was not associated with increased abundance of GS or hexokinase 2. However, AMPKγ3(R200Q) enhanced UDP-glucose pyrophosphorylase 2 (UGP2) expression approximately threefold. Although UGP2 is not generally considered a rate-limiting enzyme for glycogen synthesis, our model suggests that UGP2 plays an important role in increasing flux to glycogen synthase. Moreover, we have shown that the capacity for glycogen storage is more closely related to the AMPKγ3(R200Q) mutation than activity. PMID:27302990

  15. Compound heterozygosity with a novel S222N GALT mutation leads to atypical galactosemia with loss of GALT activity in erythrocytes but little evidence of clinical disease

    Directory of Open Access Journals (Sweden)

    Benjamin Cocanougher

    2015-03-01

    Full Text Available Galactosemia is an inborn error of galactose metabolism caused by mutations in the GALT gene. Though early detection and galactose restriction prevent severe liver disease, affected individuals have persistently elevated biomarkers and often neuro-developmental symptoms. We present a teenage compound heterozygote for a known pathogenic mutation (H132Q and a novel variant of unknown significance (S222N, with nearly absent erythrocyte GALT enzyme activity but normal biomarkers and only mild anxiety despite diet non-adherence. This case is similar to a previously reported S135L mutation. In this report we investigate the novel S222N variant and critically evaluate a clinically puzzling case.

  16. Fibrodysplasia ossificans progressiva: mechanisms and models of skeletal metamorphosis

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    Frederick S. Kaplan

    2012-11-01

    Full Text Available Fibrodysplasia ossificans progressiva (FOP; MIM #135100 is a debilitating genetic disorder of connective tissue metamorphosis. It is characterized by malformation of the great (big toes during embryonic skeletal development and by progressive heterotopic endochondral ossification (HEO postnatally, which leads to the formation of a second skeleton of heterotopic bone. Individuals with these classic clinical features of FOP have the identical heterozygous activating mutation (c.617G>A; R206H in the gene encoding ACVR1 (also known as ALK2, a bone morphogenetic protein (BMP type I receptor. Disease activity caused by this ACVR1 mutation also depends on altered cell and tissue physiology that can be best understood in the context of a high-fidelity animal model. Recently, we developed such a knock-in mouse model for FOP (Acvr1R206H/+ that recapitulates the human disease, and provides a valuable new tool for testing and developing effective therapies. The FOP knock-in mouse and other models in Drosophila, zebrafish, chickens and mice provide an arsenal of tools for understanding BMP signaling and addressing outstanding questions of disease mechanisms that are relevant not only to FOP but also to a wide variety of disorders associated with regenerative medicine and tissue metamorphosis.

  17. Disruption of Transcriptional Coactivator Sub1 Leads to Genome-Wide Re-distribution of Clustered Mutations Induced by APOBEC in Active Yeast Genes.

    Science.gov (United States)

    Lada, Artem G; Kliver, Sergei F; Dhar, Alok; Polev, Dmitrii E; Masharsky, Alexey E; Rogozin, Igor B; Pavlov, Youri I

    2015-05-01

    Mutations in genomes of species are frequently distributed non-randomly, resulting in mutation clusters, including recently discovered kataegis in tumors. DNA editing deaminases play the prominent role in the etiology of these mutations. To gain insight into the enigmatic mechanisms of localized hypermutagenesis that lead to cluster formation, we analyzed the mutational single nucleotide variations (SNV) data obtained by whole-genome sequencing of drug-resistant mutants induced in yeast diploids by AID/APOBEC deaminase and base analog 6-HAP. Deaminase from sea lamprey, PmCDA1, induced robust clusters, while 6-HAP induced a few weak ones. We found that PmCDA1, AID, and APOBEC1 deaminases preferentially mutate the beginning of the actively transcribed genes. Inactivation of transcription initiation factor Sub1 strongly reduced deaminase-induced can1 mutation frequency, but, surprisingly, did not decrease the total SNV load in genomes. However, the SNVs in the genomes of the sub1 clones were re-distributed, and the effect of mutation clustering in the regions of transcription initiation was even more pronounced. At the same time, the mutation density in the protein-coding regions was reduced, resulting in the decrease of phenotypically detected mutants. We propose that the induction of clustered mutations by deaminases involves: a) the exposure of ssDNA strands during transcription and loss of protection of ssDNA due to the depletion of ssDNA-binding proteins, such as Sub1, and b) attainment of conditions favorable for APOBEC action in subpopulation of cells, leading to enzymatic deamination within the currently expressed genes. This model is applicable to both the initial and the later stages of oncogenic transformation and explains variations in the distribution of mutations and kataegis events in different tumor cells.

  18. Mosaic Activating Mutations in GNA11 and GNAQ Are Associated with Phakomatosis Pigmentovascularis and Extensive Dermal Melanocytosis

    Science.gov (United States)

    Thomas, Anna C.; Zeng, Zhiqiang; Rivière, Jean-Baptiste; O’Shaughnessy, Ryan; Al-Olabi, Lara; St.-Onge, Judith; Atherton, David J.; Aubert, Hélène; Bagazgoitia, Lorea; Barbarot, Sébastien; Bourrat, Emmanuelle; Chiaverini, Christine; Chong, W. Kling; Duffourd, Yannis; Glover, Mary; Groesser, Leopold; Hadj-Rabia, Smail; Hamm, Henning; Happle, Rudolf; Mushtaq, Imran; Lacour, Jean-Philippe; Waelchli, Regula; Wobser, Marion; Vabres, Pierre; Patton, E. Elizabeth; Kinsler, Veronica A.

    2016-01-01

    Common birthmarks can be an indicator of underlying genetic disease but are often overlooked. Mongolian blue spots (dermal melanocytosis) are usually localized and transient, but they can be extensive, permanent, and associated with extracutaneous abnormalities. Co-occurrence with vascular birthmarks defines a subtype of phakomatosis pigmentovascularis, a group of syndromes associated with neurovascular, ophthalmological, overgrowth, and malignant complications. Here, we discover that extensive dermal melanocytosis and phakomatosis pigmentovascularis are associated with activating mutations in GNA11 and GNAQ, genes that encode Gα subunits of heterotrimeric G proteins. The mutations were detected at very low levels in affected tissues but were undetectable in the blood, indicating that these conditions are postzygotic mosaic disorders. In vitro expression of mutant GNA11R183C and GNA11Q209L in human cell lines demonstrated activation of the downstream p38 MAPK signaling pathway and the p38, JNK, and ERK pathways, respectively. Transgenic mosaic zebrafish models expressing mutant GNA11R183C under promoter mitfa developed extensive dermal melanocytosis recapitulating the human phenotype. Phakomatosis pigmentovascularis and extensive dermal melanocytosis are therefore diagnoses in the group of mosaic heterotrimeric G-protein disorders, joining McCune-Albright and Sturge-Weber syndromes. These findings will allow accurate clinical and molecular diagnosis of this subset of common birthmarks, thereby identifying infants at risk for serious complications, and provide novel therapeutic opportunities. PMID:26778290

  19. Heterozygous Mutations in MAP3K7, Encoding TGF-β-Activated Kinase 1, Cause Cardiospondylocarpofacial Syndrome.

    Science.gov (United States)

    Le Goff, Carine; Rogers, Curtis; Le Goff, Wilfried; Pinto, Graziella; Bonnet, Damien; Chrabieh, Maya; Alibeu, Olivier; Nistchke, Patrick; Munnich, Arnold; Picard, Capucine; Cormier-Daire, Valérie

    2016-08-01

    Cardiospondylocarpofacial (CSCF) syndrome is characterized by growth retardation, dysmorphic facial features, brachydactyly with carpal-tarsal fusion and extensive posterior cervical vertebral synostosis, cardiac septal defects with valve dysplasia, and deafness with inner ear malformations. Whole-exome sequencing identified heterozygous MAP3K7 mutations in six distinct CSCF-affected individuals from four families and ranging in age from 5 to 37 years. MAP3K7 encodes transforming growth factor β (TGF-β)-activated kinase 1 (TAK1), which is involved in the mitogen-activated protein kinase (MAPK)-p38 signaling pathway. MAPK-p38 signaling was markedly altered when expression of non-canonical TGF-β-driven target genes was impaired. These findings support the loss of transcriptional control of the TGF-β-MAPK-p38 pathway in fibroblasts obtained from affected individuals. Surprisingly, although TAK1 is located at the crossroad of inflammation, immunity, and cancer, this study reports MAP3K7 mutations in a developmental disorder affecting mainly cartilage, bone, and heart. PMID:27426734

  20. Compound heterozygosity with a novel S222N GALT mutation leads to atypical galactosemia with loss of GALT activity in erythrocytes but little evidence of clinical disease

    OpenAIRE

    Benjamin Cocanougher; Umut Aypar; Amber McDonald; Linda Hasadsri; Bennett, Michael J; Edward Highsmith, W.; Kristin D'Aco

    2015-01-01

    Galactosemia is an inborn error of galactose metabolism caused by mutations in the GALT gene. Though early detection and galactose restriction prevent severe liver disease, affected individuals have persistently elevated biomarkers and often neuro-developmental symptoms. We present a teenage compound heterozygote for a known pathogenic mutation (H132Q) and a novel variant of unknown significance (S222N), with nearly absent erythrocyte GALT enzyme activity but normal biomarkers and only mild a...

  1. EPIDEMIOLOGY OF ACTIVATED PROTEIN C RESISTANCE AND FACTOR V LEIDEN MUTATION IN THE MEDITERRANEAN REGION

    Directory of Open Access Journals (Sweden)

    Mehrez Mehrez M. Jadaon

    2011-09-01

    Full Text Available Venous thromboembolic disorders (VTE are serious disorders with high morbidity and mortality rates. Many genetic and acquired risk factors were identified to cause VTE The most common genetic risk factor is Factor V Leiden mutation (FVL. FVL was found in high percentage of populations of Caucasian origin but was almost absent in non-Caucasians. It was also reported in populations living in North Africa and the Middle East.  This review article briefly explains FVL and how it causes VTE, the distribution of FVL worldwide, and then it elaborates on the epidemiology of FVL in the Mediterranean Region and how this brought speculations that FVL might have originated in the Eastern Mediterranean area.

  2. Epidemiology of Activated Protein C Resistance and Factor V Leiden Mutation in the Mediterranean Region

    Science.gov (United States)

    Jadaon, Mehrez M.

    2011-01-01

    Venous thromboembolic disorders (VTE) are serious disorders with high morbidity and mortality rates. Many genetic and acquired risk factors were identified to cause VTE. The most common genetic risk factor is Factor V Leiden mutation (FVL). FVL was found in high percentage of populations of Caucasian origin but was almost absent in non-Caucasians. It was also reported in populations living in North Africa and the Middle East. This review article briefly explains FVL and how it causes VTE, the distribution of FVL worldwide, and then it elaborates on the epidemiology of FVL in the Mediterranean Region and how this brought speculations that FVL might have originated in the Eastern Mediterranean area. PMID:22224194

  3. The mtDNA NARP mutation activates the actin-Nrf2 signaling of antioxidant defenses

    International Nuclear Information System (INIS)

    An efficient handling of superoxides by antioxidant defenses is a crucial issue for cells with respiratory chain deficient mitochondria. We used human cultured skin fibroblasts to delineate the mechanism controlling the expression of antioxidant defenses in the case of a severe ATPase deficiency resulting from an 8993T>G mutation in the mitochondrial ATPase6 gene. We observed the nuclear translocation of the transcription factor Nrf2 associated with thinning of the actin stress fibers. The mobilization of the Nrf2 signaling pathway could be mimicked by a chemical blockade of the ATPase with a specific inhibitor, oligomycin. Interestingly enough, Nrf2 nuclear translocation was not observed in the case of a severe cytochrome oxidase deficiency, indicating that studying the status of this signaling pathway could throw some light on the importance of the oxidative insult associated with different respiratory chain defects

  4. Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors

    DEFF Research Database (Denmark)

    Goriely, Anne; Hansen, Ruth M S; Taylor, Indira B;

    2009-01-01

    Genes mutated in congenital malformation syndromes are frequently implicated in oncogenesis, but the causative germline and somatic mutations occur in separate cells at different times of an organism's life. Here we unify these processes to a single cellular event for mutations arising in male germ...

  5. Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations

    Directory of Open Access Journals (Sweden)

    Ludovica Ciuffreda

    2009-08-01

    Full Text Available The Raf/MEK/ERK pathway is an importantmediator of tumor cell proliferation and angiogenesis. Here, weinvestigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1 and apoptosis (Bcl-2 and survivin regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma.

  6. CYP2U1 mutations in two Iranian patients with activity induced dystonia, motor regression and spastic paraplegia

    Science.gov (United States)

    Kariminejad, A.; Schöls, L.; Schüle, R.; Tonekaboni, S.H.; Abolhassani, A.; Fadaee, M.; Rosti, R.O.; Gleeson, J.G.

    2016-01-01

    Hereditary spastic paraplegia (HSP) is a heterogeneous condition characterized by progressive spasticity and weakness in the lower limbs. It is divided into two major groups, complicated and uncomplicated, based on the presence of additional features such as intellectual disability, ataxia, seizures, peripheral neuropathy and visual problems. SPG56 is an autosomal recessive form of HSP with complicated and uncomplicated manifestations, complicated being more common. CYP2U1 gene mutations have been identified as responsible for SPG56. Intellectual disability, dystonia, subclinical sensory motor neuropathy, pigmentary degenerative maculopathy, thin corpus callosum and periventricular white-matter hyperintensities were additional features noted in previous cases of SPG56. Here we identified two novel mutations in CYP2U1 in two unrelated patients by whole exome sequencing. Both patients had complicated HSP with activity-induced dystonia, suggesting dystonia as an additional finding in SPG56. Two out of 14 previously reported patients had dystonia, and the addition of our patients suggests dystonia in a quarter of SPG56 patients. Developmental regression has not been reported in SPG56 patients so far but both of our patients developed motor regression in infancy. PMID:27292318

  7. Superantigenic activity of emm3 Streptococcus pyogenes is abrogated by a conserved, naturally occurring smeZ mutation.

    Directory of Open Access Journals (Sweden)

    Claire E Turner

    Full Text Available Streptococcus pyogenes M/emm3 strains have been epidemiologically linked with enhanced infection severity and risk of streptococcal toxic shock syndrome (STSS, a syndrome triggered by superantigenic stimulation of T cells. Comparison of S. pyogenes strains causing STSS demonstrated that emm3 strains were surprisingly less mitogenic than other emm-types (emm1, emm12, emm18, emm28, emm87, emm89 both in vitro and in vivo, indicating poor superantigenic activity. We identified a 13 bp deletion in the superantigen smeZ gene of all emm3 strains tested. The deletion led to a premature stop codon in smeZ, and was not present in other major emm-types tested. Expression of a functional non-M3-smeZ gene successfully enhanced mitogenic activity in emm3 S. pyogenes and also restored mitogenic activity to emm1 and emm89 S. pyogenes strains where the smeZ gene had been disrupted. In contrast, the M3-smeZ gene with the 13 bp deletion could not enhance or restore mitogenicity in any of these S. pyogenes strains, confirming that M3-smeZ is non-functional regardless of strain background. The mutation in M3-smeZ reduced the potential for M3 S. pyogenes to induce cytokines in human tonsil, but not during invasive infection of superantigen-sensitive mice. Notwithstanding epidemiological associations with STSS and disease severity, emm3 strains have inherently poor superantigenicity that is explained by a conserved mutation in smeZ.

  8. The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity.

    Science.gov (United States)

    Mhaske, Pallavi V; Levit, Noah A; Li, Leping; Wang, Hong-Zhan; Lee, Jack R; Shuja, Zunaira; Brink, Peter R; White, Thomas W

    2013-06-15

    Mutations in the human gene encoding connexin 26 (Cx26 or GJB2) cause either nonsyndromic deafness or syndromic deafness associated with skin diseases. That distinct clinical disorders can be caused by different mutations within the same gene suggests that different channel activities influence the ear and skin. Here we use three different expression systems to examine the functional characteristics of two Cx26 mutations causing either mild (Cx26-D50A) or lethal (Cx26-A88V) keratitis-ichthyosis-deafness (KID) syndrome. In either cRNA-injected Xenopus oocytes, transfected HeLa cells, or transfected primary human keratinocytes, we show that both Cx26-D50A and Cx26-A88V form active hemichannels that significantly increase membrane current flow compared with wild-type Cx26. This increased membrane current accelerated cell death in low extracellular calcium solutions and was not due to increased mutant protein expression. Elevated mutant hemichannel currents could be blocked by increased extracellular calcium concentration. These results show that these two mutations exhibit a shared gain of functional activity and support the hypothesis that increased hemichannel activity is a common feature of human Cx26 mutations responsible for KID syndrome.

  9. Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Flueck, Christa E.; Mullis, Primus E. [Pediatric Endocrinology, Diabetology and Metabolism, Department of Clinical Research, University of Bern, Tiefenaustrasse 120c, CH 3004 Bern (Switzerland); Pandey, Amit V., E-mail: amit@pandeylab.org [Pediatric Endocrinology, Diabetology and Metabolism, Department of Clinical Research, University of Bern, Tiefenaustrasse 120c, CH 3004 Bern (Switzerland)

    2010-10-08

    Research highlights: {yields} Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). {yields} Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. {yields} We are reporting that mutations in POR may reduce CYP3A4 activity. {yields} POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. {yields} Reduction of CYP3A4 activity may cause increased risk of drug toxicities/adverse drug reactions in patients with POR mutations. -- Abstract: Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.

  10. Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity.

    Science.gov (United States)

    Styles, Nathan A; Shonsey, Erin M; Falany, Josie L; Guidry, Amber L; Barnes, Stephen; Falany, Charles N

    2016-07-01

    Bile acid CoA:amino acid N-acyltransferase (BAAT) is the terminal enzyme in the synthesis of bile salts from cholesterol and catalyzes the conjugation of taurine or glycine to bile acid CoA thioesters to form bile acid N-acylamidates. BAAT has a dual localization to the cytosol and peroxisomes, possibly due to an inefficient carboxy-terminal peroxisomal targeting signal (PTS), -serine-glutamine-leucine (-SQL). Mutational analysis was used to define the role of the carboxy terminus in peroxisomal localization and kinetic activity. Amidation activity of BAAT and BAAT lacking the final two amino acids (AAs) (BAAT-S) were similar, whereas the activity of BAAT with a canonical PTS sequence (BAAT-SKL) was increased >2.5-fold. Kinetic analysis of BAAT and BAAT-SKL showed that BAAT-SKL had a lower Km for taurine and glycine as well as a greater Vmax There was no difference in the affinity for cholyl-CoA. In contrast to BAAT, BAAT-SKL forms bile acid N-acylamidates with β-alanine. BAAT-S immunoprecipitated when incubated with peroxisomal biogenesis factor 5 (Pex5) and rabbit anti-Pex5 antibodies; however, deleting the final 12 AAs prevented coimmunoprecipitation with Pex5, indicating the Pex5 interaction involves more than the -SQL sequence. These results indicate that even small changes in the carboxy terminus of BAAT can have significant effects on activity and substrate specificity. PMID:27230263

  11. A Mutation in Caenorhabditis elegans NDUF-7 Activates the Mitochondrial Stress Response and Prolongs Lifespan via ROS and CED-4.

    Science.gov (United States)

    Rauthan, Manish; Ranji, Parmida; Abukar, Ragda; Pilon, Marc

    2015-06-01

    The mevalonate pathway is responsible for the synthesis of cholesterol, coenzyme Q, and prenyl groups essential for small GTPase modification and function, and for the production of dolichols important for protein glycosylation. Statins, i.e., cholesterol-lowering drugs that inhibit the rate-limiting enzyme in the mevalonate pathway, HMG-CoA reductase, are lethal to Caenorhabditis elegans even though this animal lacks the branch of the mevalonate pathway that leads to cholesterol synthesis. To better understand the effects of statins that are not related to cholesterol, we have adopted the strategy of isolating statin-resistant C. elegans mutants. Previously, we showed that such mutants often have gain-of-function mutations in ATFS-1, a protein that activates the mitochondrial unfolded protein response. Here, we describe the isolation of a statin-resistant mutant allele of the NDUF-7 protein, which is a component of complex I in the mitochondrial electron transport chain. The novel nduf-7(et19) mutant also exhibits constitutive and ATFS-1-dependent activation of the mitochondrial unfolded protein response (UPR(mt)) and prolonged life span, both of which are mediated through production of ROS. Additionally, lifespan extension, but not activation, of the mitochondrial unfolded protein response was dependent on the pro-apoptotic gene ced-4. We conclude that the nduf-7(et19) mutant allele causes an increase in reactive oxygen species that activate ATFS-1, hence UPR(mt)-mediated statin resistance, and extends life span via CED-4.

  12. Mutational changes in the courtship activity of male guppies (Poecilia reticulata) after X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Werner, M.; Schroeder, J.H.

    1980-07-01

    The courtship activity of male F2 descendants of irradiated and control guppies, Poecilia reticulata, of the inbred strain Istanbul was compared. The results of Spieser and Schroeder (1978), who found a decrease in courtship activity of descendants of irradiated guppies, were confirmed under more natural conditions.

  13. Laboratory Activities to Support Student Understanding of the Molecular Mechanisms of Mutation & Natural Selection

    Science.gov (United States)

    Hubler, Tina; Adams, Patti; Scammell, Jonathan

    2015-01-01

    The molecular basis of evolution is an important and challenging concept for students to understand. In a previous article, we provided some of the scientific background necessary to teach this topic. This article features a series of laboratory activities demonstrating that molecular events can alter the genomes of organisms. These activities are…

  14. Fatal infantile cardiac glycogenosis with phosphorylase kinase deficiency and a mutation in the gamma2-subunit of AMP-activated protein kinase.

    Science.gov (United States)

    Akman, Hasan O; Sampayo, James N; Ross, Fiona A; Scott, John W; Wilson, Gregory; Benson, Lee; Bruno, Claudio; Shanske, Sara; Hardie, D Grahame; Dimauro, Salvatore

    2007-10-01

    A 10-wk-old infant girl with severe hypertrophy of the septal and atrial walls by cardiac ultrasound, developed progressive ventricular wall thickening and died of aspiration pneumonia at 5 mo of age. Postmortem examination revealed ventricular hypertrophy and massive atrial wall thickening due to glycogen accumulation. A skeletal muscle biopsy showed increased free glycogen and decreased activity of phosphorylase b kinase (PHK). The report of a pathogenic mutation (R531Q) in the gene (PRKAG2) encoding the gamma2 subunit of AMP-activated protein kinase (AMPK) in three infants with congenital hypertrophic cardiomyopathy, glycogen storage, and "pseudo PHK deficiency" prompted us to screen this gene in our patient. We found a novel (R384T) heterozygous mutation in PRKAG2, affecting an arginine residue in the N-terminal AMP-binding domain. Like R531Q, this mutation reduces the binding of AMP and ATP to the isolated nucleotide-binding domains, and prevents activation of the heterotrimer by metabolic stress in intact cells. The mutation was not found in DNA from the patient's father, the only available parent, and is likely to have arisen de novo. Our studies confirm that mutations in PRKAG2 can cause fatal infantile cardiomyopathy, often associated with apparent PHK deficiency.

  15. Active site mutants of Escherichia coli dethiobiotin synthetase: effects of mutations on enzyme catalytic and structural properties.

    Science.gov (United States)

    Yang, G; Sandalova, T; Lohman, K; Lindqvist, Y; Rendina, A R

    1997-04-22

    Five active site residues, Thr11, Glu12, Lys15, Lys37, and Ser41, implicated by the protein crystal structure studies of Escherichia coli DTBS, were mutated to determine their function in catalysis and substrate binding. Nine mutant enzymes, T11V, E12A, E12D, K15Q, K37L, K37Q, K37R, S41A, and S41C, were overproduced in an E. coli strain lacking a functional endogenous DTBS gene and purified to homogeneity. Replacement of Thr11 with valine resulted in a 24,000-fold increase in the Km(ATP) with little or no change in the Kd(ATP), KM(DAPA) and DTBS k(cat), suggesting an essential role for this residue in the steady-state affinity for ATP. The two Glu12 mutants showed essentially wild-type DTBS activity (slightly elevated k(cat)'s). Unlike wild-type DTBS, E12A had the same apparent KM(DAPA) at subsaturating and saturating ATP concentrations, indicating a possible role for Glu12 in the binding synergy between DAPA and ATP. The mutations in Lys15 and Lys37 resulted in loss of catalytic activity (0.01% and cat) for K15Q and the Lys37 mutant enzymes, respectively) and higher KM's for both DAPA (40-fold and >100-fold higher than wild-type for the K15Q and Lys37 mutant enzymes, respectively) and ATP (1800-fold and >10-fold higher than wild-type for K15Q and the K37 mutant enzymes, respectively). These results strongly suggest that Lys15 and Lys37 are crucial to both catalysis and substrate binding. S41A and S41C had essentially the same k(cat) as wild-type and had moderate increases in the DAPA and ATP KM and Kd (ATP) values. Replacement of Ser41 with cysteine resulted in larger effects than replacement with alanine. These data suggest that the H-bond between N7 of DAPA and the Ser41 side chain is not very important for catalysis. The catalytic behavior of these mutant enzymes was also studied by pulse-chase experiments which produced results consistent with the steady-state kinetic analyses. X-ray crystallographic studies of four mutant enzymes, S41A, S41C, K37Q, and K37L

  16. Pathogenic LRRK2 mutations, through increased kinase activity, produce enlarged lysosomes with reduced degradative capacity and increase ATP13A2 expression.

    Science.gov (United States)

    Henry, Anastasia G; Aghamohammadzadeh, Soheil; Samaroo, Harry; Chen, Yi; Mou, Kewa; Needle, Elie; Hirst, Warren D

    2015-11-01

    Lysosomal dysfunction plays a central role in the pathogenesis of several neurodegenerative disorders, including Parkinson's disease (PD). Several genes linked to genetic forms of PD, including leucine-rich repeat kinase 2 (LRRK2), functionally converge on the lysosomal system. While mutations in LRRK2 are commonly associated with autosomal-dominant PD, the physiological and pathological functions of this kinase remain poorly understood. Here, we demonstrate that LRRK2 regulates lysosome size, number and function in astrocytes, which endogenously express high levels of LRRK2. Expression of LRRK2 G2019S, the most common pathological mutation, produces enlarged lysosomes and diminishes the lysosomal capacity of these cells. Enlarged lysosomes appears to be a common phenotype associated with pathogenic LRRK2 mutations, as we also observed this effect in cells expressing other LRRK2 mutations; R1441C or Y1699C. The lysosomal defects associated with these mutations are dependent on both the catalytic activity of the kinase and autophosphorylation of LRRK2 at serine 1292. Further, we demonstrate that blocking LRRK2's kinase activity, with the potent and selective inhibitor PF-06447475, rescues the observed defects in lysosomal morphology and function. The present study also establishes that G2019S mutation leads to a reduction in lysosomal pH and increased expression of the lysosomal ATPase ATP13A2, a gene linked to a parkinsonian syndrome (Kufor-Rakeb syndrome), in brain samples from mouse and human LRRK2 G2019S carriers. Together, these results demonstrate that PD-associated LRRK2 mutations perturb lysosome function in a kinase-dependent manner, highlighting the therapeutic promise of LRRK2 kinase inhibitors in the treatment of PD. PMID:26251043

  17. Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities

    Energy Technology Data Exchange (ETDEWEB)

    Kajikawa, Takao; Kataoka, Kunishige [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Sakurai, Takeshi, E-mail: tsakurai@se.kanazawa-u.ac.jp [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Proton transfer pathway to dioxygen in CueO was identified. Black-Right-Pointing-Pointer Glu506 is the key amino acid to transport proton. Black-Right-Pointing-Pointer The Ala mutation at Glu506 formed a compensatory proton transfer pathway. Black-Right-Pointing-Pointer The Ile mutation at Glu506 shut down the hydrogen bond network. -- Abstract: CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase.

  18. Pulmonary adenocarcinoma in situ: analyses of a large series with reference to smoking, driver mutations, and receptor tyrosine kinase pathway activation.

    Science.gov (United States)

    Sato, Seijiro; Motoi, Noriko; Hiramatsu, Miyako; Miyauchi, Eisaku; Ono, Hiroshi; Saito, Yuichi; Nagano, Hiroko; Ninomiya, Hironori; Inamura, Kentaro; Uehara, Hirofumi; Mun, Mingyon; Sakao, Yukinori; Okumura, Sakae; Tsuchida, Masanori; Ishikawa, Yuichi

    2015-07-01

    Lung adenocarcinomas in situ (AISs) often occur in individuals who have never smoked, although smoking is one of the main causes of lung cancer. To characterize AIS and, in particular, determine how AIS might be related to smoking, we collected a large number of AIS cases and examined clinicopathologic features, EGFR and KRAS mutation status, and activation status of receptor tyrosine kinase downstream signal pathways, including pAkt, pERK, and pStat3, using immunohistochemistry. We identified 110 AISs (36 smokers and 74 nonsmokers) among 1549 adenocarcinomas resected surgically during 1995 to 2010. Between the AIS of smokers and nonsmokers, only the sex ratio was significantly different; all the other clinicopathologic factors including TTF-1 and driver mutations were not significantly different: EGFR and KRAS mutation rates (smokers:nonsmokers) were 61:58 (%) (P=0.7) and 6.1:1.4 (%) (P=0.2), respectively, whereas, in invasive adenocarcinomas, the rates were 41:69 (%) (P80% were positive, with no significant differences between smokers and nonsmokers with AIS. Mucinous AIS (n=8) rarely harbored KRAS mutations and expressed significantly less pStat3 (Plineage, driver mutations, and receptor tyrosine kinase pathway activation. Our results suggest that smoking is not a major cause of AIS. Rather, smoking may play a role in progression of AIS to invasive adenocarcinoma with AIS features. PMID:25970685

  19. Segmental overgrowth syndrome due to an activating PIK3CA mutation identified in affected muscle tissue by exome sequencing

    DEFF Research Database (Denmark)

    Rasmussen, Maria; Sunde, Lone; Weigert, Karen Petra;

    2014-01-01

    Mosaic PIK3CA-mutations have been described in an increasing number of overgrowth syndromes. We describe a patient with a previously unreported segmental overgrowth syndrome with the mutation, PIKCA3 c.3140A>G (p.His1047Arg) in affected tissue diagnosed by exome sequencing. This PIK3CA-associated......Mosaic PIK3CA-mutations have been described in an increasing number of overgrowth syndromes. We describe a patient with a previously unreported segmental overgrowth syndrome with the mutation, PIKCA3 c.3140A>G (p.His1047Arg) in affected tissue diagnosed by exome sequencing. This PIK3CA...

  20. Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

    Directory of Open Access Journals (Sweden)

    Brewster Aaron S

    2010-08-01

    Full Text Available Abstract Background The mini-chromosome maintenance protein (MCM complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7, the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM, six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp. We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

  1. Mutational Analysis of the Absolutely Conserved B8Gly: Consequence on Foldability and Activity of Insulin

    Institute of Scientific and Technical Information of China (English)

    Zhan-Yun GUO; Zhou ZHANG; Xiao-Yuan JIA; Yue-Hua TANG; You-Min FENG

    2005-01-01

    B8Gly is absolutely conserved in insulins during evolution. Moreover, its corresponding position is always occupied by a Gly residue in other members of insulin superfamily. Previous work showed that Ala replacement of B8Gly significantly decreased both the activity and the foldability of insulin. However,the effects of substitution are complicated, and different replacements sometimes cause significantly different results. To analyze the effects of B8 replacement by different amino acids, three new insulin/single-chain insulin mutants with B8Gly replaced by Ser, Thr or Leu were prepared by protein engineering, and both their foldability and activity were analyzed. In general, replacement of B8Gly by other amino acids causes significant detriment to the foldability of single-chain insulin: the conformations of the three B8 mutants are essentially different from that of wild-type molecules as revealed by circular dichroism; their disulfide stabilities in redox buffer are significantly decreased; their in vitro refolding efficiencies are decreased approximately two folds; the structural stabilities of the mutants with Ser or Thr substitution are decreased significantly,while Leu substitution has little effect as measured by equilibrium guanidine denaturation. As far as biological activity is concerned, Ser replacement of B8Gly has only a moderate effect: its insulin receptor-binding activity is 23% of native insulin. But Thr or Leu replacement produces significant detriment: the receptorbinding potencies of the two mutants are less than 0.2% of native insulin. The present results suggest that Gly is likely the only applicable natural amino acid for the B8 position of insulin where both foldability and activity are concerned.

  2. Chemical models for cytochrome P450 as a biomimetic metabolic activation system in mutation assays.

    Science.gov (United States)

    Inami, Keiko; Mochizuki, Masataka

    2002-08-26

    DNA damage is a critical factor in carcinogenesis. The Ames assay is a short-term test that screens for DNA-damaging agents. To be detected in the assay, most carcinogens require oxidation by cytochrome P450, a component of the liver homogenate preparation (S9 mix) that is traditionally used to metabolize promutagens to an active form in vitro. A combination of iron(III) porphyrin plus an oxidant activates many promutagens by mimicking cytochrome P450 metabolism. We previously reported that the mutagenicity of the N-nitrosodialkylamines was detected following reaction with tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (Fe(F(5)P)Cl) plus tert-butyl hydroperoxide (t-BuOOH), which yielded the same alcohols and aldehydes as the enzymatic reaction. In the present study, to extend the scope of biomimetic models, we tested the mutagenicity of other carcinogens exposed to chemical oxidation systems.We investigated the optimal assay conditions for the models in Salmonella typhimurium TA1538, a strain sensitive to frame-shift mutagens. We activated 2-aminofluorene (AF), benzo[a]pyrene (B[a]P), a tryptophane pyrolysate 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), and 2-acetylaminofluorene (AAF) with Fe(F(5)P)Cl plus an oxidant-t-BuOOH, m-chloroperoxybenzoic acid (mCPBA), or magnesium monoperoxyphthalate (MPPT)-and we noted the effect of three solvents-acetonitrile (CH(3)CN),1,4-dioxane, and N,N-dimethylformamide (DMF)-on AF activation. All the promutagens became mutagenic in the presence of Fe(F(5)P)Cl plus an oxidant, with the effectiveness of the oxidant varying with the chemical. Aromatic amines, for example, showed the strongest mutagenicity with t-BuOOH whereas polycyclic hydrocarbons showed the strongest mutagenicity with mCPBA. All the promutagens were mutagenic in the presence of Fe(F(5)P)Cl plus MPPT. For AF activation, the order of effectiveness of the solvents was CH(3)CN>1,4-dioxane>DMF. The results suggested that these systems would serve as

  3. Allosteric modulation and constitutive activity of fusion proteins between the adenosine A1 receptor and different 351Cys-mutated Gi α-subunits

    NARCIS (Netherlands)

    Klaasse, E.; Ligt, R.A.F.de; Roerink, S.F.; Lorenzen, A.; Milligan, G.; Leurs, R.; IJzerman, A.P.

    2004-01-01

    We studied fusion proteins between the human adenosine A1 receptor and different 351Cys-mutated Gi1 α-subunits (A1-Giα) with respect to two important concepts in receptor pharmacology, i.e. allosteric modulation and constitutive activity/inverse agonism. The aim of our study was twofold. We first an

  4. Effects of Somatic Mutations in the C-Terminus of Insulin-Like Growth Factor 1 Receptor on Activity and Signaling

    Directory of Open Access Journals (Sweden)

    Barbara P. Craddock

    2012-01-01

    Full Text Available The insulin-like growth factor I receptor (IGF1R is overexpressed in several forms of human cancer, and it has emerged as an important target for anticancer drug design. Cancer genome sequencing efforts have recently identified three somatic mutations in IGF1R: A1374V, a deletion of S1278 in the C-terminal tail region of the receptor, and M1255I in the C-terminal lobe of the kinase catalytic domain. The possible effects of these mutations on IGF1R activity and biological function have not previously been tested. Here, we tested the effects of the mutations on the in vitro biochemical activity of IGF1R and on major IGF1R signaling pathways in mammalian cells. While the mutations do not affect the intrinsic tyrosine kinase activity of the receptor, we demonstrate that the basal (unstimulated levels of MAP kinase and Akt activation are increased in the mutants (relative to wild-type IGF1R. We hypothesize that the enhanced signaling potential of these mutants is due to changes in protein-protein interactions between the IGF1R C-terminus and cellular substrates or modulators.

  5. Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Hansen, Dennis K.; Webb, Helen; Nielsen, Jonas Willum;

    2015-01-01

    Mutational analysis of Sulfolobus solfataricus class II α-mannosidase was focused on side chains that interact with the hydroxyls of the-1 mannosyl of the substrate (Asp-534) or form ligands to the active site divalent metal ion (His-228 and His-533) judged from crystal structures of homologous...... enzymes. D534A and D534N appeared to be completely inactive. When compared to the wild-type enzyme, the mutant enzymes in general showed only small changes in KM for the substrate, p-nitrophenyl-α-mannoside, but elevated activation constants, KA, for the divalent metal ion (Co2+, Zn2+, Mn2+, or Cd2......+). Some mutant enzyme forms displayed an altered preference for the metal ion compared to that of the wild type-enzyme. Furthermore, the H228Q, H533E, and H533Q enzymes were inhibited at increasing Zn2+ concentrations. The catalytic rate was reduced for all enzymes compared to that of the wild-type enzyme...

  6. Constitutive activation of the Saccharomyces cerevisiae transcriptional regulator Ste12p by mutations at the amino-terminus.

    Science.gov (United States)

    Crosby, J A; Konopka, J B; Fields, S

    2000-11-01

    The transcriptional activator Ste12p is required for the expression of genes induced by mating pheromone in the yeast Saccharomyces cerevisiae. We identified mutations in the amino-terminal DNA-binding domain of Ste12p that lead to constitutively high-level transcription of pheromone-induced genes. The behaviour of these mutant proteins is consistent with an enhanced DNA-binding ability. Cells carrying these hyperactive proteins retain their sensitivity to pheromone treatment, and their phenotype is largely dependent on the presence of at least one of the MAP kinases (Fus3p or Kss1p) and the scaffold protein Ste5p. Deletion of either FUS3 or KSS1 leads to a marked increase in Ste12p activity, consistent with a negative regulatory role for Fus3p, similar to that described for Kss1p. The properties of the constitutive mutants support the idea that the pheromone response pathway plays a role in basal as well as pheromone-induced transcription. PMID:11054817

  7. A novel mutation in the β-spectrin gene causes the activation of a cryptic 5'-splice site and the creation of a de novo 3'-splice site.

    Science.gov (United States)

    Salas, Pilar Carrasco; Rosales, José Miguel Lezana; Milla, Carmen Palma; Montiel, Javier López; Siles, Juan López

    2015-01-01

    The analysis of genes involved in hereditary spherocytosis, by next-generation sequencing in two patients with clinical diagnosis of the disease, showed the presence of the c.1795+1G>A mutation in the SPTB gene. cDNA amplification then revealed the occurrence of a consequent aberrant mRNA isoform produced from the activation of a cryptic 5'-splice site and the creation of a newly 3'-splice site. The mechanisms by which these two splice sites are used as a result of the same mutation should be analyzed in depth in further studies.

  8. Mutational analysis of GlnB residues critical for NifA activation in Azospirillum brasilense.

    Science.gov (United States)

    Inaba, Juliana; Thornton, Jeremy; Huergo, Luciano Fernandes; Monteiro, Rose Adele; Klassen, Giseli; Pedrosa, Fábio de Oliveira; Merrick, Mike; de Souza, Emanuel Maltempi

    2015-02-01

    PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A. brasilense contains two PII encoding genes, named glnB and glnZ. In this paper, glnB was mutagenised in order to identify amino acid residues involved in GlnB signaling. Two variants were obtained by random mutagenesis, GlnBL13P and GlnBV100A and a site directed mutant, GlnBY51F, was obtained. Their ability to complement nitrogenase activity of glnB mutant strains of A. brasilense were determined. The variant proteins were also overexpressed in Escherichia coli, purified and characterized biochemically. None of the GlnB variant forms was able to restore nitrogenase activity in glnB mutant strains of A. brasilense LFH3 and 7628. The purified GlnBY51F and GlnBL13P proteins could not be uridylylated by GlnD, whereas GlnBV100A was uridylylated but at only 20% of the rate for wild type GlnB. Biochemical and computational analyses suggest that residue Leu13, located in the α helix 1 of GlnB, is important to maintain GlnB trimeric structure and function. The substitution V100A led to a lower affinity for ATP binding. Together the results suggest that NifA activation requires uridylylated GlnB bound to ATP.

  9. Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex Structure-Activity Relationships

    Energy Technology Data Exchange (ETDEWEB)

    J Beierlein; N Karri; A Anderson

    2011-12-31

    Several antifolates, including trimethoprim (TMP) and a series of propargyl-linked analogues, bind dihydrofolate reductase from Bacillus anthracis (BaDHFR) with lower affinity than is typical in other bacterial species. To guide lead optimization for BaDHFR, we explored a new approach to determine structure-activity relationships whereby the enzyme is altered and the analogues remain constant, essentially reversing the standard experimental design. Active site mutants of the enzyme, Ba(F96I)DHFR and Ba(Y102F)DHFR, were created and evaluated with enzyme inhibition assays and crystal structures. The affinities of the antifolates increase up to 60-fold with the Y102F mutant, suggesting that interactions with Tyr 102 are critical for affinity. Crystal structures of the enzymes bound to TMP and propargyl-linked inhibitors reveal the basis of TMP resistance and illuminate the influence of Tyr 102 on the lipophilic linker between the pyrimidine and aryl rings. Two new inhibitors test and validate these conclusions and show the value of the technique for providing new directions during lead optimization.

  10. The missense Thr211Pro mutation in the factor X activation peptide of a bleeding patient causes molecular defect in the clotting cascade.

    Science.gov (United States)

    Ding, Qiulan; Shen, Yiping; Yang, Likui; Wang, Xuefeng; Rezaie, Alireza R

    2013-07-01

    Factor X (FX) is a vitamin K-dependent coagulation zymogen, which upon activation to factor Xa assembles into the prothrombinase complex to activate prothrombin to thrombin. FX can be activated by either factor VIIa-tissue factor or factor IXa-factor VIIIa in extrinsic and intrinsic pathways, respectively. In this study, we identified a bleeding patient with moderate FX deficiency who exhibits a clotting defect only in the intrinsic pathway. Exome sequencing revealed that the patient carries a novel homozygous missense mutation that results in substitution of Thr211 with Pro in the activation peptide of FX. Thr211 is the site of an O-linked glycosylation in the activation peptide of FX. We postulated that the lack of this post-translational modification specifically impacts the activation of FX by intrinsic Xase, thereby impairing thrombin generation in the subject. To test this hypothesis, we expressed both wild-type FX and FX containing this mutation in mammalian cells and following the purification of the zymogens to homogeneity characterized their properties in both purified and plasma-based assay systems. Analysis of the results suggests that Thr211 to Pro substitution renders the FX mutant a poor substrate for both physiological activators, however, at physiological concentration of the substrate, the clotting defect manifest itself only in the intrinsic pathway, thus explaining the bleeding phenotype for the patient carrying this mutation. PMID:23677006

  11. A point mutation in a silencer module reduces the promoter activity for the human mercaptopyruvate sulfurtransferase.

    Science.gov (United States)

    Nagahara, Noriyuki; Sreeja, V G; Li, Qing; Shimizu, Takako; Tsuchiya, Terumasa; Fujii-Kuriyama, Yoshiaki

    2004-11-01

    A promoter region of human mercaptopyruvate sulfurtransferase (MST) [EC 2.8.1.2] is G+C-rich and TATA-less, showing features of a house-keeping gene. In the core promoter, a GC box (-284:GGGGCGTGGC:-275) and an initiator (-219:TTATATG:-225) are found. A cap site hunting analysis for human liver cDNA revealed four possible transcriptional start sites, nucleotides -223, -159, -35 and -25. Point mutagenesis and deletion studies suggest that a module of the silencer element is -394:GCTG:-391. A replacement of -391G to C lost the silencer function; on the other hand, a replacement of -394G to T or C, -393C to T or -392T to G markedly reduced the promoter activity. PMID:15507321

  12. Gain-of-function mutations in the Toll-like Receptor pathway: TPL2-mediated ERK1/ERK2 MAPK activation, a path to tumorigenesis in lymphoid neoplasms?

    Directory of Open Access Journals (Sweden)

    Simon eRousseau

    2016-05-01

    Full Text Available Lymphoid neoplasms form a family of cancers affecting B-cells, T-cells and NK cells. The Toll-Like Receptor (TLR signalling adapter molecule MYD88 is the most frequently mutated gene in these neoplasms. This signalling adaptor relays signals from TLRs to downstream effector pathways such as the Nuclear Factor kappa B (NFB and Mitogen Activated Protein Kinase (MAPK pathways to regulate innate immune responses (Kawai and Akira, 2010. Gain-of-function mutations such as MYD88[L265P] activate downstream signalling pathways in absence of cognate ligands for TLRs, resulting in increased cellular proliferation and survival. This article reports an analysis of non-synonymous somatic mutations found in the TLR signaling network in lymphoid neoplasms. In accordance with previous reports, mutations map to MYD88 pro-inflammatory signaling and not TRIF-mediated Type I IFN production. Interestingly, the analysis of somatic mutations found downstream of the core TLR-signaling network uncovered a strong association with the ERK1/2 MAPK cascade. In support of this analysis, heterologous expression of MYD88[L265P] in HEK 293 cells led to ERK1/2 MAPK phosphorylation in addition to NFB activation. Moreover, this activation is dependent on the protein kinase Tumour Promoting Locus-2 (TPL-2, activated downstream of the IKK complex. Activation of ERK1/2 would then lead to activation, amongst others, of MYC and hnRNP A1, two proteins previously shown to contribute to tumour formation in lymphoid neoplasms. Taken together, this analysis suggests that TLR-mediated tumorigenesis occurs via the TPL2-mediated ERK1/2 activation. Therefore, the hypothesis proposed is that inhibition of ERK1/2 MAPK activation would prevent tumour growth downstream of MYD88[L265]. It will be interesting to test whether pharmacological inhibitors of this pathway show efficacy in primary tumour cells derived from hematologic malignancies such as Waldenstrom’s Macroglobulinemia, where the

  13. Dietary restriction-resistant human tumors harboring the PIK3CA-activating mutation H1047R are sensitive to metformin

    Science.gov (United States)

    Cufí, Sílvia; Corominas-Faja, Bruna; Lopez-Bonet, Eugeni; Bonavia, Rosa; Pernas, Sonia; López, Isabel álvarez; Dorca, Joan; Martínez, Susana; López, Norberto Batista; Fernández, Severina Domínguez; Cuyàs, Elisabet; Visa, Joana; Rodríguez-Gallego, Esther; Quirantes-Piné, Rosa; Segura-Carretero, Antonio; Joven, Jorge; Martin-Castillo, Begoña; Menendez, Javier A.

    2013-01-01

    Cancer cells expressing constitutively active phosphatidylinositol-3 kinase (PI3K) are proliferative regardless of the absence of insulin, and they form dietary restriction (DR)-resistant tumors in vivo. Because the binding of insulin to its receptors activates the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling cascade, activating mutations in the PIK3CA oncogene may determine tumor response to DR-like pharmacological strategies targeting the insulin and mTOR pathways. The anti-diabetic drug metformin is a stereotypical DR mimetic that exerts its anti-cancer activity through a dual mechanism involving insulin-related (systemic) and mTOR-related (cell-autonomous) effects. However, it remains unclear whether PIK3CA-activating mutations might preclude the anti-cancer activity of metformin in vivo. To model the oncogenic PIK3CA-driven early stages of cancer, we used the clonal breast cancer cell line MCF10DCIS.com, which harbors the gain-of-function H1047R hot-spot mutation in the catalytic domain of the PI3KCA gene and has been shown to form DR-refractory xenotumors. To model PIK3CA-activating mutations in late stages of cancer, we took advantage of the isogenic conversion of a PIK3CA-wild-type tumor into a PIK3CA H1047R-mutated tumor using the highly metastatic colorectal cancer cell line SW48. MCF10DCIS.com xenotumors, although only modestly affected by treatment with oral metformin (approximately 40% tumor growth inhibition), were highly sensitive to the intraperitoneal (i.p.) administration of metformin, the anti-cancer activity of which increased in a time-dependent manner and reached >80% tumor growth inhibition by the end of the treatment. Metformin treatment via the i.p. route significantly reduced the proliferation factor mitotic activity index (MAI) and decreased tumor cellularity in MCF10DCIS.com cancer tissues. Whereas SW48-wild-type (PIK3CA+/+) cells rapidly formed metformin-refractory xenotumors in mice, ad libitum access to water containing

  14. Mutations within the putative active site of heterodimeric deoxyguanosine kinase block the allosteric activation of the deoxyadenosine kinase subunit.

    Science.gov (United States)

    Park, Inshik; Ives, David H

    2002-03-31

    Replacement of the Asp-84 residue of the deoxyguanosine kinase subunit of the tandem deoxyadenosine kinase/ deoxyguanosine kinase (dAK/dGK) from Lactobacillus acidophilus R-26 by Ala, Asn, or Glu produced increased Km values for deoxyguanosine on dGK. However, it did not seem to affect the binding of Mg-ATP. The Asp-84 dGK replacements had no apparent effect on the binding of deoxyadenosine by dAK. However, the mutant dGKs were no longer inhibited by dGTP, normally a potent distal endproduct inhibitor of dGK. Moreover, the allosteric activation of dAK activity by dGTP or dGuo was lost in the modified heterodimeric dAK/dGK enzyme. Therefore, it seems very likely that Asp-84 participates in dGuo binding at the active site of the dGK subunit of dAK/dGK from Lactobacillus acidophilus R-26.

  15. The Antiviral Activity of Approved and Novel Drugs against HIV-1 Mutations Evaluated under the Consideration of Dose-Response Curve Slope.

    Directory of Open Access Journals (Sweden)

    Shuai Chang

    Full Text Available This study was designed to identify common HIV-1 mutation complexes affecting the slope of inhibition curve, and to propose a new parameter incorporating both the IC50 and the slope to evaluate phenotypic resistance.Utilizing site-directed mutagenesis, we constructed 22 HIV-1 common mutation complexes. IC50 and slope of 10 representative approved drugs and a novel agent against these mutations were measured to determine the resistance phenotypes. The values of new parameter incorporating both the IC50 and the slope of the inhibition curve were calculated, and the correlations between parameters were assessed.Depending on the class of drug, there were intrinsic differences in how the resistance mutations affected the drug parameters. All of the mutations resulted in large increases in the IC50s of nucleoside reverse transcriptase inhibitors. The effects of the mutations on the slope were the most apparent when examining their effects on the inhibition of non-nucleoside reverse transcriptase inhibitors and protease inhibitors. For example, some mutations, such as V82A, had no effect on IC50, but reduced the slope. We proposed a new concept, termed IIPatoxic, on the basis of IC50, slope and the maximum limiting concentrations of the drug. The IIPatoxic values of 10 approved drugs and 1 novel agent were calculated, and were closely related to the IIPmax values (r > 0.95, p < 0.001.This study confirms that resistance mutations cannot be accurately assessed by IC50 alone, because it tends to underestimate the degree of resistance. The slope parameter is of very importance in the measurement of drug resistance and the effect can be applied to more complex patterns of resistance. This is the most apparent when testing the effects of the mutations on protease inhibitors activity. We also propose a new index, IIPatoxic, which incorporates both the IC50 and the slope. This new index could complement current IIP indices, thereby enabling predict the

  16. The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation

    OpenAIRE

    Zarbock Ralf; Woischnik Markus; Sparr Christiane; Thurm Tobias; Kern Sunčana; Kaltenborn Eva; Hector Andreas; Hartl Dominik; Liebisch Gerhard; Schmitz Gerd; Griese Matthias

    2012-01-01

    Abstract Background Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We al...

  17. EPIDEMIOLOGY OF ACTIVATED PROTEIN C RESISTANCE AND FACTOR V LEIDEN MUTATION IN THE MEDITERRANEAN REGION

    Directory of Open Access Journals (Sweden)

    Mehrez Mehrez M. Jadaon

    2011-01-01

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    Venous thromboembolic disorders (VTE are serious disorders with high morbidity and mortality rates. Many genetic and acquired risk factors were identified to cause VTE The most common genetic risk factor is Factor V Leiden mutation (FVL. FVL was found in high percentage of populations of Caucasian origin but was almost absent in non-Caucasians. It was

  18. Acquired resistance mechanisms to tyrosine kinase inhibitors in lung cancer with activating epidermal growth factor receptor mutation--diversity, ductility, and destiny.

    Science.gov (United States)

    Suda, Kenichi; Mizuuchi, Hiroshi; Maehara, Yoshihiko; Mitsudomi, Tetsuya

    2012-12-01

    Lung cancers that harbor somatic activating mutations in the gene for the epidermal growth factor receptor (EGFR) depend on mutant EGFR for their proliferation and survival; therefore, lung cancer patients with EGFR mutations often dramatically respond to orally available EGFR tyrosine kinase inhibitors (TKIs). However, emergence of acquired resistance is virtually inevitable, thus limiting improvement in patient outcomes. To elucidate and overcome this acquired resistance, multidisciplinary basic and clinical investigational approaches have been applied, using in vitro cell line models or samples obtained from lung cancer patients treated with EGFR-TKIs. These efforts have revealed several acquired resistance mechanisms and candidates, including EGFR secondary mutations (T790M and other rare mutations), MET amplification, PTEN downregulation, CRKL amplification, high-level HGF expression, FAS-NFκB pathway activation, epithelial-mesenchymal transition, and conversion to small cell lung cancer. Interestingly, cancer cells harbor potential destiny and ductility together in acquiring resistance to EGFR-TKIs, as shown in in vitro acquired resistance models. Molecular mechanisms of "reversible EGFR-TKI tolerance" that occur in early phase EGFR-TKI exposure have been identified in cell line models. Furthermore, others have reported molecular markers that can predict response to EGFR-TKIs in clinical settings. Deeper understanding of acquired resistance mechanisms to EGFR-TKIs, followed by the development of molecular target drugs that can overcome the resistance, might turn this fatal disease into a chronic disorder. PMID:22736441

  19. Effect of mutation of two critical glutamic acid residues on the activity and stability of human carboxypeptidase M and characterization of its signal for glycosylphosphatidylinositol anchoring.

    Science.gov (United States)

    Tan, Fulong; Balsitis, Scott; Black, Judy K; Blöchl, Andrea; Mao, Ji-Fang; Becker, Robert P; Schacht, David; Skidgel, Randal A

    2003-03-01

    Human carboxypeptidase (CP) M was expressed in baculovirus-infected insect cells in a glycosylphosphatidylinositol-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidylinositol anchoring, was secreted at high levels into the medium. Both forms had lower molecular masses (50 kDa) than native placental CPM (62 kDa), indicating minimal glycosylation. The predicted glycosylphosphatidylinositol-anchor attachment site was investigated by mutation of Ser(406) to Ala, Thr or Pro and expression in HEK-293 and COS-7 cells. The wild-type and S406A and S406T mutants were expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, but the S406P mutant was not and was retained in a perinuclear location. The roles of Glu(260) and Glu(264) in CPM were investigated by site-directed mutagenesis. Mutation of Glu(260) to Gln had minimal effects on kinetic parameters, but decreased heat stability, whereas mutation to Ala reduced the k(cat)/ K(m) by 104-fold and further decreased stability. In contrast, mutation of Glu(264) to Gln resulted in a 10000-fold decrease in activity, but the enzyme still bound to p-aminobenzoylarginine-Sepharose and was resistant to trypsin treatment, indicating that the protein was folded properly. These results show that Glu(264) is the critical catalytic glutamic acid and that Glu(260) probably stabilizes the conformation of the active site.

  20. Fusing simulation and experiment: The effect of mutations on the structure and activity of the influenza fusion peptide

    Science.gov (United States)

    Lousa, Diana; Pinto, Antónia R. T.; Victor, Bruno L.; Laio, Alessandro; Veiga, Ana S.; Castanho, Miguel A. R. B.; Soares, Cláudio M.

    2016-01-01

    During the infection process, the influenza fusion peptide (FP) inserts into the host membrane, playing a crucial role in the fusion process between the viral and host membranes. In this work we used a combination of simulation and experimental techniques to analyse the molecular details of this process, which are largely unknown. Although the FP structure has been obtained by NMR in detergent micelles, there is no atomic structure information in membranes. To answer this question, we performed bias-exchange metadynamics (BE-META) simulations, which showed that the lowest energy states of the membrane-inserted FP correspond to helical-hairpin conformations similar to that observed in micelles. BE-META simulations of the G1V, W14A, G12A/G13A and G4A/G8A/G16A/G20A mutants revealed that all the mutations affect the peptide’s free energy landscape. A FRET-based analysis showed that all the mutants had a reduced fusogenic activity relative to the WT, in particular the mutants G12A/G13A and G4A/G8A/G16A/G20A. According to our results, one of the major causes of the lower activity of these mutants is their lower membrane affinity, which results in a lower concentration of peptide in the bilayer. These findings contribute to a better understanding of the influenza fusion process and open new routes for future studies. PMID:27302370

  1. Fusing simulation and experiment: The effect of mutations on the structure and activity of the influenza fusion peptide.

    Science.gov (United States)

    Lousa, Diana; Pinto, Antónia R T; Victor, Bruno L; Laio, Alessandro; Veiga, Ana S; Castanho, Miguel A R B; Soares, Cláudio M

    2016-01-01

    During the infection process, the influenza fusion peptide (FP) inserts into the host membrane, playing a crucial role in the fusion process between the viral and host membranes. In this work we used a combination of simulation and experimental techniques to analyse the molecular details of this process, which are largely unknown. Although the FP structure has been obtained by NMR in detergent micelles, there is no atomic structure information in membranes. To answer this question, we performed bias-exchange metadynamics (BE-META) simulations, which showed that the lowest energy states of the membrane-inserted FP correspond to helical-hairpin conformations similar to that observed in micelles. BE-META simulations of the G1V, W14A, G12A/G13A and G4A/G8A/G16A/G20A mutants revealed that all the mutations affect the peptide's free energy landscape. A FRET-based analysis showed that all the mutants had a reduced fusogenic activity relative to the WT, in particular the mutants G12A/G13A and G4A/G8A/G16A/G20A. According to our results, one of the major causes of the lower activity of these mutants is their lower membrane affinity, which results in a lower concentration of peptide in the bilayer. These findings contribute to a better understanding of the influenza fusion process and open new routes for future studies. PMID:27302370

  2. Hypertension-linked mutation of α-adducin increases CFTR surface expression and activity in HEK and cultured rat distal convoluted tubule cells.

    Directory of Open Access Journals (Sweden)

    Anna Mondini

    Full Text Available The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats, an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus hypertensive model carrying the F316Y adducin mutation, compared to MNS (Milan Normotensive Strain rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.

  3. BRAFV600E mutation, TIMP-1 upregulation, and NF-κB activation: closing the loop on the papillary thyroid cancer trilogy.

    Science.gov (United States)

    Bommarito, Alessandra; Richiusa, Pierina; Carissimi, Elvira; Pizzolanti, Giuseppe; Rodolico, Vito; Zito, Giovanni; Criscimanna, Angela; Di Blasi, Francesco; Pitrone, Maria; Zerilli, Monica; Amato, Marco C; Spinelli, Gaetano; Carina, Valeria; Modica, Giuseppe; Latteri, M Adelfio; Galluzzo, Aldo; Giordano, Carla

    2011-12-01

    BRAF(V600E) is the most common mutation found in papillary thyroid carcinoma (PTC). Tissue inhibitor of metalloproteinases (TIMP-1) and nuclear factor (NF)-κB have been shown to play an important role in thyroid cancer. In particular, TIMP-1 binds its receptor CD63 on cell surface membrane and activates Akt signaling pathway, which is eventually responsible for its anti-apoptotic activity. The aim of our study was to evaluate whether interplay among these three factors exists and exerts a functional role in PTCs. To this purpose, 56 PTC specimens were analyzed for BRAF(V600E) mutation, TIMP-1 expression, and NF-κB activation. We found that BRAF(V600E) mutation occurs selectively in PTC nodules and is associated with hyperactivation of NF-κB and upregulation of both TIMP-1 and its receptor CD63. To assess the functional relationship among these factors, we first silenced BRAF gene in BCPAP cells, harboring BRAF(V600E) mutation. We found that silencing causes a marked decrease in TIMP-1 expression and NF-κB binding activity, as well as decreased invasiveness. After treatment with specific inhibitors of MAPK pathway, we found that only sorafenib was able to increase IκB-α and reduce both TIMP-1 expression and Akt phosphorylation in BCPAP cells, indicating that BRAF(V600E) activates NF-κB and this pathway is MEK-independent. Taken together, our findings demonstrate that BRAF(V600E) causes upregulation of TIMP-1 via NF-κB. TIMP-1 binds then its surface receptor CD63, leading eventually to Akt activation, which in turn confers antiapoptotic behavior and promotion of cell invasion. The recognition of this functional trilogy provides insight on how BRAF(V600E) determines cancer initiation, progression, and invasiveness in PTC, also identifying new therapeutic targets for the treatment of highly aggressive forms.

  4. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity.

    Science.gov (United States)

    Cruz-Bermúdez, Alberto; Vicente-Blanco, Ramiro J; Hernández-Sierra, Rosana; Montero, Mayte; Alvarez, Javier; González Manrique, Mar; Blázquez, Alberto; Martín, Miguel Angel; Ayuso, Carmen; Garesse, Rafael; Fernández-Moreno, Miguel A

    2016-01-01

    The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.

  5. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity.

    Directory of Open Access Journals (Sweden)

    Alberto Cruz-Bermúdez

    Full Text Available The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids. Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.

  6. Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity

    Science.gov (United States)

    Cruz-Bermúdez, Alberto; Vicente-Blanco, Ramiro J.; Hernández-Sierra, Rosana; Montero, Mayte; Alvarez, Javier; González Manrique, Mar; Blázquez, Alberto; Martín, Miguel Angel; Ayuso, Carmen; Garesse, Rafael; Fernández-Moreno, Miguel A.

    2016-01-01

    The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations. PMID:26784702

  7. Effects of T592 phosphomimetic mutations on tetramer stability and dNTPase activity of SAMHD1 can not explain the retroviral restriction defect.

    Science.gov (United States)

    Bhattacharya, Akash; Wang, Zhonghua; White, Tommy; Buffone, Cindy; Nguyen, Laura A; Shepard, Caitlin N; Kim, Baek; Demeler, Borries; Diaz-Griffero, Felipe; Ivanov, Dmitri N

    2016-01-01

    SAMHD1, a dNTP triphosphohydrolase, contributes to interferon signaling and restriction of retroviral replication. SAMHD1-mediated retroviral restriction is thought to result from the depletion of cellular dNTP pools, but it remains controversial whether the dNTPase activity of SAMHD1 is sufficient for restriction. The restriction ability of SAMHD1 is regulated in cells by phosphorylation on T592. Phosphomimetic mutations of T592 are not restriction competent, but appear intact in their ability to deplete cellular dNTPs. Here we use analytical ultracentrifugation, fluorescence polarization and NMR-based enzymatic assays to investigate the impact of phosphomimetic mutations on SAMHD1 tetramerization and dNTPase activity in vitro. We find that phosphomimetic mutations affect kinetics of tetramer assembly and disassembly, but their effects on tetramerization equilibrium and dNTPase activity are insignificant. In contrast, the Y146S/Y154S dimerization-defective mutant displays a severe dNTPase defect in vitro, but is indistinguishable from WT in its ability to deplete cellular dNTP pools and to restrict HIV replication. Our data suggest that the effect of T592 phosphorylation on SAMHD1 tetramerization is not likely to explain the retroviral restriction defect, and we hypothesize that enzymatic activity of SAMHD1 is subject to additional cellular regulatory mechanisms that have not yet been recapitulated in vitro. PMID:27511536

  8. Mating-type suppression of the DNA-repair defect of the yeast rad6 delta mutation requires the activity of genes in the RAD52 epistasis group.

    Science.gov (United States)

    Yan, Y X; Schiestl, R H; Prakash, L

    1995-06-01

    The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MATa rad6 delta strain can be suppressed by the MAT alpha 2 gene carried on a multicopy plasmid. The a1-alpha 2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-alpha 2 suppression of the rad6 delta mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 delta mutant into the RAD52 recombinational repair pathway. The a1-alpha 2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.

  9. T-cell independent, B-cell receptor-mediated induction of telomerase activity differs among IGHV mutation-based subgroups of chronic lymphocytic leukemia patients

    OpenAIRE

    Damle, Rajendra N.; Temburni, Sonal; Banapour, Taraneh; Paul, Santanu; Mongini, Patricia K. A.; Allen, Steven L.; Kolitz, Jonathan E.; Rai, Kanti R; Chiorazzi, Nicholas

    2012-01-01

    Although B-cell chronic lymphocytic leukemia (B-CLL) clones with unmutated IGHV genes (U-CLL) exhibit greater telomerase activity than those with mutated IGHV genes (M-CLL), the extent to which B-cell receptor (BCR) triggering contributes to telomerase up-regulation is not known. Therefore, we studied the effect of BCR stimulation on modulating telomerase activity. The multivalent BCR ligand, dextran conjugated anti-μ mAb HB57 (HB57-dex), increased telomerase activity and promoted cell surviv...

  10. Triazole resistance mediated by mutations of a conserved active site tyrosine in fungal lanosterol 14α-demethylase.

    Science.gov (United States)

    Sagatova, Alia A; Keniya, Mikhail V; Wilson, Rajni K; Sabherwal, Manya; Tyndall, Joel D A; Monk, Brian C

    2016-01-01

    Emergence of fungal strains showing resistance to triazole drugs can make treatment of fungal disease problematic. Triazole resistance can arise due to single mutations in the drug target lanosterol 14α-demethylase (Erg11p/CYP51). We have determined how commonly occurring single site mutations in pathogenic fungi affect triazole binding using Saccharomyces cerevisiae Erg11p (ScErg11p) as a target surrogate. The mutations Y140F/H were introduced into full-length hexahistidine-tagged ScErg11p. Phenotypes and high-resolution X-ray crystal structures were determined for the mutant enzymes complexed with short-tailed (fluconazole and voriconazole) or long-tailed (itraconazole and posaconazole) triazoles and wild type enzyme complexed with voriconazole. The mutations disrupted a water-mediated hydrogen bond network involved in binding of short-tailed triazoles, which contain a tertiary hydroxyl not present in long-tailed triazoles. This appears to be the mechanism by which resistance to these short chain azoles occurs. Understanding how these mutations affect drug affinity will aid the design of azoles that overcome resistance. PMID:27188873

  11. Cerebral sinus thrombosis in a patient with active ulcerative colitis and double heterozygosity for Factor V Leiden and prothrombin gene mutations

    Directory of Open Access Journals (Sweden)

    Yakaryilmaz Fahri

    2009-01-01

    Full Text Available Inflammatory bowel diseases are associated with increased risk for thrombotic complications, In patients with ulcerative colitis (UC cerebral sinus venous thrombosis (CSVT is an extremely rare complication. We report a patient with active UC and CSVT. The patient was heterozygous for Factor V Leiden and G20210A prothrombin gene mutations without other identifiable precipitating factors. This patient highlights the need for investigating the patients with UC with thrombotic complications for other thrombophilic states.

  12. Meta-Analysis of First-Line Therapies in Advanced Non–Small-Cell Lung Cancer Harboring EGFR-Activating Mutations

    OpenAIRE

    Haaland, Benjamin; Tan, Pui San; Castro, Gilberto de; Lopes, Gilberto

    2014-01-01

    Introduction: Tyrosine kinase inhibitors gefitinib, erlotinib, and afatinib have been compared with chemotherapy as first-line therapies for patients with advanced non–small-cell lung cancer harboring epidermal growth factor receptor–activating mutations. This meta-analysis compares gefitinib, erlotinib, afatinib, and chemotherapy. Methods: Literature search was performed using relevant keywords. Direct and indirect meta-estimates were generated using log-linear mixed-effects models, with ran...

  13. Positive selection of mutations leading to loss or reduction of transcriptional activity of PrfA, the central regulator of Listeria monocytogenes virulence

    OpenAIRE

    Herler, M.; Bubert, A.; Goetz, M.; Vega, Yolanda; Vazquez-Boland, Jose A; Goebel, Werner

    2001-01-01

    Transcription factor PrfA controls the expression of virulence genes essential for Listeria monocytogenes pathogenesis. To gain insight into the structure-function relationship of PrfA, we devised a positive-selection system to isolate mutations reducing or abolishing transcriptional activity. The system is based on the observation that the listerial iap gene, encoding the p60 protein, is lethal if overexpressed in Bacillus subtilis. A plasmid in which the iap gene is placed under the control...

  14. A comparative study of drug resistance mechanism associated with active site and non-active site mutations: I388N and D425G mutants of acetyl-coenzyme-A carboxylase.

    Science.gov (United States)

    Zhu, Xiao-Lei; Yang, Guang-Fu

    2012-03-01

    A major concern in the development of acetyl-CoA carboxylase-inhibiting (ACCase; EC 6.4.1.2) herbicides is the emergence of resistance as a result of the selection of distinct mutations within the CT domain. Mutations associated with resistance have been demonstrated to include both active sites and non-active sites, including Ile-1781-Leu, Trp- 2027-Cys, Ile-2041-Asn, Asp-2078-Gly, and Gly-2096-Ala (numbered according to the Alopecurus myosuroides plastid ACCase). In the present study, extensive computational simulations, including molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM/PBSA) calculations, were carried out to compare the molecular mechanisms of active site mutation (I388N) and non-active site mutation (D425G) in Alopecurus myosuroides resistance to some commercial herbicides targeting ACCase, including haloxyfop (HF), diclofop (DF) and fenoxaprop (FR). All of the computational model and energetic results indicated that both I388N and D425G mutations have effects on the conformational change of the binding pocket. The π-π interaction between ligand and Phe377 and Tyr161' residues, which make an important contribution to the binding affinity, was decreased after mutation. As a result, the mutant-type ACCase has a lower affinity for the inhibitor than the wild-type enzyme, which accounts for the molecular basis of herbicidal resistance. The structural and mechanistic insights obtained from the present study will deepen our understanding of the interactions between ACCase and herbicides, which provides a molecular basis for the future design of a promising inhibitor with low resistance risk. PMID:22242795

  15. Registered report: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate

    Science.gov (United States)

    Fiehn, Oliver; Showalter, Megan Reed; Schaner-Tooley, Christine E

    2016-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from “The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate” by Ward and colleagues, published in Cancer Cell in 2010 (Ward et al., 2010). The experiments that will be replicated are those reported in Figures 2, 3 and 5. Ward and colleagues demonstrate the mutations in isocitrate dehydrogenase 2 (IDH2), commonly found in acute myeloid leukemia (AML), abrogate the enzyme’s wild-type activity and confer to the mutant neomorphic activity that produces the oncometabolite 2-hydroxyglutarate (2-HG) (Figures 2 and 3). They then show that elevated levels of 2-HG are correlated with mutations in IDH1 and IDH2 in AML patient samples (Figure 5). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published by eLife. DOI: http://dx.doi.org/10.7554/eLife.12626.001 PMID:26943899

  16. Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

    Science.gov (United States)

    Yamada, T; Takeuchi, S; Fujita, N; Nakamura, A; Wang, W; Li, Q; Oda, M; Mitsudomi, T; Yatabe, Y; Sekido, Y; Yoshida, J; Higashiyama, M; Noguchi, M; Uehara, H; Nishioka, Y; Sone, S; Yano, S

    2013-09-12

    Despite initial dramatic response, epidermal growth factor receptor (EGFR) mutant lung cancer patients always acquire resistance to EGFR-tyrosine kinase inhibitors (TKIs). Gatekeeper T790M mutation in EGFR is the most prevalent genetic alteration underlying acquired resistance to EGFR-TKI, and EGFR mutant lung cancer cells are reported to be addictive to EGFR/Akt signaling even after acquired T790M mutation. Here, we focused on Akt kinase-interacting protein1 (Aki1), a scaffold protein of PI3K (phosphoinositide 3-kinase)/PDK1 (3-phosphoinositide-dependent protein kinase)/Akt that determines receptor signal selectivity for non-mutated EGFR, and assessed its role in EGFR mutant lung cancer with or without gatekeeper T790M mutation. Cell line-based assays showed that Aki1 constitutively associates with mutant EGFR in lung cancer cells with (H1975) or without (PC-9 and HCC827) T790M gatekeeper mutation. Silencing of Aki1 induced apoptosis of EGFR mutant lung cancer cells. Treatment with Aki1 siRNA dramatically inhibited growth of H1975 cells in a xenograft model. Moreover, silencing of Aki1 further potentiated growth inhibitory effect of new generation EGFR-TKIs against H1975 cells in vitro. Aki1 was frequently expressed in tumor cells of EGFR mutant lung cancer patients (53/56 cases), including those with acquired resistance to EGFR-TKI treatment (7/7 cases). Our data suggest that Aki1 may be a critical mediator of survival signaling from mutant EGFR to Akt, and may therefore be an ideal target for EGFR mutant lung cancer patients, especially those with acquired EGFR-TKI resistance due to EGFR T790M gatekeeper mutation. PMID:23045273

  17. Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations

    OpenAIRE

    Yamada, Tadaaki; Takeuchi, Shinji; Fujita, Naoya; Nakamura, Akito; Wang, Wei; Li, Qi; Oda, Makoto; Mitsudomi, Tetsuya; Yatabe, Yasushi; Sekido, Yoshitaka; Yoshida, Junji; Higashiyama, Masahiko; Noguchi, Masayuki; Uehara, Hisanori; Nishioka, Yasuhiko

    2013-01-01

    Despite initial dramatic response, epidermal growth factor receptor (EGFR) mutant lung cancer patients always acquire resistance to EGFR-tyrosine kinase inhibitors (TKIs). Gatekeeper T790M mutation in EGFR is the most prevalent genetic alteration underlying acquired resistance to EGFR-TKI, and EGFR mutant lung cancer cells are reported to be addictive to EGFR/Akt signaling even after acquired T790M mutation. Here, we focused on Akt kinase-interacting protein1 (Aki1), a scaffold protein of PI3...

  18. p53 mutations in non-small-cell lung cancers occurring in individuals without a past history of active smoking.

    OpenAIRE

    Takagi, Y.; Osada, H.; Kuroishi, T; Mitsudomi, T.; Kondo, M.; Niimi, T.; Saji, S; Gazdar, A. F.; Minna, J D; Takahashi, T.

    1998-01-01

    Accumulating evidence suggests that the p53 gene is a good target for molecular epidemiological studies. We previously reported an association between the presence of p53 mutations and lifetime cigarette consumption. Although over 675 p53 mutations have been reported in lung cancers in the literature thus far, very little is known about the nature of such changes in lung cancers in the absence of a smoking background. In the present study, we therefore analysed 69 non-small-cell lung cancer s...

  19. The Domain II S4-S5 Linker in Nav1.9: A Missense Mutation Enhances Activation, Impairs Fast Inactivation, and Produces Human Painful Neuropathy.

    Science.gov (United States)

    Han, Chongyang; Yang, Yang; de Greef, Bianca T A; Hoeijmakers, Janneke G J; Gerrits, Monique M; Verhamme, Camiel; Qu, Jian; Lauria, Giuseppe; Merkies, Ingemar S J; Faber, Catharina G; Dib-Hajj, Sulayman D; Waxman, Stephen G

    2015-06-01

    Painful small fiber neuropathy is a challenging medical condition with no effective treatment. Non-genetic causes can be identified in one half of the subjects. Gain-of-function variants of sodium channels Nav1.7 and Nav1.8 have recently been associated with painful small fiber neuropathy. More recently, mutations of sodium channel Nav1.9 have been linked to human pain disorders, with two gain-of-function mutations found in patients with painful small fiber neuropathy. Here we report a novel Nav1.9 mutation, a glycine 699 substitution by arginine (G699R) in the domain II S4-S5 linker, identified in a patient with painful small fiber neuropathy. In this study, we assayed the mutant channels by voltage-clamp in superior cervical ganglion neurons, which do not produce endogenous Nav1.8 or Nav1.9 currents, and provide a novel platform where Nav1.9 is expressed at relatively high levels. Voltage-clamp analysis showed that the mutation hyperpolarizes (-10.1 mV) channel activation, depolarizes (+6.3 mV) steady-state fast inactivation, slows deactivation, and enhances ramp responses compared with wild-type Nav1.9 channels. Current-clamp analysis showed that the G699R mutant channels render dorsal root ganglion neurons hyperexcitable, via depolarized resting membrane potential, reduced current threshold and increased evoked firing. These observations show that the domain II S4-S5 linker plays an important role in the gating of Nav1.9 and demonstrates that a mutation in this linker is linked to a common pain disorder.

  20. Computational simulations of structural role of the active-site W374C mutation of acetyl-coenzyme-A carboxylase: multi-drug resistance mechanism.

    Science.gov (United States)

    Zhu, Xiao-Lei; Yang, Wen-Chao; Yu, Ning-Xi; Yang, Sheng-Gang; Yang, Guang-Fu

    2011-03-01

    Herbicides targeting grass plastidic acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) are selectively effective against graminicides. The intensive worldwide use of this herbicide family has selected for resistance genes in a number of grass weed species. Recently, the active-site W374C mutation was found to confer multi-drug resistance toward haloxyfop (HF), fenoxaprop (FR), Diclofop (DF), and clodinafop (CF) in A. myosuroides. In order to uncover the resistance mechanism due to W374C mutation, the binding of above-mentioned four herbicides to both wild-type and the mutant-type ACCase was investigated in the current work by molecular docking and molecular dynamics (MD) simulations. The binding free energies were calculated by molecular mechanics-Poisson-Boltzmann surface area (MM/PBSA) method. The calculated binding free energy values for four herbicides were qualitatively consistent with the experimental order of IC(50) values. All the computational model and energetic results indicated that the W374C mutation has great effects on the conformational change of the binding pocket and the ligand-protein interactions. The most significant conformational change was found to be associated with the aromatic amino acid residues, such as Phe377, Tyr161' and Trp346. As a result, the π-π interaction between the ligand and the residue of Phe377 and Tyr161', which make important contributions to the binding affinity, was decreased after mutation and the binding affinity for the inhibitors to the mutant-type ACCase was less than that to the wild-type enzyme, which accounts for the molecular basis of herbicidal resistance. The structural role and mechanistic insights obtained from computational simulations will provide a new starting point for the rational design of novel inhibitors to overcome drug resistance associated with W374C mutation. PMID:20499260

  1. Vigorous physical activity impairs myocardial function in patients with arrhythmogenic right ventricular cardiomyopathy and in mutation positive family members

    DEFF Research Database (Denmark)

    Saberniak, Jørg; Hasselberg, Nina E; Borgquist, Rasmus;

    2014-01-01

    patients and 45 mutation-positive family members. Athletes were defined as subjects with ≥4 h vigorous exercise/week [≥1440 metabolic equivalents (METs × minutes/week)] during a minimum of 6 years. Athlete definition was fulfilled in 37/110 (34%) subjects. We assessed right ventricular (RV) and left...

  2. The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils from patients with polycythemia vera

    OpenAIRE

    Pieri, Lisa; Bogani, Costanza; Guglielmelli, Paola; Zingariello, Maria; Rana, Rosa Alba; Bartalucci, Niccolò; Bosi, Alberto; Vannucchi, Alessandro M.

    2009-01-01

    The JAK2 (V617F) mutation is found in almost all patients with polycythemia vera and an important fraction of patients with essential thrombocythemia and primary myelofibrosis. This study shows that basophil counts are increased in JAK2 (V617F)-positive patients, and that the basophils contain an increased number of granules. See related article on page 1484.

  3. A thrombomodulin mutation that impairs active protein C generation is detrimental in severe pneumonia-derived gram-negative sepsis (melioidosis.

    Directory of Open Access Journals (Sweden)

    Liesbeth M Kager

    2014-04-01

    Full Text Available BACKGROUND: During severe (pneumosepsis inflammatory and coagulation pathways become activated as part of the host immune response. Thrombomodulin (TM is involved in a range of host defense mechanisms during infection and plays a pivotal role in activation of protein C (PC into active protein C (APC. APC has both anticoagulant and anti-inflammatory properties. In this study we investigated the effects of impaired TM-mediated APC generation during melioidosis, a common form of community-acquired Gram-negative (pneumosepsis in South-East Asia caused by Burkholderia (B. pseudomallei. METHODOLOGY/PRINCIPAL FINDINGS: (WT mice and mice with an impaired capacity to activate protein C due to a point mutation in their Thbd gene (TMpro/pro mice were intranasally infected with B. pseudomallei and sacrificed after 24, 48 or 72 hours for analyses. Additionally, survival studies were performed. When compared to WT mice, TMpro/pro mice displayed a worse survival upon infection with B. pseudomallei, accompanied by increased coagulation activation, enhanced lung neutrophil influx and bronchoalveolar inflammation at late time points, together with increased hepatocellular injury. The TMpro/pro mutation had limited if any impact on bacterial growth and dissemination. CONCLUSION/SIGNIFICANCE: TM-mediated protein C activation contributes to protective immunity after infection with B. pseudomallei. These results add to a better understanding of the regulation of the inflammatory and procoagulant response during severe Gram-negative (pneumosepsis.

  4. Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

    Directory of Open Access Journals (Sweden)

    Masahiro Sato

    2015-08-01

    Full Text Available Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1 encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4, which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP and human Cas9 mRNAs, 65% (24/37 of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24 showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.

  5. Rare loss-of-function mutation in complement component C3 provides insight into molecular and pathophysiological determinants of complement activity

    OpenAIRE

    Sfyroera, Georgia; Ricklin, Daniel; Reis, Edimara S.; Chen, Hui; Wu, Emilia L.; Kaznessis, Yiannis N.; Ekdahl, Kristina N.; Nilsson, Bo; Lambris, John D.

    2015-01-01

    The plasma protein C3 is a central element in the activation and effector functions of the complement system. A hereditary dysfunction of C3 that prevents complement activation via the alternative pathway (AP) was described previously in a Swedish family, but its genetic cause and molecular consequences have remained elusive. Here we provide these missing links by pinpointing the dysfunction to a point mutation in the β-chain of C3 (c.1180T>C; p.Met373Thr). In the patient’s plasma, AP activit...

  6. Modifications of laccase activities of copper efflux oxidase, CueO by synergistic mutations in the first and second coordination spheres of the type I copper center.

    Science.gov (United States)

    Kataoka, Kunishige; Kogi, Hiroki; Tsujimura, Seiya; Sakurai, Takeshi

    2013-02-15

    The redox potential of type I copper in the Escherichia coli multicopper oxidase CueO was shifted in the positive or negative direction as a result of the single, double, and triple mutations in the first and second coordination spheres: the formation of the NH···S(-)(Cys500 ligand) hydrogen bond, the breakdown of the NH(His443 ligand)···O(-)(Asp439) hydrogen bond, and the substitution of the Met510 ligand for the non-coordinating Leu or coordinating Gln. Laccase activities of CueO were maximally enhanced 140-fold by virtue of the synergistic effect of mild mutations at and at around the ligand groups to type I copper.

  7. Modifications of laccase activities of copper efflux oxidase, CueO by synergistic mutations in the first and second coordination spheres of the type I copper center.

    Science.gov (United States)

    Kataoka, Kunishige; Kogi, Hiroki; Tsujimura, Seiya; Sakurai, Takeshi

    2013-02-15

    The redox potential of type I copper in the Escherichia coli multicopper oxidase CueO was shifted in the positive or negative direction as a result of the single, double, and triple mutations in the first and second coordination spheres: the formation of the NH···S(-)(Cys500 ligand) hydrogen bond, the breakdown of the NH(His443 ligand)···O(-)(Asp439) hydrogen bond, and the substitution of the Met510 ligand for the non-coordinating Leu or coordinating Gln. Laccase activities of CueO were maximally enhanced 140-fold by virtue of the synergistic effect of mild mutations at and at around the ligand groups to type I copper. PMID:23337502

  8. Galactosemia caused by a point mutation that activates cryptic donor splice site in the galactose-1-phosphate uridyltransferase gene

    Energy Technology Data Exchange (ETDEWEB)

    Wadelius, C.; Lagerkvist, A. (Univ. Hospital, Uppsala (Sweden) Uppsala Univ. (Sweden)); Molin, A.K.; Larsson, A. (Univ. Hospital, Uppsala (Sweden)); Von Doebeln, U. (Karolinska Institute, Stockholm (Sweden))

    1993-08-01

    Galactosemia affects 1/84,000 in Sweden and is manifested in infancy when the child is exposed to galactose in the diet. If untreated there is a risk of severe early symptoms and, even with a lactose-free diet, late symptoms such as mental retardation and ovarial dysfunction may develop. In classical galactosemia, galactose-1-phosphate uridyltransferase (GALT) (EC 2.7.7.12) is defective and the normal cDNA sequence of this enzyme has been characterized. Recently eight mutations leading to galactosemia were published. Heparinized venous blood was drawn from a patient with classical galactosemia. In the cDNA from the patient examined, an insertion of 54 bp was found at position 1087. Amplification of the relevant genomic region of the patient's DNA was performed. Exon-intron boundaries and intronic sequences thus determined revealed that the 54-bp insertion was located immediately downstream of exon 10. It was further found that the patient was heterozygous for a point mutation, changing a C to a T (in 5 of 9 clones) at the second base in the intron downstream of the insertion. This alteration creates a sequence which, as well as the ordinary splice site, differs in only two positions from the consensus sequence. It was found that the mutation occurred in only one of the 20 alleles from galactosemic patients and in none of the 200 alleles from normal controls. The mutation is inherited from the mother, who also was found to express the 54-bp-long insertion at the mRNA level. Sequences from the 5[prime] end of the coding region were determined after genomic amplification, revealing a sequence identical to that reported. The mutation on the paternal allele has not been identified. 9 refs., 1 fig.

  9. Calreticulin Mutations in Myeloproliferative Neoplasms

    Directory of Open Access Journals (Sweden)

    Noa Lavi

    2014-10-01

    Full Text Available With the discovery of the JAK2V617F mutation in patients with Philadelphia chromosome-negative (Ph− myeloproliferative neoplasms (MPNs in 2005, major advances have been made in the diagnosis of MPNs, in understanding of their pathogenesis involving the JAK/STAT pathway, and finally in the development of novel therapies targeting this pathway. Nevertheless, it remains unknown which mutations exist in approximately one-third of patients with non-mutated JAK2 or MPL essential thrombocythemia (ET and primary myelofibrosis (PMF. At the end of 2013, two studies identified recurrent mutations in the gene encoding calreticulin (CALR using whole-exome sequencing. These mutations were revealed in the majority of ET and PMF patients with non-mutated JAK2 or MPL but not in polycythemia vera patients. Somatic 52-bp deletions (type 1 mutations and recurrent 5-bp insertions (type 2 mutations in exon 9 of the CALR gene (the last exon encoding the C-terminal amino acids of the protein calreticulin were detected and found always to generate frameshift mutations. All detected mutant calreticulin proteins shared a novel amino acid sequence at the C-terminal. Mutations in CALR are acquired early in the clonal history of the disease, and they cause activation of JAK/STAT signaling. The CALR mutations are the second most frequent mutations in Ph− MPN patients after the JAK2V617F mutation, and their detection has significantly improved the diagnostic approach for ET and PMF. The characteristics of the CALR mutations as well as their diagnostic, clinical, and pathogenesis implications are discussed in this review.

  10. Antithrombin Cambridge II(A384S) mutation frequency and antithrombin activity levels in 120 of deep venous thrombosis and 150 of cerebral infarction patients in a single center in Southern China.

    Science.gov (United States)

    Zhang, Guang-sen; Tang, Yang-ming; Tang, Mei-qing; Qing, Zi-Ju; Shu, Chang; Tang, Xiang-qi; Deng, Ming-yang; Tan, Li-ming

    2010-09-01

    Antithrombin Cambridge II(A384S) mutation shows a relatively high frequency in western population. Some studies suggest that the mutation is an independent genetic risk factor both for deep vein thrombosis (DVT) and for arterial thrombosis, but whether the mutation has racial difference or has a general significance for thrombophilia remains unclear. In this study we performed an analysis of the prevalence of the mutation in Chinese southern population; Also, the antithrombin activity levels were evaluated in each investigated individual. The studies included 120 patients with DVT, 150 patients with cerebral infarction, and 110 controls. The mutation was detected using polymerase chain reaction/PvuII restrictive fragment length polymorphism procedures. Antithrombin activity assay was done using chromogenic substrate method. The results showed that no antithrombin Cambridge II mutation was detected in all three groups (DVT, cerebral infarction and controls), the incidence was 0/380. Plasma antithrombin activity was 91.37% +/- 16.15% in the DVT patients and 102.68% +/- 13.10% in the controls; the antithrombin activity was significantly reduced in the DVT group (P Cambridge II mutation has a racial difference, and may not be a valuable risk factor of thrombophilia in Asian population, and antithrombin deficiency remains a major genetic risk factor for DVT patients in China.

  11. Multicentric Carpotarsal Osteolysis Is Caused by Mutations Clustering in the Amino-Terminal Transcriptional Activation Domain of MAFB

    OpenAIRE

    Zankl, Andreas; Duncan, Emma L.; Leo, Paul J.; Clark, Graeme R.; Glazov, Evgeny A.; Addor, Marie-Claude; Herlin, Troels; Kim, Chong Ae; Leheup, Bruno P.; McGill, Jim; McTaggart, Steven; Mittas, Stephen; Mitchell, Anna L.; Mortier, Geert R.; Robertson, Stephen P.

    2012-01-01

    Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated s...

  12. In Vitro Activities of Telithromycin, Linezolid, and Quinupristin-Dalfopristin against Streptococcus pneumoniae with Macrolide Resistance Due to Ribosomal Mutations

    OpenAIRE

    Farrell, David J.; Morrissey, Ian; Bakker, Sarah; Buckridge, Sylvie; Felmingham, David

    2004-01-01

    To date, 86 of 7,746 macrolide-resistant Streptococcus pneumoniae isolates from 1999 to 2002 PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) surveillance studies were negative for methylase and efflux mechanisms. Mutations in 23S rRNA or the genes encoding riboprotein L4 or L22 were found in 77 of 86 isolates. Six isolates were resistant to quinupristin-dalfopristin and two were resistant to linezolid, while telithromycin demonstrated good act...

  13. A P-loop Mutation in G[alpha] Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Bosch, Dustin E.; Willard, Francis S.; Ramanujam, Ravikrishna; Kimple, Adam J.; Willard, Melinda D.; Naqvi, Naweed I.; Siderovski, David P. (UNC); (Singapore)

    2012-10-23

    Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active G{alpha}{beta}{gamma} heterotrimer relies on nucleotide cycling by the G{alpha} subunit: exchange of GTP for GDP activates G{alpha}, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting G{alpha} to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of G{alpha} subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that G{alpha}(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon G{alpha}{sub i1}(G42R) binding to GDP {center_dot} AlF{sub 4}{sup -} or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. G{alpha}(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with G{beta}{gamma} and GoLoco motifs in any nucleotide state. The corresponding G{alpha}{sub q}(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the G{alpha} subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two G{alpha} mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.

  14. Mechanism of the Quorum-Quenching Lactonase (AiiA) from Bacillus thuringiensis. 2. Substrate Modeling and Active Site Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Momb, Jessica; Wang, Canhui; Liu, Dali; Thomas, Pei W.; Petsko, Gregory A.; Guo, Hua; Ringe, Dagmar; Fast, Walter (UNM); (Brandeis); (Texas)

    2008-12-02

    The N-acyl-l-homoserine lactone hydrolases (AHL lactonases) have attracted considerable attention because of their ability to quench AHL-mediated quorum-sensing pathways in Gram-negative bacteria and because of their relation to other enzymes in the metallo-{beta}-lactamase superfamily. To elucidate the detailed catalytic mechanism of AHL lactonase, mutations are made on residues that presumably contribute to substrate binding and catalysis. Steady-state kinetic studies are carried out on both the wild-type and mutant enzymes using a spectrum of substrates. Two mutations, Y194F and D108N, present significant effects on the overall catalysis. On the basis of a high-resolution structural model of the enzyme-product complex, a hybrid quantum mechanical/molecular mechanical method is used to model the substrate binding orientation and to probe the effect of the Y194F mutation. Combining all experimental and computational results, we propose a detailed mechanism for the ring-opening hydrolysis of AHL substrates as catalyzed by the AHL lactonase from Bacillus thuringiensis. Several features of the mechanism that are also found in related enzymes are discussed and may help to define an evolutionary thread that connects the hydrolytic enzymes of this mechanistically diverse superfamily.

  15. FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

    Science.gov (United States)

    Peterlongo, Paolo; Catucci, Irene; Colombo, Mara; Caleca, Laura; Mucaki, Eliseos; Bogliolo, Massimo; Marin, Maria; Damiola, Francesca; Bernard, Loris; Pensotti, Valeria; Volorio, Sara; Dall'Olio, Valentina; Meindl, Alfons; Bartram, Claus; Sutter, Christian; Surowy, Harald; Sornin, Valérie; Dondon, Marie-Gabrielle; Eon-Marchais, Séverine; Stoppa-Lyonnet, Dominique; Andrieu, Nadine; Sinilnikova, Olga M.; Mitchell, Gillian; James, Paul A.; Thompson, Ella; Marchetti, Marina; Verzeroli, Cristina; Tartari, Carmen; Capone, Gabriele Lorenzo; Putignano, Anna Laura; Genuardi, Maurizio; Medici, Veronica; Marchi, Isabella; Federico, Massimo; Tognazzo, Silvia; Matricardi, Laura; Agata, Simona; Dolcetti, Riccardo; Puppa, Lara Della; Cini, Giulia; Gismondi, Viviana; Viassolo, Valeria; Perfumo, Chiara; Mencarelli, Maria Antonietta; Baldassarri, Margherita; Peissel, Bernard; Roversi, Gaia; Silvestri, Valentina; Rizzolo, Piera; Spina, Francesca; Vivanet, Caterina; Tibiletti, Maria Grazia; Caligo, Maria Adelaide; Gambino, Gaetana; Tommasi, Stefania; Pilato, Brunella; Tondini, Carlo; Corna, Chiara; Bonanni, Bernardo; Barile, Monica; Osorio, Ana; Benitez, Javier; Balestrino, Luisa; Ottini, Laura; Manoukian, Siranoush; Pierotti, Marco A.; Renieri, Alessandra; Varesco, Liliana; Couch, Fergus J.; Wang, Xianshu; Devilee, Peter; Hilbers, Florentine S.; van Asperen, Christi J.; Viel, Alessandra; Montagna, Marco; Cortesi, Laura; Diez, Orland; Balmaña, Judith; Hauke, Jan; Schmutzler, Rita K.; Papi, Laura; Pujana, Miguel Angel; Lázaro, Conxi; Falanga, Anna; Offit, Kenneth; Vijai, Joseph; Campbell, Ian; Burwinkel, Barbara; Kvist, Anders; Ehrencrona, Hans; Mazoyer, Sylvie; Pizzamiglio, Sara; Verderio, Paolo; Surralles, Jordi; Rogan, Peter K.; Radice, Paolo

    2015-01-01

    Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28–12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04–12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09–13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer. PMID:26130695

  16. FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor.

    Science.gov (United States)

    Peterlongo, Paolo; Catucci, Irene; Colombo, Mara; Caleca, Laura; Mucaki, Eliseos; Bogliolo, Massimo; Marin, Maria; Damiola, Francesca; Bernard, Loris; Pensotti, Valeria; Volorio, Sara; Dall'Olio, Valentina; Meindl, Alfons; Bartram, Claus; Sutter, Christian; Surowy, Harald; Sornin, Valérie; Dondon, Marie-Gabrielle; Eon-Marchais, Séverine; Stoppa-Lyonnet, Dominique; Andrieu, Nadine; Sinilnikova, Olga M; Mitchell, Gillian; James, Paul A; Thompson, Ella; Marchetti, Marina; Verzeroli, Cristina; Tartari, Carmen; Capone, Gabriele Lorenzo; Putignano, Anna Laura; Genuardi, Maurizio; Medici, Veronica; Marchi, Isabella; Federico, Massimo; Tognazzo, Silvia; Matricardi, Laura; Agata, Simona; Dolcetti, Riccardo; Della Puppa, Lara; Cini, Giulia; Gismondi, Viviana; Viassolo, Valeria; Perfumo, Chiara; Mencarelli, Maria Antonietta; Baldassarri, Margherita; Peissel, Bernard; Roversi, Gaia; Silvestri, Valentina; Rizzolo, Piera; Spina, Francesca; Vivanet, Caterina; Tibiletti, Maria Grazia; Caligo, Maria Adelaide; Gambino, Gaetana; Tommasi, Stefania; Pilato, Brunella; Tondini, Carlo; Corna, Chiara; Bonanni, Bernardo; Barile, Monica; Osorio, Ana; Benitez, Javier; Balestrino, Luisa; Ottini, Laura; Manoukian, Siranoush; Pierotti, Marco A; Renieri, Alessandra; Varesco, Liliana; Couch, Fergus J; Wang, Xianshu; Devilee, Peter; Hilbers, Florentine S; van Asperen, Christi J; Viel, Alessandra; Montagna, Marco; Cortesi, Laura; Diez, Orland; Balmaña, Judith; Hauke, Jan; Schmutzler, Rita K; Papi, Laura; Pujana, Miguel Angel; Lázaro, Conxi; Falanga, Anna; Offit, Kenneth; Vijai, Joseph; Campbell, Ian; Burwinkel, Barbara; Kvist, Anders; Ehrencrona, Hans; Mazoyer, Sylvie; Pizzamiglio, Sara; Verderio, Paolo; Surralles, Jordi; Rogan, Peter K; Radice, Paolo

    2015-09-15

    Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer. PMID:26130695

  17. A mutation in the tuft mouse disrupts TET1 activity and alters the expression of genes that are crucial for neural tube closure

    Directory of Open Access Journals (Sweden)

    Keith S. K. Fong

    2016-05-01

    Full Text Available Genetic variations affecting neural tube closure along the head result in malformations of the face and brain. Neural tube defects (NTDs are among the most common birth defects in humans. We previously reported a mouse mutant called tuft that arose spontaneously in our wild-type 3H1 colony. Adult tuft mice present midline craniofacial malformations with or without an anterior cephalocele. In addition, affected embryos presented neural tube closure defects resulting in insufficient closure of the anterior neuropore or exencephaly. Here, through whole-genome sequencing, we identified a nonsense mutation in the Tet1 gene, which encodes a methylcytosine dioxygenase (TET1, co-segregating with the tuft phenotype. This mutation resulted in premature termination that disrupts the catalytic domain that is involved in the demethylation of cytosine. We detected a significant loss of TET enzyme activity in the heads of tuft embryos that were homozygous for the mutation and had NTDs. RNA-Seq transcriptome analysis indicated that multiple gene pathways associated with neural tube closure were dysregulated in tuft embryo heads. Among them, the expressions of Cecr2, Epha7 and Grhl2 were significantly reduced in some embryos presenting neural tube closure defects, whereas one or more components of the non-canonical WNT signaling pathway mediating planar cell polarity and convergent extension were affected in others. We further show that the recombinant mutant TET1 protein was capable of entering the nucleus and affected the expression of endogenous Grhl2 in IMCD-3 (inner medullary collecting duct cells. These results indicate that TET1 is an epigenetic determinant for regulating genes that are crucial to closure of the anterior neural tube and its mutation has implications to craniofacial development, as presented by the tuft mouse.

  18. T-cell independent, B-cell receptor-mediated induction of telomerase activity differs among IGHV mutation-based subgroups of chronic lymphocytic leukemia patients.

    Science.gov (United States)

    Damle, Rajendra N; Temburni, Sonal; Banapour, Taraneh; Paul, Santanu; Mongini, Patricia K A; Allen, Steven L; Kolitz, Jonathan E; Rai, Kanti R; Chiorazzi, Nicholas

    2012-09-20

    Although B-cell chronic lymphocytic leukemia (B-CLL) clones with unmutated IGHV genes (U-CLL) exhibit greater telomerase activity than those with mutated IGHV genes (M-CLL), the extent to which B-cell receptor (BCR) triggering contributes to telomerase up-regulation is not known. Therefore, we studied the effect of BCR stimulation on modulating telomerase activity. The multivalent BCR ligand, dextran conjugated anti-μ mAb HB57 (HB57-dex), increased telomerase activity and promoted cell survival and proliferation preferentially in U-CLL cases, whereas the PI3K/Akt inhibitor LY294002 blocked HB57-dex induced telomerase activation. Although both U-CLL and M-CLL clones exhibited similar membrane proximal signaling responses to HB57-dex, telomerase activity and cell proliferation, when inducible in M-CLL, differed. B-CLL cells stimulated using bivalent F(ab')(2) -goat anti-μ antibody (goat anti-μ) exhibited higher membrane proximal response in U-CLL than M-CLL cells, whereas telomerase activity, cell survival, and proliferation were induced to lower levels than those induced by HB57-dex. In normal B lymphocytes, HB57-dex induced less protein phosphorylation but more cell proliferation and survival than goat anti-μ. Although both anti-BCR stimuli induced comparable telomerase activity, normal CD5(+) B cells preferentially exhibited higher hTERT positivity than their CD5(-) counterparts. These findings provide an understanding of how BCR-mediated signals impact telomerase modulation in IGHV mutation-based subgroups of B-CLL and normal B cells. PMID:22875913

  19. A novel familial mutation in the PCSK1 gene that alters the oxyanion hole residue of proprotein convertase 1/3 and impairs its enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Michael Wilschanski

    Full Text Available Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1 gene, encoding the neuroendocrine convertase 1 precursor (PC1/3 which was recently reported as a cause of Congenital Diarrhea Disorder (CDD. The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.

  20. Normal hematopoiesis and lack of β-catenin activation in osteoblasts of patients and mice harboring Lrp5 gain-of-function mutations

    DEFF Research Database (Denmark)

    Galán-Díez, Marta; Isa, Adiba; Ponzetti, Marco;

    2016-01-01

    of hematopoiesis and leukemogenic properties of β-catenin activation in osteoblasts, that lead to development of acute myeloid leukemia (AML). Using mice with gain-of-function (GOF) Lrp5 alleles (Lrp5(A214V)) that recapitulate the human high bone mass (HBM) phenotype, as well as patients with the T253I HBM Lrp5......Osteoblasts are emerging regulators of myeloid malignancies since genetic alterations in them, such as constitutive activation of β-catenin, instigate their appearance. The LDL receptor-related protein 5 (LRP5), initially proposed to be a co-receptor for Wnt proteins, in fact favors bone formation...... patients showed normal hematopoiesis, normal percentage of myeloid cells, and lack of anemia. We conclude that Lrp5 GOF mutations do not activate β-catenin signaling in osteoblasts. As a result, myeloid lineage differentiation is normal in HBM patients and mice. This article is part of a Special Issue...

  1. Mutations in galactosemia

    Energy Technology Data Exchange (ETDEWEB)

    Reichardt, J.K.V. [Univ. of Southern California School of Medicine, Los Angeles, CA (United States)

    1995-10-01

    This Letter raises four issues concerning two papers on galactosemia published in the March 1995 of the Journal. First, table 2 in the paper by Elsas et al. incorrectly attributes seven galactose-l-phosphate uridyl transferase (GALT) mutations (S135L, L195P, K285N, N314D, R333W, R333G, and K334R). The table also fails to mention that others have reported the same two findings attributed to {open_quotes}Leslie et al.; Elsas et al. and in press{close_quotes} and {open_quotes}Leslie et al.; Elsas et al.{close_quotes} The first finding on the prevalence of the Q188R galactosemia mutation in the G/G Caucasian population has also been described by Ng et al., and the second finding on the correlation of the N314D GALT mutation with the Duarte variant was reported by Lin et al. Second, Elsas et al. suggest that the E203K and N314D mutations may {open_quotes}produce intra-allelic complementation when in cis{close_quotes}. This speculation is supported by the activity data of individual III-2 but is inconsistent with the activities of three other individuals I-1, II-1, and III-1 of the same pedigree. The GALT activity measured in these three individuals suggests a dominant negative effect of E203K in E203K-N314D chromosomes, since they all have less than normal activity. Thus, the preponderance of the data in this paper is at odds with the authors speculation. It is worth recalling that Lin et al. also identified four N314D GALT mutations on 95 galactosemic chromosomes examined. A similar situation also appears to be the case in proband III-1 (with genotype E203K-N314D/IVSC) in the Elsas et al. paper. 9 refs.

  2. Gene mutations in hepatocellular adenomas

    DEFF Research Database (Denmark)

    Raft, Marie B; Jørgensen, Ernö N; Vainer, Ben

    2015-01-01

    is associated with bi-allelic mutations in the TCF1 gene and morphologically has marked steatosis. β-catenin activating HCA has increased activity of the Wnt/β-catenin pathway and is associated with possible malignant transformation. Inflammatory HCA is characterized by an oncogene-induced inflammation due....... This review offers an overview of the reported gene mutations associated with hepatocellular adenomas together with a discussion of the diagnostic and prognostic value....

  3. Increased Variation in Adh Enzyme Activity in Drosophila Mutation-Accumulation Experiment Is Not Due to Transposable Elements at the Adh Structural Gene

    Science.gov (United States)

    Aquadro, C. F.; Tachida, H.; Langley, C. H.; Harada, K.; Mukai, T.

    1990-01-01

    We present here a molecular analysis of the region surrounding the structural gene encoding alcohol dehydrogenase (Adh) in 47 lines of Drosophila melanogaster that have each accumulated mutations for 300 generations. While these lines show a significant increase in variation of alcohol dehydrogenase enzyme activity compared to control lines, we found no restriction map variation in a 13-kb region including the complete Adh structural gene and roughly 5 kb of both 5' and 3' sequences. Thus, the rapid accumulation of ADH activity variation after 28,200 allele generations does not appear to have been due to the mobilization of transposable elements into or out of the Adh structural gene region. PMID:1963870

  4. Amino acid sequence of toxin XI of the scorpion Buthus occitanus tunetanus. Evidence of a mutation having an important effect upon neurotoxic activity.

    Science.gov (United States)

    Sampieri, F; Habersetzer-Rochat, C; Martin, M F; Kopeyan, C; Rochat, H

    1987-02-01

    The complete amino acid sequence of toxin XI of the North African scorpion Buthus occitanus tunetanus has been elucidated by automatic sequencing of the reduced and alkylated toxin and of the peptides obtained after tryptic cleavage restricted to arginyl bonds. This toxin is structurally homologous to toxin II of Androctonus australis Hector, the most active among the alpha-toxins, but is far less potent, both in vivo and in vitro. This work points out 12 mutations, many of which are conservative. Nevertheless, the most striking difference is the replacement of the lysine residue at position 58, known to be important in the activity of AaH toxin II, by a valine residue. Thus, it seems that the presence of a positive charge at this location facilitates the interactions between the receptor on the sodium channel and the alpha-type toxins.

  5. Mutations in Subunits of the Activating Signal Cointegrator 1 Complex Are Associated with Prenatal Spinal Muscular Atrophy and Congenital Bone Fractures

    Science.gov (United States)

    Knierim, Ellen; Hirata, Hiromi; Wolf, Nicole I.; Morales-Gonzalez, Susanne; Schottmann, Gudrun; Tanaka, Yu; Rudnik-Schöneborn, Sabine; Orgeur, Mickael; Zerres, Klaus; Vogt, Stefanie; van Riesen, Anne; Gill, Esther; Seifert, Franziska; Zwirner, Angelika; Kirschner, Janbernd; Goebel, Hans Hilmar; Hübner, Christoph; Stricker, Sigmar; Meierhofer, David; Stenzel, Werner; Schuelke, Markus

    2016-01-01

    Transcriptional signal cointegrators associate with transcription factors or nuclear receptors and coregulate tissue-specific gene transcription. We report on recessive loss-of-function mutations in two genes (TRIP4 and ASCC1) that encode subunits of the nuclear activating signal cointegrator 1 (ASC-1) complex. We used autozygosity mapping and whole-exome sequencing to search for pathogenic mutations in four families. Affected individuals presented with prenatal-onset spinal muscular atrophy (SMA), multiple congenital contractures (arthrogryposis multiplex congenita), respiratory distress, and congenital bone fractures. We identified homozygous and compound-heterozygous nonsense and frameshift TRIP4 and ASCC1 mutations that led to a truncation or the entire absence of the respective proteins and cosegregated with the disease phenotype. Trip4 and Ascc1 have identical expression patterns in 17.5-day-old mouse embryos with high expression levels in the spinal cord, brain, paraspinal ganglia, thyroid, and submandibular glands. Antisense morpholino-mediated knockdown of either trip4 or ascc1 in zebrafish disrupted the highly patterned and coordinated process of α-motoneuron outgrowth and formation of myotomes and neuromuscular junctions and led to a swimming defect in the larvae. Immunoprecipitation of the ASC-1 complex consistently copurified cysteine and glycine rich protein 1 (CSRP1), a transcriptional cofactor, which is known to be involved in spinal cord regeneration upon injury in adult zebrafish. ASCC1 mutant fibroblasts downregulated genes associated with neurogenesis, neuronal migration, and pathfinding (SERPINF1, DAB1, SEMA3D, SEMA3A), as well as with bone development (TNFRSF11B, RASSF2, STC1). Our findings indicate that the dysfunction of a transcriptional coactivator complex can result in a clinical syndrome affecting the neuromuscular system. PMID:26924529

  6. MPL mutations in myeloproliferative disorders

    DEFF Research Database (Denmark)

    Beer, Philip A.; Campbell, Peter J.; Scott, Linda M.;

    2008-01-01

    Activating mutations of MPL exon 10 have been described in a minority of patients with idiopathic myelofibrosis (IMF) or essential thrombocythemia (ET), but their prevalence and clinical significance are unclear. Here we demonstrate that MPL mutations outside exon 10 are uncommon in platelet c......DNA and identify 4 different exon 10 mutations in granulocyte DNA from a retrospective cohort of 200 patients with ET or IMF. Allele-specific polymerase chain reaction was then used to genotype 776 samples from patients with ET entered into the PT-1 studies. MPL mutations were identified in 8.5% of JAK2 V617F......(-) patients and a single V617F(+) patient. Patients carrying the W515K allele had a significantly higher allele burden than did those with the W515L allele, suggesting a functional difference between the 2 variants. Compared with V617F(+) ET patients, those with MPL mutations displayed lower hemoglobin...

  7. Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations12

    Science.gov (United States)

    Ciuffreda, Ludovica; Del Bufalo, Donatella; Desideri, Marianna; Di Sanza, Cristina; Stoppacciaro, Antonella; Ricciardi, Maria Rosaria; Chiaretti, Sabina; Tavolaro, Simona; Benassi, Barbara; Bellacosa, Alfonso; Foà, Robin; Tafuri, Agostino; Cognetti, Francesco; Anichini, Andrea; Zupi, Gabriella; Milella, Michele

    2009-01-01

    The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma. PMID:19649202

  8. Mutation of isocitrate dehydrogenase 1 induces glioma cell proliferation via nuclear factor-κB activation in a hypoxia-inducible factor 1-α dependent manner.

    Science.gov (United States)

    Wang, Guoliang; Sai, Ke; Gong, Fanghe; Yang, Qunying; Chen, Furong; Lin, Jian

    2014-05-01

    Recently, mutations of the isocitrate dehydrogenase (IDH) 1 gene, which specifically occur in the majority of low-grade and secondary high-grade gliomas, have drawn particular attention of neuro-oncologists. Mutations of the IDH1 gene have been proposed to have significant roles in the tumorigenesis, progression and prognosis of gliomas. However, the molecular mechanism of the role of IDH1 mutants in gliomagenesis remains to be elucidated. The present study, showed that forced expression of an IDH1 mutant, of which the 132th amino acid residue arginine is substituted by histidine (IDH1R132H), promoted cell proliferation in cultured cells, while wild-type IDH1 overexpression had no effect on cell proliferation. Consistent with previous studies, it was also observed that expression of hypoxia-inducible factor 1-α (HIF1-α) was upregulated in IDH1R132H expressing cells with the induction of vascular endothelial growth factor (VEGF) expression. However, knockdown of VEGF via small RNA interference had no significant influence on the cell proliferation induced by overexpression of IDH1R132H, implying that another signaling pathway may be involved. Next, forced expression of IDH1R132H was found to activate nuclear factor-κB (NF-κB), since the inhibitory IκB protein (IκBα) was highly phosphorylated and the NF-κB p65 subunit was translocated into the nucleus. Notably, knockdown of HIF1-α significantly blocked NF-κB activation, which was induced by the overexpression of IDH1 mutants. In addition, expression of IDH1 mutants markedly induced the NF-κB target gene expression, including cyclin D1 and E and c-myc, which were involved in the regulation of cell proliferation. In conclusion, it was demonstrated that the IDH1 mutant activated NF-κB in a HIF1-α‑dependent manner and was involved in the regulation of cell proliferation.

  9. The prevalent deep intronic c. 639+919 G>A GLA mutation causes pseudoexon activation and Fabry disease by abolishing the binding of hnRNPA1 and hnRNP A2/B1 to a splicing silencer

    DEFF Research Database (Denmark)

    Palhais, Bruno; Dembic, Maja; Sabaratnam, Rugivan;

    2016-01-01

    with the ESS is also able to inhibit inclusion of an unrelated pseudoexon in the FGB gene, and that also in the FGB context inactivation of the ESS by the c.639+919 G>A mutation causes pseudoexon activation, underscoring the universal nature of the ESS. Finally, we demonstrate that splice switching......Fabry disease is an X-linked recessive inborn disorder of the glycosphingolipid metabolism, caused by total or partial deficiency of the lysosomal α-galactosidase A enzyme due to mutations in the GLA gene. The prevalent c.639+919 G>A mutation in GLA leads to pathogenic insertion of a 57bp...... pseudoexon sequence from intron 4, which is responsible for the cardiac variant phenotype. In this study we investigate the splicing regulatory mechanism leading to GLA pseudoexon activation. Splicing analysis of GLA minigenes revealed that pseudoexon activation is influenced by cell-type. We demonstrate...

  10. Analysis of the epidermal growth factor receptor specific transcriptome: effect of receptor expression level and an activating mutation

    DEFF Research Database (Denmark)

    Pedersen, Mikkel W; Pedersen, Nina; Damstrup, Lars;

    2005-01-01

    moderately expressed or overexpressed at an in-itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes...... by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1...

  11. Effects of vaccinia virus uracil DNA glycosylase catalytic site and deoxyuridine triphosphatase deletion mutations individually and together on replication in active and quiescent cells and pathogenesis in mice

    Directory of Open Access Journals (Sweden)

    Moss Bernard

    2008-12-01

    Full Text Available Abstract Background Low levels of uracil in DNA result from misincorporation of dUMP or cytosine deamination. Vaccinia virus (VACV, the prototype poxvirus, encodes two enzymes that can potentially reduce the amount of uracil in DNA. Deoxyuridine triphosphatase (dUTPase hydrolyzes dUTP, generating dUMP for biosynthesis of thymidine nucleotides while decreasing the availability of dUTP for misincorporation; uracil DNA glycosylase (UNG cleaves uracil N-glycosylic bonds in DNA initiating base excision repair. Studies with actively dividing cells showed that the VACV UNG protein is required for DNA replication but the UNG catalytic site is not, whereas the dUTPase gene can be deleted without impairing virus replication. Recombinant VACV with an UNG catalytic site mutation was attenuated in vivo, while a dUTPase deletion mutant was not. However, the importance of the two enzymes for replication in quiescent cells, their possible synergy and roles in virulence have not been fully assessed. Results VACV mutants lacking the gene encoding dUTPase or with catalytic site mutations in UNG and double UNG/dUTPase mutants were constructed. Replication of UNG and UNG/dUTPase mutants were slightly reduced compared to wild type or the dUTPase mutant in actively dividing cells. Viral DNA replication was reduced about one-third under these conditions. After high multiplicity infection of quiescent fibroblasts, yields of wild type and mutant viruses were decreased by 2-logs with relative differences similar to those observed in active fibroblasts. However, under low multiplicity multi-step growth conditions in quiescent fibroblasts, replication of the dUTPase/UNG mutant was delayed and 5-fold lower than that of either single mutant or parental virus. This difference was exacerbated by 1-day serial passages on quiescent fibroblasts, resulting in 2- to 3-logs lower titer of the double mutant compared to the parental and single mutant viruses. Each mutant was more

  12. Point mutation in the NF2 gene of HEI-193 human schwannoma cells results in the expression of a merlin isoform with attenuated growth suppressive activity

    Energy Technology Data Exchange (ETDEWEB)

    Lepont, Pierig; Stickney, John T.; Foster, Lauren A.; Meng, Jin-Jun; Hennigan, Robert F. [Department of Cell and Cancer Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States); Ip, Wallace [Department of Cell and Cancer Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States)], E-mail: wallace.ip@uc.edu

    2008-01-01

    Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by the formation of bilateral schwannomas of the eighth cranial nerve. Although the protein product of the NF2 gene (merlin) is a classical tumor suppressor, the mechanism by which merlin suppresses cell proliferation is not fully understood. The availability of isolated tumor cells would facilitate a better understanding of the molecular function of merlin, but primary schwannoma cells obtained from patients grow slowly and do not yield adequate numbers for biochemical analysis. In this study, we have examined the NF2 mutation in HEI-193 cells, an immortalized cell line derived from the schwannoma of an NF2 patient. Previous work showed that the NF2 mutation in HEI-193 cells causes a splicing defect in the NF2 transcript. We have confirmed this result and further identified the resultant protein product as an isoform of merlin previously designated as isoform 3. The level of isoform 3 proteins in HEI-193 cells is comparable to the levels of merlin isoforms 1 and 2 in normal human Schwann cells and several other immortalized cell lines. In contrast to many mutant forms of merlin, isoform 3 is as resistant to proteasomal degradation as isoforms 1 and 2 and can interact with each of these isoforms in vivo. Cell proliferation assays showed that, in NF2{sup -/-} mouse embryonic fibroblasts, exogenously expressed merlin isoform 3 does exhibit growth suppressive activity although it is significantly lower than that of identically expressed merlin isoform 1. These results indicate that, although HEI-193 cells have undetectable levels of merlin isoforms 1 and 2, they are, in fact, not a merlin-null model because they express the moderately active growth suppressive merlin isoform 3.

  13. Dual role of G-runs and hnRNP F in the regulation of a mutation-activated pseudoexon in the fibrinogen gamma-chain transcript.

    Directory of Open Access Journals (Sweden)

    Valeria Rimoldi

    Full Text Available Most pathological pseudoexon inclusion events originate from single activating mutations, suggesting that many intronic sequences are on the verge of becoming exons. However, the precise mechanisms controlling pseudoexon definition are still largely unexplored. Here, we investigated the cis-acting elements and trans-acting regulatory factors contributing to the regulation of a previously described fibrinogen gamma-chain (FGG pseudoexon, which is activated by a deep-intronic mutation (IVS6-320A>T. This pseudoexon contains several G-run elements, which may be bound by heterogeneous nuclear ribonucleoproteins (hnRNPs F and H. To explore the effect of these proteins on FGG pseudoexon inclusion, both silencing and overexpression experiments were performed in eukaryotic cells. While hnRNP H did not significantly affect pseudoexon splicing, hnRNP F promoted pseudoexon inclusion, indicating that these two proteins have only partially redundant functions. To verify the binding of hnRNP F and the possible involvement of other trans-acting splicing modulators, pulldown experiments were performed on the region of the pseudoexon characterized by both a G-run and enrichment for exonic splicing enhancers. This 25-bp-long region strongly binds hnRNP F/H and weakly interacts with Serine/Arginine-rich protein 40, which however was demonstrated to be dispensable for FGG pseudoexon inclusion in overexpression experiments. Deletion analysis, besides confirming the splicing-promoting role of the G-run within this 25-bp region, demonstrated that two additional hnRNP F binding sites might instead function as silencer elements. Taken together, our results indicate a major role of hnRNP F in regulating FGG pseudoexon inclusion, and strengthen the notion that G-runs may function either as splicing enhancers or silencers of the same exon.

  14. Dual role of G-runs and hnRNP F in the regulation of a mutation-activated pseudoexon in the fibrinogen gamma-chain transcript.

    Science.gov (United States)

    Rimoldi, Valeria; Soldà, Giulia; Asselta, Rosanna; Spena, Silvia; Stuani, Cristiana; Buratti, Emanuele; Duga, Stefano

    2013-01-01

    Most pathological pseudoexon inclusion events originate from single activating mutations, suggesting that many intronic sequences are on the verge of becoming exons. However, the precise mechanisms controlling pseudoexon definition are still largely unexplored. Here, we investigated the cis-acting elements and trans-acting regulatory factors contributing to the regulation of a previously described fibrinogen gamma-chain (FGG) pseudoexon, which is activated by a deep-intronic mutation (IVS6-320A>T). This pseudoexon contains several G-run elements, which may be bound by heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H. To explore the effect of these proteins on FGG pseudoexon inclusion, both silencing and overexpression experiments were performed in eukaryotic cells. While hnRNP H did not significantly affect pseudoexon splicing, hnRNP F promoted pseudoexon inclusion, indicating that these two proteins have only partially redundant functions. To verify the binding of hnRNP F and the possible involvement of other trans-acting splicing modulators, pulldown experiments were performed on the region of the pseudoexon characterized by both a G-run and enrichment for exonic splicing enhancers. This 25-bp-long region strongly binds hnRNP F/H and weakly interacts with Serine/Arginine-rich protein 40, which however was demonstrated to be dispensable for FGG pseudoexon inclusion in overexpression experiments. Deletion analysis, besides confirming the splicing-promoting role of the G-run within this 25-bp region, demonstrated that two additional hnRNP F binding sites might instead function as silencer elements. Taken together, our results indicate a major role of hnRNP F in regulating FGG pseudoexon inclusion, and strengthen the notion that G-runs may function either as splicing enhancers or silencers of the same exon.

  15. Griscelli syndrome: characterization of a new mutation and rescue of T-cytotoxic activity by retroviral transfer of RAB27A gene.

    Science.gov (United States)

    Bizario, João C S; Feldmann, Jérôme; Castro, Fabíola A; Ménasché, Gaël; Jacob, Cristina M A; Cristofani, L; Casella, Erasmo B; Voltarelli, Júlio C; de Saint-Basile, Geneviève; Espreafico, Enilza M

    2004-07-01

    Griscelli syndrome (GS) is caused by mutations in the MYO5A (GS1), RAB27A (GS2), or MLPH (GS3) genes, all of which lead to a similar pigmentary dilution. In addition, GS1 patients show primary neurological impairment, whereas GS2 patients present immunodeficiency and periods of lymphocyte proliferation and activation, leading to their infiltration in many organs, such as the nervous system, causing secondary neurological damage. We report the diagnosis of GS2 in a 4-year-old child with haemophagocytic syndrome, immunodeficiency, and secondary neurological disorders. Typical melanosome accumulation was found in skin melanocytes and pigment clumps were observed in hair shafts. Two heterozygous mutant alleles of the RAB27A gene were found, a C-T transition (C352T) that leads to Q118stop and a G-C transversion on the exon 5 splicing donor site (G467+1C). Functional assays showed increased cellular activation and decreased cytotoxic activity of NK and CD8+ T cells, associated with defective lytic granules release. Myosin-Va expression and localization in the patient lymphocytes were also analyzed. Most importantly, we show that cytotoxic activity of the patient's CD8+ T lymphocytes can be rescued in vitro by RAB27A gene transfer mediated by a recombinant retroviral vector, a first step towards a potential treatment of the acute phase of GS2 by RAB27A transduced lymphocytes. PMID:15163896

  16. An activating mutation reveals a second binding mode of the integrin α2 I domain to the GFOGER motif in collagens.

    Directory of Open Access Journals (Sweden)

    Federico Carafoli

    Full Text Available The GFOGER motif in collagens (O denotes hydroxyproline represents a high-affinity binding site for all collagen-binding integrins. Other GxOGER motifs require integrin activation for maximal binding. The E318W mutant of the integrin α2β1 I domain displays a relaxed collagen specificity, typical of an active state. E318W binds more strongly than the wild-type α2 I domain to GMOGER, and forms a 2:1 complex with a homotrimeric, collagen-like, GFOGER peptide. Crystal structure analysis of this complex reveals two E318W I domains, A and B, bound to a single triple helix. The E318W I domains are virtually identical to the collagen-bound wild-type I domain, suggesting that the E318W mutation activates the I domain by destabilising the unligated conformation. E318W I domain A interacts with two collagen chains similarly to wild-type I domain (high-affinity mode. E318W I domain B makes favourable interactions with only one collagen chain (low-affinity mode. This observation suggests that single GxOGER motifs in the heterotrimeric collagens V and IX may support binding of activated integrins.

  17. Mutational analysis of the activator of late transcription, Alt , in the lactococcal bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Pedersen, Margit; Hammer, Karin

    2007-01-01

    An activator protein, Alt, synthesized during the early state of lytic infection is required for transcription of the late operon in the lactococcal phage TP901-1. In order to identify amino acid residues in the Alt protein required for activation of the TP901-1 late promoter, Plate, hydroxylamin...

  18. Functional characterization of somatic mutations in cancer using network-based inference of protein activity | Office of Cancer Genomics

    Science.gov (United States)

    Identifying the multiple dysregulated oncoproteins that contribute to tumorigenesis in a given patient is crucial for developing personalized treatment plans. However, accurate inference of aberrant protein activity in biological samples is still challenging as genetic alterations are only partially predictive and direct measurements of protein activity are generally not feasible.

  19. Whole-exome sequencing identifies mutations of TBC1D1 encoding a Rab-GTPase-activating protein in patients with congenital anomalies of the kidneys and urinary tract (CAKUT).

    Science.gov (United States)

    Kosfeld, Anne; Kreuzer, Martin; Daniel, Christoph; Brand, Frank; Schäfer, Anne-Kathrin; Chadt, Alexandra; Weiss, Anna-Carina; Riehmer, Vera; Jeanpierre, Cécile; Klintschar, Michael; Bräsen, Jan Hinrich; Amann, Kerstin; Pape, Lars; Kispert, Andreas; Al-Hasani, Hadi; Haffner, Dieter; Weber, Ruthild G

    2016-01-01

    Congenital anomalies of the kidneys and urinary tract (CAKUT) are genetically highly heterogeneous leaving most cases unclear after mutational analysis of the around 30 causative genes known so far. Assuming that phenotypes frequently showing dominant inheritance, such as CAKUT, can be caused by de novo mutations, de novo analysis of whole-exome sequencing data was done on two patient-parent-trios to identify novel CAKUT genes. In one case, we detected a heterozygous de novo frameshift variant in TBC1D1 encoding a Rab-GTPase-activating protein regulating glucose transporter GLUT4 translocation. Sequence analysis of 100 further CAKUT cases yielded three novel or rare inherited heterozygous TBC1D1 missense variants predicted to be pathogenic. TBC1D1 mutations affected Ser237-phosphorylation or protein stability and thereby act as hypomorphs. Tbc1d1 showed widespread expression in the developing murine urogenital system. A mild CAKUT spectrum phenotype, including anomalies observed in patients carrying TBC1D1 mutations, was found in kidneys of some Tbc1d1 (-/-) mice. Significantly reduced Glut4 levels were detected in kidneys of Tbc1d1 (-/-) mice and the dysplastic kidney of a TBC1D1 mutation carrier versus controls. TBC1D1 and SLC2A4 encoding GLUT4 were highly expressed in human fetal kidney. The patient with the truncating TBC1D1 mutation showed evidence for insulin resistance. These data demonstrate heterozygous deactivating TBC1D1 mutations in CAKUT patients with a similar renal and ureteral phenotype, and provide evidence that TBC1D1 mutations may contribute to CAKUT pathogenesis, possibly via a role in glucose homeostasis. PMID:26572137

  20. Modulation of Bacillus thuringiensis Phosphatidylinositol-Specific Phospholipase C Activity by Mutations in the Putative Dimerization Interface

    Energy Technology Data Exchange (ETDEWEB)

    Shi, X.; Shao, C; Zhang, X; Zambonelli, C; Redfield, A; Head, J; Seaton, B; Roberts, M

    2009-01-01

    Cleavage of phosphatidylinositol (PI) to inositol 1,2-(cyclic)-phosphate (cIP) and cIP hydrolysis to inositol 1-phosphate by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C are activated by the enzyme binding to phosphatidylcholine (PC) surfaces. Part of this reflects improved binding of the protein to interfaces. However, crystallographic analysis of an interfacially impaired phosphatidylinositol-specific phospholipase (W47A/W242A) suggested protein dimerization might occur on the membrane. In the W47A/W242A dimer, four tyrosine residues from one monomer interact with the same tyrosine cluster of the other, forming a tight dimer interface close to the membrane binding regions. We have constructed mutant proteins in which two or more of these tyrosine residues have been replaced with serine. Phospholipid binding and enzymatic activity of these mutants have been examined to assess the importance of these residues to enzyme function. Replacing two tyrosines had small effects on enzyme activity. However, removal of three or four tyrosine residues weakened PC binding and reduced PI cleavage by the enzyme as well as PC activation of cIP hydrolysis. Crystal structures of Y247S/Y251S in the absence and presence of myo-inositol as well as Y246S/Y247S/Y248S/Y251S indicate that both mutant proteins crystallized as monomers, were very similar to one another, and had no change in the active site region. Kinetic assays, lipid binding, and structural results indicate that either (i) a specific PC binding site, critical for vesicle activities and cIP activation, has been impaired, or (ii) the reduced dimerization potential for Y246S/Y247S/Y248S and Y246S/Y247S/Y248S/Y251S is responsible for their reduced catalytic activity in all assay systems.

  1. Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and provides insights into complement activation and regulation

    OpenAIRE

    Martínez-Barricarte, Rubén; Heurich, Meike; Valdés-Cañedo, Francisco; Vázquez-Martul, Eduardo; Torreira, Eva; Montes, Tamara; Tortajada, Agustín; Pinto, Sheila; López-Trascasa, Margarita; Morgan, B. Paul; Llorca, Óscar; Harris, Claire L.; Rodríguez de Córdoba, Santiago

    2010-01-01

    Dense deposit disease (DDD) is a severe renal disease characterized by accumulation of electron-dense material in the mesangium and glomerular basement membrane. Previously, DDD has been associated with deficiency of factor H (fH), a plasma regulator of the alternative pathway (AP) of complement activation, and studies in animal models have linked pathogenesis to the massive complement factor 3 (C3) activation caused by this deficiency. Here, we identified a unique DDD pedigree that associate...

  2. The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites.

    Science.gov (United States)

    Fernández-Calero, Tamara; Flouriot, Gilles; Marín, Mónica

    2016-01-01

    Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene.

  3. Ultraviolet-B sensitivities in Japanese lowland rice [Oryza sativa] cultivars: Cyclobutane pyrimidine dimer photolyase activity and gene mutation

    International Nuclear Information System (INIS)

    There is a cultivar difference in the response to ultraviolet-B (UVB: 280-320 um) in rice (Oryza saliva L.). Among Japanese lowland rice cultivars, Sasanishiki, a leading Japanese rice cultivar, is resistant to the damaging effects of UVB while Norin 1, a close relative, is less resistant. We found previously that Norin 1 was deficient in cyclobutane pyrimidine dimer (CPD) photorepair ability and suggested that the UVB sensitivity in rice depends largely on CPD photorepair ability. In order to verify that suggestion, we examined the correlation between UVB sensitivity and CPD photolyase activity in 17 rice cultivars of progenitors and relatives in breeding of UV-resistant Sasanishiki and UV- sensitive Norin 1. The amino acid at position 126 of the deduced amino acid sequence of CPD photolyase in cultivars including such as Norin 1 was found to be arginine, the CPD photolyase activities of which were lower. The amino acid at that position in cultivars including such as Sasanishiki was glutamine. Furthermore, cultivars more resistant to UVB were found to exhibit higher photolyase activities than less resistant cultivars. These results emphasize that single amino acid alteration from glutamine to arginine leads to a deficit of CPD photolyase activity and that CPD photolyase activity is one of the main factors determining UVB sensitivity in rice

  4. 贲门癌端粒酶活性表达及与p53基因突变关系的研究%Relationship of telomerase activity and p53 gene mutation in cardiac cancer

    Institute of Scientific and Technical Information of China (English)

    Jingruo li; Mengquan li; Jiangtao Li; Juntao Bao; Yunhang Zhang

    2007-01-01

    Objective: To study the relationship of the telomerase activity and the p53 gene mutation in cardiac cancer.Methods: Telomerase activity and the p53 gene mutation were detected in 46 case of cardiac cancer, peri-cancerous and 30 case of normal mucosa by TRAP-ELISA and PCR-SSCP. Results: The rate of expression of telomerase activity in cardiac cancer, peri-cancerous and normal mucosa were 82.61% (38/46), 43.48% (20/46) and 13.33% (4/30) respectively. The rate of Exon5→8 of p53 gene mutation were 39.13% (18/46), 4.35% (2/46) and 0.00% respectively. There was significant differ ence between group cancer and without cancer (P < 0.01). Mean of (A) value of telomerase is 1.89 ± 0.41 in cancer group and were 1.49 ± 0.43, 0.54 ± 0.45 respectively in peri-canvcerous and normal mucosa, there were significant differences in cancer group and group of without cancer (P < 0.05). The rate of p53 gene mutations in group of expression of telomerase activity was 44.74% (17/38), and 12.50% (1/8) in without expression of telomerase activity. There were significant differences between the two groups. Conclusion: The rate of expression of telomerase activity and mean of (A) value of telomerase in cardiac cancer were obviously higher than without cancer, which indicating telomerase activity was closely related with the occurrence of cardiac cancer. P53 gene mutation in cardiac cancer were higher than the tissue of without cancer, and the rate of p53 gene mutation in telomerase activity were obviously higher than the group of without cancer. This shows the p53 gene mutation can loss of function of suppressing cancer and prompt telomerase activity and cause the cardiac cancer.

  5. The Detection of Genotıxic Activity of The Deltamethrin and Permethrin by Somatic Mutation and Recombination Test With Drosophila Melanogaster

    Directory of Open Access Journals (Sweden)

    F. Azize BUDAK

    2005-06-01

    Full Text Available In this study, different doses of deltamethrin from pyretyroide insecticides and permethrine from organophosphate insecticides have been analysied for their genotoxic effects by somatic mutation and recombination test (SMART in Drosophila melanogaster. Lethal doses of chemicals used were determined. Trans-heterozygous larvae for two genetic markers mwh and flr3 were evaluated for 25 ppm, 50 ppm and 75 ppm concentrations of the insecticides. A possitive correlation was observed among total mutations, the number of wings having mutations and percentage survival. Mutations observed were classified according to their size and type per wing and the dataevaluated by statistical analysis. Deltamethrin was found more toxic and mutagenic than permethrine.

  6. Disease-causing missense mutations affect enzymatic activity, stability and oligomerization of glutaryl-CoA dehydrogenase (GCDH)

    DEFF Research Database (Denmark)

    Keyser, B.; Muhlhausen, C.; Dickmanns, A.;

    2008-01-01

    revealed that all mutants were enzymatically inactive with the exception of p.Met263Val which showed 10% activity of the expressed wild-type enzyme. Western blot and pulse-chase analyses demonstrated that the amount of expressed p.Arg402Trp protein was significantly reduced compared with cells expressing...

  7. In vitro mutation techniques and phyllanthus niruri L. tissue culture for secondary metabolites improvement having an antiplasmodial activity

    International Nuclear Information System (INIS)

    The research undertaken within the IAEA projects framework ZAI/6/009 and ZAI/5/016 consists in the checking of the antiplasmodial activity allotted to Phyllanthus niruri and to the bioguided isolement of the secondary metabolites produced by irradiation and in vitro culture. The preliminary ex-vivo and in vitro tests by the lactate dehydrogenase (LDH) method showed an antiplasmodial activity extract ethanolic of the whole plant higher than that of aqueous decoct. The leaves aqueous extract presented a significant antiplasmodial activity in vitro (IC50 = 14 ±1 dg/ml) by the radioactive microdilution technique (3HP). The preliminary chemical screening tests of the whole plant, leaves and roots revealed the presence of Alkaloids, gallic Tannins, Flavonoides, Coumarines, of Terpenoides and Steroids. The secondary metabolites present in the extracts ethanolic of the whole plant were separated by the analytical HPLC. The chromatogram obtained gave a majority peak at 26 min. Among the raised signals from various fractions of the majority peak, the spectra MS/MS (of fragmentation) of the ions responsible of the peak in m/Z 353 revealed the bicharged ions with a molecular weight around 660. This major molecule was purified and isolated at 27.97 min. by the coupling LC/MS. A molecule of mass 448 correspondent to that of Quercitrin (Flavonoid) was among the molecules separated and identified from leaves ethanolic extract by coupling LC/MS. The ethanolic extracts calli of 1 month old revealed a significant antiplasmodial activity (IC50 = 16.3 ± 2.5 dg/ml). The calli aqueous extracts of 3 months old obtained from explants irradiated at the dose of 1Gy showed an activity higher compared to the extracts of the whole plant and calli formed from no irradiated and irradiated explants at 4Gy and 7,5 Gy. In addition, the alkaloids, catechic tanins, saponins and terpenoids were identified in the calli from irradiated explants extracts. (author)

  8. Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

    International Nuclear Information System (INIS)

    Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of LmnaH222P/H222P mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in LmnaH222P/H222P mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male LmnaH222P/H222P mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of LmnaH222P/H222P mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left ventricular fractional

  9. Shutoff and agonist-triggered internalization of protease-activated receptor 1 can be separated by mutation of putative phosphorylation sites in the cytoplasmic tail.

    Science.gov (United States)

    Hammes, S R; Shapiro, M J; Coughlin, S R

    1999-07-20

    The thrombin receptor PAR1 becomes rapidly phosphorylated upon activation by either thrombin or exogenous SFLLRN agonist peptide. Substitution of alanine for all serine and threonine residues in the receptor's cytoplasmic carboxyl-terminal tail ablated phosphorylation and yielded a receptor defective in both shutoff and agonist-triggered internalization. These observations suggested that activation-dependent phosphorylation of PAR1's cytoplasmic tail is required for both shutoff and agonist-triggered internalization. To identify the phosphorylation site(s) that are necessary for these functions, we generated three mutant receptors in which alanine was substituted for serine and threonine residues in the amino-terminal, middle, and carboxyl-terminal thirds of PAR1's cytoplasmic tail. When stably expressed in fibroblasts, all three mutated receptors were rapidly phosphorylated in response to agonist, while a mutant in which all serines and threonines in the cytoplasmic tail were converted to alanines was not. This result suggests that phosphorylation can occur at multiple sites in PAR1's cytoplasmic tail. Alanine substitutions in the N-terminal and C-terminal portions of the tail had no effect on either receptor shutoff or agonist-triggered internalization. By contrast, alanine substitutions in the "middle" serine cluster between Ser(391) and Ser(406) yielded a receptor with considerably slower shutoff of signaling after thrombin activation than the wild type. Surprisingly, this same mutant was indistinguishable from the wild type in agonist-triggered internalization and degradation. Overexpression of G protein-coupled receptor kinase 2 (GRK2) and GRK3 "suppressed" the shutoff defect of the S --> A (391-406) mutant, consistent with this defect being due to altered receptor phosphorylation. These results suggest that specific phosphorylation sites are required for rapid receptor shutoff, but phosphorylation at multiple alternative sites is sufficient for agonist

  10. Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

    Energy Technology Data Exchange (ETDEWEB)

    Muchir, Antoine, E-mail: a.muchir@institut-myologie.org [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Wu, Wei [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Sera, Fusako; Homma, Shunichi [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Worman, Howard J., E-mail: hjw14@columbia.edu [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States)

    2014-10-03

    Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna{sup H222P/H222P} mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna{sup H222P/H222P} mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna{sup H222P/H222P} mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna{sup H222P/H222P} mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left

  11. Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis.

    Science.gov (United States)

    Bag, Satyabrata; Das, Bhabatosh; Dasgupta, Shreya; Bhadra, Rupak K

    2014-08-01

    Rel(Mtb), a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel(Mtb) has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel(Mtb) gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel(Mtb) and its truncated derivatives were expressed from an arabinose inducible promoter (P(BAD)), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188-430 residues) containing fragment are sufficient for Rel(Mtb) (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel(Mtb) by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.

  12. CK1δ in lymphoma: gene expression and mutation analyses and validation of CK1δ kinase activity for therapeutic application

    Directory of Open Access Journals (Sweden)

    Brigitte Sophia Winkler

    2015-02-01

    Full Text Available The prognosis of lymphoid neoplasms has improved considerably during the last decades. However, treatment response for some lymphoid neoplasms is still poor, indicating the need for new therapeutic approaches. One promising new strategy is the inhibition of kinases regulating key signal transduction pathways, which are of central importance in tumorigenesis. Kinases of the CK1 family may represent an attractive drug target since CK1 expression and/or activity are associated with the pathogenesis of malignant diseases. Over the last years efforts were taken to develop highly potent and selective CK1-specific inhibitor compounds and their therapeutic potential has now to be proved in pre-clinical trials. Therefore, we analyzed expression and mutational status of CK1δ in several cell lines representing established lymphoma entities, and also measured the mRNA expression level in primary lymphoma tissue as well as non-neoplastic blood cells. For a selection of lymphoma cell lines we furthermore determined CK1δ kinase activity and demonstrated therapeutic potential of CK1-specific inhibitors as a putative therapeutic option in the treatment of lymphoid neoplasms.

  13. Occipital horn syndrome and classical Menkes syndrome caused by deep intronic mutations, leading to the activation of ATP7A pseudo-exon

    DEFF Research Database (Denmark)

    Yasmeen, Saiqa; Lund, Katrine; De Paepe, Anne;

    2014-01-01

    Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene. Whereas most of the patients exhibit a severe classical form, about 9% of the patients exhibit a milder form of Menkes disease. The mildest form is called occipital horn syndrome (OHS). Mutations in...

  14. A novel mutation in the β-spectrin gene causes the activation of a cryptic 5′-splice site and the creation of a de novo 3′-splice site

    Science.gov (United States)

    Salas, Pilar Carrasco; Rosales, José Miguel Lezana; Milla, Carmen Palma; Montiel, Javier López; Siles, Juan López

    2015-01-01

    The analysis of genes involved in hereditary spherocytosis, by next-generation sequencing in two patients with clinical diagnosis of the disease, showed the presence of the c.1795+1G>A mutation in the SPTB gene. cDNA amplification then revealed the occurrence of a consequent aberrant mRNA isoform produced from the activation of a cryptic 5′-splice site and the creation of a newly 3′-splice site. The mechanisms by which these two splice sites are used as a result of the same mutation should be analyzed in depth in further studies. PMID:27081538

  15. Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach

    Science.gov (United States)

    Dutta, Sruti; Choudhury, Debi; Roy, Sumana; Dattagupta, Jiban Kanti; Biswas, Sampa

    2016-01-01

    Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we have generated three different mutants of papain (I86F, I86L and I86A) by replacing the residue I86 in its pro-peptide region, which blocks the specificity determining S2-subsite of the catalytic cleft of the protease in its zymogen form with a view to investigate the effect of mutation on the catalytic activity of the protease. Steady-state enzyme kinetic analyses of the corresponding mutant proteases with specific peptide substrates show significant alteration of substrate specificity—I86F and I86L have 2.7 and 29.1 times higher kcat/Km values compared to the wild-type against substrates having Phe and Leu at P2 position, respectively, while I86A shows lower catalytic activity against majority of the substrates tested. Far-UV CD scan and molecular mass analyses of the mature form of the mutant proteases reveal similar CD spectra and intact masses to that of the wild-type. Crystal structures of zymogens of I86F and I86L mutants suggest that subtle reorganization of active site residues, including water, upon binding of the pro-peptide may allow the enzyme to achieve discriminatory substrate selectivity and catalytic efficiency. However, accurate and reliable predictions on alteration of substrate specificity require atomic resolution structure of the catalytic domain after zymogen activation, which remains a challenging task. In this study we demonstrate that through single amino acid substitution in pro-peptide, it is possible to modify the substrate specificity of papain and hence the pro-peptide of a protease can also be a useful target for altering its catalytic activity/specificity. PMID:27352302

  16. Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity-A Structural and Biochemical Approach.

    Science.gov (United States)

    Dutta, Sruti; Choudhury, Debi; Roy, Sumana; Dattagupta, Jiban Kanti; Biswas, Sampa

    2016-01-01

    Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we have generated three different mutants of papain (I86F, I86L and I86A) by replacing the residue I86 in its pro-peptide region, which blocks the specificity determining S2-subsite of the catalytic cleft of the protease in its zymogen form with a view to investigate the effect of mutation on the catalytic activity of the protease. Steady-state enzyme kinetic analyses of the corresponding mutant proteases with specific peptide substrates show significant alteration of substrate specificity-I86F and I86L have 2.7 and 29.1 times higher kcat/Km values compared to the wild-type against substrates having Phe and Leu at P2 position, respectively, while I86A shows lower catalytic activity against majority of the substrates tested. Far-UV CD scan and molecular mass analyses of the mature form of the mutant proteases reveal similar CD spectra and intact masses to that of the wild-type. Crystal structures of zymogens of I86F and I86L mutants suggest that subtle reorganization of active site residues, including water, upon binding of the pro-peptide may allow the enzyme to achieve discriminatory substrate selectivity and catalytic efficiency. However, accurate and reliable predictions on alteration of substrate specificity require atomic resolution structure of the catalytic domain after zymogen activation, which remains a challenging task. In this study we demonstrate that through single amino acid substitution in pro-peptide, it is possible to modify the substrate specificity of papain and hence the pro-peptide of a protease can also be a useful target for altering its catalytic activity/specificity. PMID:27352302

  17. A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

    Directory of Open Access Journals (Sweden)

    Gao Chen

    2012-02-01

    Full Text Available Abstract Background The human papillomavirus (HPV E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. Results CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. Conclusions These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

  18. Mutations in the signal transducer and activator of transcription 3 (STAT3) and diagnostic guidelines for the Hyper-IgE Syndrome

    Science.gov (United States)

    Woellner, Cristina; Gertz, E. Michael; Schäffer, Alejandro A.; Lagos, Macarena; Perro, Mario; Glocker, Erik-Oliver; Pietrogrande, Maria C.; Cossu, Fausto; Franco, José L.; Matamoros, Nuria; Pietrucha, Barbara; Heropolitańska-Pliszka, Edyta; Yeganeh, Mehdi; Moin, Mostafa; Español, Teresa; Ehl, Stephan; Gennery, Andrew R.; Abinun, Mario; Bręborowicz, Anna; Niehues, Tim; Kilic, Sara Sebnem; Junker, Anne; Turvey, Stuart E.; Plebani, Alessandro; Sánchez, Berta; Garty, Ben-Zion; Pignata, Claudio; Cancrini, Caterina; Litzman, Jiri; Sanal, Özden; Baumann, Ulrich; Bacchetta, Rosa; Hsu, Amy P.; Davis, Joie N.; Hammarström, Lennart; Davies, E. Graham; Eren, Efrem; Arkwright, Peter D.; Moilanen, Jukka S.; Viemann, Dorothee; Khan, Sujoy; Maródi, László; Cant, Andrew J.; Freeman, Alexandra F.; Puck, Jennifer M.; Holland, Steven M.; Grimbacher, Bodo

    2010-01-01

    Background The hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by infections of the lung and skin, elevated serum IgE, and involvement of the soft and tissues. Recently, HIES has been associated with heterozygous dominant-negative mutations in STAT3 and severe reductions of Th17 cells. Objective To determine whether there is a correlation between the genotype and phenotype of HIES patients and to establish diagnostic criteria to distinguish between STAT3 mutated and STAT3 wild-type patients. Methods We collected clinical data, determined Th17 cell numbers, and sequenced STAT3 100 patients with a strong clinical suspicion of HIES and serum IgE >1000 IU/mL. explored diagnostic criteria by using a machine-learning approach to identify which features best predict a STAT3 mutation. Results In 64 patients we identified 31 different STAT3 mutations, 18 of which are novel. These included mutations at splice sites and outside the previously implicated DNA-binding and SH2 domains. A combination of five clinical features predicted STAT3 mutations with 85% accuracy. Th17 cells were profoundly reduced in patients harboring STAT3 mutations, while 10 out of 13 patients without mutations had low (1000 IU/mL plus a weighted score of clinical features >30 based on recurrent pneumonia, newborn rash, pathologic bone fractures, characteristic face, and high palate. Probable: Above plus lack of Th17 cells or a family history for definitive HIES. Definitive: Above plus a dominant-negative heterozygous mutation in STAT3. PMID:20159255

  19. Q344ter mutation causes mislocalization of rhodopsin molecules that are catalytically active: a mouse model of Q344ter-induced retinal degeneration.

    Directory of Open Access Journals (Sweden)

    Francis Concepcion

    Full Text Available Q344ter is a naturally occurring rhodopsin mutation in humans that causes autosomal dominant retinal degeneration through mechanisms that are not fully understood, but are thought to involve an early termination that removed the trafficking signal, QVAPA, leading to its mislocalization in the rod photoreceptor cell. To better understand the disease mechanism(s, transgenic mice that express Q344ter were generated and crossed with rhodopsin knockout mice. Dark-reared Q344ter(rho+/- mice exhibited retinal degeneration, demonstrating that rhodopsin mislocalization caused photoreceptor cell death. This degeneration is exacerbated by light-exposure and is correlated with the activation of transducin as well as other G-protein signaling pathways. We observed numerous sub-micrometer sized vesicles in the inter-photoreceptor space of Q344ter(rho+/- and Q344ter(rho-/- retinas, similar to that seen in another rhodopsin mutant, P347S. Whereas light microscopy failed to reveal outer segment structures in Q344ter(rho-/- rods, shortened and disorganized rod outer segment structures were visible using electron microscopy. Thus, some Q344ter molecules trafficked to the outer segment and formed disc structures, albeit inefficiently, in the absence of full length wildtype rhodopsin. These findings helped to establish the in vivo role of the QVAPA domain as well as the pathways leading to Q344ter-induced retinal degeneration.

  20. TERT promoter mutations are highly recurrent in SHH subgroup medulloblastoma

    NARCIS (Netherlands)

    M. Remke (Marc); E.A. Ramaswamy; M. Peacock (Munro); D.J.H. Shih (David J.); C. Koelsche (Christian); P.A. Northcott (Paul A.); N. Hill (Nadia); S. Cavalli (Silvia); M. Kool (Marcel); X. Wang (Xin); S. Mack (Stephen); M. Barszczyk (Mark); A.S. Morrissy (A. Sorana); X. Wu (Xiaochong); S. Agnihotri (Sameer); P. Luu (Phan); D. Jones (David); L. Garzia (Livia); A.M. Dubuc (Adrian); N. Zhukova (Nataliya); R. Vanner (Robert); J.M. Kros (Johan); P.J. French (Pim); E.G. van Meir (Erwin); R. Vibhakar (Rajeev); K. Zitterbart (Karel); J.A. Chan (Jennifer); L. Bognár (László); A. Klekner (Almos); B. Lach (Boleslaw); S. Jung; F. Saad (Fred); L.M. Liau (Linda); S. Albrecht (Steffen); M. Zollo (Maurizio); M.K. Cooper (Michael); R.C. Thompson (Reid); O. Delattre (Olivier); F. Bourdeaut (Franck); F.F. Doz (François); M. Garami (Miklós); P. Hauser (Peter); C.G. Carlotti (Carlos); T.E. Van Meter (Timothy); L. Massimi (Luca); D. Fults (Daniel); L.W. Pomeroy (Laura); T. Kumabe (Toshiro); Y.S. Ra (Young Shin); J.R. Leonard (Jeffrey); S.K. Elbabaa (Samer); J. Mora (Jaume); J.B. Rubin (Joshua); Y.-J. Cho (Yoon-Jae); R.E. McLendon (Roger); D.D. Bigner (Darell); C.G. Eberhart (Charles); M. Fouladi (Maryam); R.J. Wechsler-Reya (Robert); R. Faria (Rui); S.E. Croul (Sidney); A. Huang (Anding); E. Bouffet (Eric); C.E. Hawkins (Cynthia); M. Dirks (Maaike); W.A. Weiss (William); U. Schüller (Ulrich); A. Pollack (Aaron); P. Rutkowski (Piotr); D. Meyronet (David); A. Jouvet (Anne); M. Fèvre-Montange (Michelle); N. Jabado (Nada); M. Perek-Polnik (Marta); W.A. Grajkowska (Wieslawa); S.-K. Kim (Seung-Ki); J.T. Rutka (James); E. Malkin (Elissa); U. Tabori (Uri); S.M. Pfister (Stefan); A. Korshunov (Andrey); A. von Deimling (Andreas); M.D. Taylor (Michael)

    2013-01-01

    textabstractTelomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought

  1. Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

    Science.gov (United States)

    Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

    2016-04-01

    R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low

  2. Sustained receptor activation and hyperproliferation in response to granulocyte colony-stimulating factor (G-CSF) in mice with a severe congenital neutropenia/acute myeloid leukemia-derived mutation in the G-CSF receptor gene.

    Science.gov (United States)

    Hermans, M H; Antonissen, C; Ward, A C; Mayen, A E; Ploemacher, R E; Touw, I P

    1999-02-15

    In approximately 20% of cases of severe congenital neutropenia (SCN), mutations are found in the gene encoding the granulocyte colony-stimulating factor receptor (G-CSF-R). These mutations introduce premature stop codons, which result in truncation of 82-98 COOH-terminal amino acids of the receptor. SCN patients who develop secondary myelodysplastic syndrome and acute myeloid leukemia almost invariably acquired a GCSFR mutation, suggesting that this genetic alteration represents a key step in leukemogenesis. Here we show that an equivalent mutation targeted in mice (gcsfr-Delta715) results in the selective expansion of the G-CSF- responsive progenitor (G-CFC) compartment in the bone marrow. In addition, in vivo treatment of gcsfr-Delta715 mice with G-CSF results in increased production of neutrophils leading to a sustained neutrophilia. This hyperproliferative response to G-CSF is accompanied by prolonged activation of signal transducer and activator of transcription (STAT) complexes and extended cell surface expression of mutant receptors due to defective internalization. In view of the continuous G-CSF treatment of SCN patients, these data provide insight into why progenitor cells expressing truncated receptors clonally expand in vivo, and why these cells may be targets for additional genetic events leading to leukemia. PMID:9989983

  3. Sustained Receptor Activation and Hyperproliferation in Response to Granulocyte Colony-stimulating Factor (G-CSF) in Mice with a Severe Congenital Neutropenia/Acute Myeloid Leukemia–derived Mutation in the G-CSF Receptor Gene

    Science.gov (United States)

    Hermans, Mirjam H.A.; Antonissen, Claudia; Ward, Alister C.; Mayen, Angelique E.M.; Ploemacher, Rob E.; Touw, Ivo P.

    1999-01-01

    In approximately 20% of cases of severe congenital neutropenia (SCN), mutations are found in the gene encoding the granulocyte colony-stimulating factor receptor (G-CSF–R). These mutations introduce premature stop codons, which result in truncation of 82–98 COOH-terminal amino acids of the receptor. SCN patients who develop secondary myelodysplastic syndrome and acute myeloid leukemia almost invariably acquired a GCSFR mutation, suggesting that this genetic alteration represents a key step in leukemogenesis. Here we show that an equivalent mutation targeted in mice (gcsfr-Δ715) results in the selective expansion of the G-CSF– responsive progenitor (G-CFC) compartment in the bone marrow. In addition, in vivo treatment of gcsfr-Δ715 mice with G-CSF results in increased production of neutrophils leading to a sustained neutrophilia. This hyperproliferative response to G-CSF is accompanied by prolonged activation of signal transducer and activator of transcription (STAT) complexes and extended cell surface expression of mutant receptors due to defective internalization. In view of the continuous G-CSF treatment of SCN patients, these data provide insight into why progenitor cells expressing truncated receptors clonally expand in vivo, and why these cells may be targets for additional genetic events leading to leukemia. PMID:9989983

  4. A myosin activator improves actin assembly and sarcomere function of human-induced pluripotent stem cell-derived cardiomyocytes with a troponin T point mutation.

    Science.gov (United States)

    Broughton, K M; Li, J; Sarmah, E; Warren, C M; Lin, Y-H; Henze, M P; Sanchez-Freire, V; Solaro, R J; Russell, B

    2016-07-01

    We have investigated cardiac myocytes derived from human-induced pluripotent stem cells (iPSC-CMs) from two normal control and two family members expressing a mutant cardiac troponin T (cTnT-R173W) linked to dilated cardiomyopathy (DCM). cTnT is a regulatory protein of the sarcomeric thin filament. The loss of this basic charge, which is strategically located to control tension, has consequences leading to progressive DCM. iPSC-CMs serve as a valuable platform for understanding clinically relevant mutations in sarcomeric proteins; however, there are important questions to be addressed with regard to myocyte adaptation that we model here by plating iPSC-CMs on softer substrates (100 kPa) to create a more physiologic environment during recovery and maturation of iPSC-CMs after thawing from cryopreservation. During the first week of culture of the iPSC-CMs, we have determined structural and functional characteristics as well as actin assembly dynamics. Shortening, actin content, and actin assembly dynamics were depressed in CMs from the severely affected mutant at 1 wk of culture, but by 2 wk differences were less apparent. Sarcomeric troponin and myosin isoform composition were fetal/neonatal. Furthermore, the troponin complex, reconstituted with wild-type cTnT or recombinant cTnT-R173W, depressed the entry of cross-bridges into the force-generating state, which can be reversed by the myosin activator omecamtiv mecarbil. Therapeutic doses of this drug increased both contractility and the content of F-actin in the mutant iPSC-CMs. Collectively, our data suggest the use of a myosin activation reagent to restore function within patient-specific iPSC-CMs may aid in understanding and treating this familial DCM. PMID:27199119

  5. A single mutation in the 15S rRNA gene confers nonsense suppressor activity and interacts with mRF1 the release factor in yeast mitochondria

    Directory of Open Access Journals (Sweden)

    Ali Gargouri

    2015-08-01

    Full Text Available We have determined the nucleotide sequence of the mim3-1 mitochondrial ribosomal suppressor, acting on ochre mitochondrial mutations and one frameshift mutation in Saccharomyces cerevisiae. The 15s rRNA suppressor gene contains a G633 to C transversion. Yeast mitochondrial G633 corresponds to G517 of the E.coli 15S rRNA, which is occupied by an invariant G in all known small rRNA sequences. Interestingly, this mutation has occurred at the same position as the known MSU1 mitochondrial suppressor which changes G633 to A. The suppressor mutation lies in a highly conserved region of the rRNA, known in E.coli as the 530-loop, interacting with the S4, S5 and S12 ribosomal proteins. We also show an interesting interaction between the mitochondrial mim3-1 and the nuclear nam3-1 suppressors, both of which have the same action spectrum on mitochondrial mutations: nam3-1 abolishes the suppressor effect when present with mim3-1 in the same haploid cell. We discuss these results in the light of the nature of Nam3, identified by [1] as the yeast mitochondrial translation release factor. A hypothetical mechanism of suppression by "ribosome shifting" is also discussed in view of the nature of mutations suppressed and not suppressed.

  6. Transgenic Animal Mutation Assays

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Ph.D.D.A.B.T.

    2005-01-01

    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  7. ENU mutagenesis screening for dominant behavioral mutations based on normal control data obtained in home-cage activity, open-field, and passive avoidance tests.

    Science.gov (United States)

    Wada, Yumiko; Furuse, Tamio; Yamada, Ikuko; Masuya, Hiroshi; Kushida, Tomoko; Shibukawa, Yoko; Nakai, Yuji; Kobayashi, Kimio; Kaneda, Hideki; Gondo, Yoichi; Noda, Tetsuo; Shiroishi, Toshihiko; Wakana, Shigeharu

    2010-01-01

    To establish the cutoff values for screening ENU-induced behavioral mutations, normal variations in mouse behavioral data were examined in home-cage activity (HA), open-field (OF), and passive-avoidance (PA) tests. We defined the normal range as one that included more than 95% of the normal control values. The cutoffs were defined to identify outliers yielding values that deviated from the normal by less than 5% for C57BL/6J, DBA/2J, DBF(1), and N(2) (DXDB) progenies. Cutoff values for G1-phenodeviant (DBF(1)) identification were defined based on values over +/- 3.0 SD from the mean of DBF(1) for all parameters assessed in the HA and OF tests. For the PA test, the cutoff values were defined based on whether the mice met the learning criterion during the 2nd (at a shock intensity of 0.3 mA) or the 3rd (at a shock intensity of 0.15 mA) retention test. For several parameters, the lower outliers were undetectable as the calculated cutoffs were negative values. Based on the cutoff criteria, we identified 275 behavioral phenodeviants among 2,646 G1 progeny. Of these, 64 were crossed with wild-type DBA/2J individuals, and the phenotype transmission was examined in the G2 progeny using the cutoffs defined for N(2) mice. In the G2 mice, we identified 15 novel dominant mutants exhibiting behavioral abnormalities, including hyperactivity in the HA or OF tests, hypoactivity in the OF test, and PA deficits. Genetic and detailed behavioral analysis of these ENU-induced mutants will provide novel insights into the molecular mechanisms underlying behavior.

  8. Maize Mutator transposon

    Institute of Scientific and Technical Information of China (English)

    Yijun WANG; Mingliang XU; Dexiang DENG; Yunlong BIAN

    2008-01-01

    Transposable elements are widely distributed in eukaryotes. Due to its high copy numbers, high forward mutation rate and preferential insertion into low-copy DNA sequences, among others, the Mutator system has been widely used as a mutagen in genomic research. The discovery, classification, transposition specificity and epige-netic regulation of Mutator transposons were described. The application of Mutator tagging in plant genomic research was also presented. The role of Mu-like elements in genome evolution was briefly depicted. Moreover, the direction of Mutator transposon research in the future was discussed.

  9. Mutations in the control of virulence sensor gene from Streptococcus pyogenes after infection in mice lead to clonal bacterial variants with altered gene regulatory activity and virulence.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Mayfield

    Full Text Available The cluster of virulence sensor (CovS/responder (CovR two-component operon (CovRS regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448, containing wild-type (WT CovRS (5448/CovR+S+, or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS- was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection.

  10. Long QT 1 mutation KCNQ1A344V increases local anesthetic sensitivity of the slowly activating delayed rectifier potassium current

    DEFF Research Database (Denmark)

    Siebrands, Cornelia C; Binder, Stephan; Eckhoff, Ulrike;

    2006-01-01

    was to investigate whether the LQT1 mutation A344V in the S6 region of KCNQ1, at a position corresponding to the local anesthetic binding site in HERG, may render drug insensitive KCNQ1 channels into a toxicologically relevant target of these pharmacologic agents. This may suggest that LQTS constitutes not only...... a prolongation of the cardiac action potential duration and induction of early afterdepolarizations by the mutation A344V that were aggravated by local anesthetic intoxication. CONCLUSIONS: The results indicate that certain forms of the LQTS may constitute a specific pharmacogenetic risk factor for regional...

  11. Mutations in C4orf26, encoding a peptide with in vitro hydroxyapatite crystal nucleation and growth activity, cause amelogenesis imperfecta.

    Science.gov (United States)

    Parry, David A; Brookes, Steven J; Logan, Clare V; Poulter, James A; El-Sayed, Walid; Al-Bahlani, Suhaila; Al Harasi, Sharifa; Sayed, Jihad; Raïf, El Mostafa; Shore, Roger C; Dashash, Mayssoon; Barron, Martin; Morgan, Joanne E; Carr, Ian M; Taylor, Graham R; Johnson, Colin A; Aldred, Michael J; Dixon, Michael J; Wright, J Tim; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

    2012-09-01

    Autozygosity mapping and clonal sequencing of an Omani family identified mutations in the uncharacterized gene, C4orf26, as a cause of recessive hypomineralized amelogenesis imperfecta (AI), a disease in which the formation of tooth enamel fails. Screening of a panel of 57 autosomal-recessive AI-affected families identified eight further families with loss-of-function mutations in C4orf26. C4orf26 encodes a putative extracellular matrix acidic phosphoprotein expressed in the enamel organ. A mineral nucleation assay showed that the protein's phosphorylated C terminus has the capacity to promote nucleation of hydroxyapatite, suggesting a possible function in enamel mineralization during amelogenesis.

  12. Identification of a new phospholipase C activity by analysis of an insertional mutation in the hemolytic phospholipase C structural gene of Pseudomonas aeruginosa.

    OpenAIRE

    Ostroff, R M; Vasil, M L

    1987-01-01

    The phospholipase C (PLC) gene of Pseudomonas aeruginosa encodes a heat-labile secreted hemolysin which is part of a Pi-regulated operon. The structural gene for PLC, plcS, was mutated in vitro by insertion of a tetracycline resistance gene cartridge. Gene replacement techniques were used to introduce the mutated plcS gene into the P. aeruginosa chromosome in place of the wild-type gene. The precise replacement of wild-type sequences by mutant sequences was confirmed by Southern hybridization...

  13. The human c-Kirsten ras gene is activated by a novel mutation in codon 13 in the breast carcinoma cell line MDA-MB231.

    OpenAIRE

    Kozma, S C; Bogaard, M E; Buser, K; Saurer, S M; Bos, J. L.; Groner, B; Hynes, N E

    1987-01-01

    We have detected amplified human Ki-ras sequences in tumorigenic NIH 3T3 cells transfected with genomic DNA from the human breast carcinoma cell line MDA-MB231. Hybridization of synthetic oligonucleotides specific for human Ki-ras sequences showed a mutation at codon 13. The polymerase chain reaction with Ki-ras specific amplimers revealed a guanosine to adenosine transition at the second position of codon 13, resulting in a substitution of glycine by aspartic acid. The codon 13 mutation is a...

  14. A Recurrent Germline Mutation in the 5'UTR of the Androgen Receptor Causes Complete Androgen Insensitivity by Activating Aberrant uORF Translation.

    Science.gov (United States)

    Hornig, Nadine C; de Beaufort, Carine; Denzer, Friederike; Cools, Martine; Wabitsch, Martin; Ukat, Martin; Kulle, Alexandra E; Schweikert, Hans-Udo; Werner, Ralf; Hiort, Olaf; Audi, Laura; Siebert, Reiner; Ammerpohl, Ole; Holterhus, Paul-Martin

    2016-01-01

    A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two unrelated patients with complete androgen insensitivity syndrome (CAIS) generating an upstream open reading frame (uORF) in the 5' untranslated region (5'-UTR) of the androgen receptor (AR) gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5'UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5'UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general.

  15. A Recurrent Germline Mutation in the 5'UTR of the Androgen Receptor Causes Complete Androgen Insensitivity by Activating Aberrant uORF Translation.

    Directory of Open Access Journals (Sweden)

    Nadine C Hornig

    Full Text Available A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two unrelated patients with complete androgen insensitivity syndrome (CAIS generating an upstream open reading frame (uORF in the 5' untranslated region (5'-UTR of the androgen receptor (AR gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5'UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5'UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general.

  16. A Recurrent Germline Mutation in the 5’UTR of the Androgen Receptor Causes Complete Androgen Insensitivity by Activating Aberrant uORF Translation

    Science.gov (United States)

    Hornig, Nadine C.; de Beaufort, Carine; Denzer, Friederike; Cools, Martine; Wabitsch, Martin; Ukat, Martin; Kulle, Alexandra E.; Schweikert, Hans-Udo; Werner, Ralf; Hiort, Olaf; Audi, Laura; Siebert, Reiner; Ammerpohl, Ole; Holterhus, Paul-Martin

    2016-01-01

    A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two unrelated patients with complete androgen insensitivity syndrome (CAIS) generating an upstream open reading frame (uORF) in the 5’ untranslated region (5’-UTR) of the androgen receptor (AR) gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5′UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5′UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general. PMID:27110943

  17. Genetic mutations in adipose triglyceride lipase and myocardial up-regulation of peroxisome proliferated activated receptor-γ in patients with triglyceride deposit cardiomyovasculopathy

    International Nuclear Information System (INIS)

    Highlights: •Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare severe heart disease. •PPARγ is up-regulated in myocardium in patients with TGCV. •Possible vicious cycle for fatty acid may be involved in pathophysiology of TGCV. -- Abstract: Adipose triglyceride lipase (ATGL, also known as PNPLA2) is an essential molecule for hydrolysis of intracellular triglyceride (TG). Genetic ATGL deficiency is a rare multi-systemic neutral lipid storage disease. Information regarding its clinical profile and pathophysiology, particularly for cardiac involvement, is still very limited. A previous middle-aged ATGL-deficient patient in our institute (Case 1) with severe heart failure required cardiac transplantation (CTx) and exhibited a novel phenotype, “Triglyceride deposit cardiomyovasculopathy (TGCV)”. Here, we tried to elucidate molecular mechanism underlying TGCV. The subjects were two cases with TGCV, including our second case who was a 33-year-old male patient (Case 2) with congestive heart failure requiring CTx. Case 2 was homozygous for a point mutation in the 5′ splice donor site of intron 5 in the ATGL, which results in at least two types of mRNAs due to splicing defects. The myocardium of both patients (Cases 1 and 2) showed up-regulation of peroxisome proliferated activated receptors (PPARs), key transcription factors for metabolism of long chain fatty acids (LCFAs), which was in contrast to these molecules’ lower expression in ATGL-targeted mice. We investigated the intracellular metabolism of LCFAs under human ATGL-deficient conditions using patients’ passaged skin fibroblasts as a model. ATGL-deficient cells showed higher uptake and abnormal intracellular transport of LCFA, resulting in massive TG accumulation. We used these findings from cardiac specimens and cell-biological experiments to construct a hypothetical model to clarify the pathophysiology of the human disorder. In patients with TGCV, even when hydrolysis of intracellular TG

  18. Genetic mutations in adipose triglyceride lipase and myocardial up-regulation of peroxisome proliferated activated receptor-γ in patients with triglyceride deposit cardiomyovasculopathy

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Ken-ichi, E-mail: khirano@cnt-osaka.com [Laboratory of Cardiovascular Disease, Novel, Non-Invasive, and Nutritional Therapeutics (CNT), Graduate School of Medicine, Osaka University, 6-2-3, Furuedai, Suita, Osaka 565-0874 (Japan); Department of Cardiovascular Medicine, Graduate School of Medicine, Osaka University, 2-2, Yamadaoka, Suita, Osaka 565-0871 (Japan); Tanaka, Tatsuya [Center for Medical Research and Education, Graduate School of Medicine, Osaka University, 2-2, Yamadaoka, Suita, Osaka 565-0871 (Japan); Ikeda, Yoshihiko [Department of Pathology, National Cerebral and Cardiovascular Center, 5-7-1 Fujishirodai, Suita 565-8565 (Japan); Yamaguchi, Satoshi [Laboratory of Cardiovascular Disease, Novel, Non-Invasive, and Nutritional Therapeutics (CNT), Graduate School of Medicine, Osaka University, 6-2-3, Furuedai, Suita, Osaka 565-0874 (Japan); Department of Cardiovascular Medicine, Graduate School of Medicine, Osaka University, 2-2, Yamadaoka, Suita, Osaka 565-0871 (Japan); Zaima, Nobuhiro [Department of Applied Biochemistry, Kinki University, 3327-204, Nakamachi, Nara 631-8505 (Japan); Kobayashi, Kazuhiro [Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 (Japan); Suzuki, Akira [Laboratory of Cardiovascular Disease, Novel, Non-Invasive, and Nutritional Therapeutics (CNT), Graduate School of Medicine, Osaka University, 6-2-3, Furuedai, Suita, Osaka 565-0874 (Japan); Department of Cardiovascular Medicine, Graduate School of Medicine, Osaka University, 2-2, Yamadaoka, Suita, Osaka 565-0871 (Japan); Sakata, Yasuhiko [Department of Cardiovascular Medicine, Graduate School of Medicine, Osaka University, 2-2, Yamadaoka, Suita, Osaka 565-0871 (Japan); Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, 1-1, Seiryo-cho, Aoba-ku, Sendai 980-8574 (Japan); and others

    2014-01-10

    Highlights: •Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare severe heart disease. •PPARγ is up-regulated in myocardium in patients with TGCV. •Possible vicious cycle for fatty acid may be involved in pathophysiology of TGCV. -- Abstract: Adipose triglyceride lipase (ATGL, also known as PNPLA2) is an essential molecule for hydrolysis of intracellular triglyceride (TG). Genetic ATGL deficiency is a rare multi-systemic neutral lipid storage disease. Information regarding its clinical profile and pathophysiology, particularly for cardiac involvement, is still very limited. A previous middle-aged ATGL-deficient patient in our institute (Case 1) with severe heart failure required cardiac transplantation (CTx) and exhibited a novel phenotype, “Triglyceride deposit cardiomyovasculopathy (TGCV)”. Here, we tried to elucidate molecular mechanism underlying TGCV. The subjects were two cases with TGCV, including our second case who was a 33-year-old male patient (Case 2) with congestive heart failure requiring CTx. Case 2 was homozygous for a point mutation in the 5′ splice donor site of intron 5 in the ATGL, which results in at least two types of mRNAs due to splicing defects. The myocardium of both patients (Cases 1 and 2) showed up-regulation of peroxisome proliferated activated receptors (PPARs), key transcription factors for metabolism of long chain fatty acids (LCFAs), which was in contrast to these molecules’ lower expression in ATGL-targeted mice. We investigated the intracellular metabolism of LCFAs under human ATGL-deficient conditions using patients’ passaged skin fibroblasts as a model. ATGL-deficient cells showed higher uptake and abnormal intracellular transport of LCFA, resulting in massive TG accumulation. We used these findings from cardiac specimens and cell-biological experiments to construct a hypothetical model to clarify the pathophysiology of the human disorder. In patients with TGCV, even when hydrolysis of intracellular TG

  19. Mutations affecting gyrase in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, J.K.; Cabrera-Juarez, E.; Albritton, W.L.; Spikes, D.; Mutschler, A.

    1985-11-01

    Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch.

  20. A nonsense mutation in mouse Tardbp affects TDP43 alternative splicing activity and causes limb-clasping and body tone defects.

    Directory of Open Access Journals (Sweden)

    Thomas Ricketts

    Full Text Available Mutations in TARDBP, encoding Tar DNA binding protein-43 (TDP43, cause amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD. Attempts to model TDP43 dysfunction in mice have used knockouts or transgenic overexpressors, which have revealed the difficulties of manipulating TDP43, whose level is tightly controlled by auto-regulation. In a complementary approach, to create useful mouse models for the dissection of TDP43 function and pathology, we have identified a nonsense mutation in the endogenous mouse Tardbp gene through screening an N-ethyl-N-nitrosourea (ENU mutant mouse archive. The mutation is predicted to cause a Q101X truncation in TDP43. We have characterised Tardbp(Q101X mice to investigate this mutation in perturbing TDP43 biology at endogenous expression levels. We found the Tardbp(Q101X mutation is homozygous embryonic lethal, highlighting the importance of TDP43 in early development. Heterozygotes (Tardbp(+/Q101X have abnormal levels of mutant transcript, but we find no evidence of the truncated protein and mice have similar full-length TDP43 protein levels as wildtype littermates. Nevertheless, Tardbp(+/Q101X mice have abnormal alternative splicing of downstream gene targets, and limb-clasp and body tone phenotypes. Thus the nonsense mutation in Tardbp causes a mild loss-of-function phenotype and behavioural assessment suggests underlying neurological abnormalities. Due to the role of TDP43 in ALS, we investigated potential interactions with another known causative gene, mutant superoxide dismutase 1 (SOD1. Tardbp(+/Q101X mice were crossed with the SOD1(G93Adl transgenic mouse model of ALS. Behavioural and physiological assessment did not reveal modifying effects on the progression of ALS-like symptoms in the double mutant progeny from this cross. In summary, the Tardbp(Q101X mutant mice are a useful tool for the dissection of TDP43 protein regulation, effects on splicing, embryonic development and neuromuscular

  1. Three kinds of mutation

    CERN Document Server

    Buan, Aslak Bakke; Thomas, Hugh

    2010-01-01

    For a finite dimensional hereditary algebra, we consider: exceptional sequences in the category of finite dimensional modules, silting objects in the bounded derived category, and m-cluster tilting objects in the m-cluster category. There are mutation operations on both the set of m-cluster tilting objects and the set of exceptional sequences. It is also possible to define a mutation operation for silting objects. We compare these three different notions of mutation.

  2. Mapping Mutations on Phylogenies

    DEFF Research Database (Denmark)

    Nielsen, Rasmus

    2005-01-01

    This chapter provides a short review of recent methodologies developed for mapping mutations on phylogenies. Mapping of mutations, or character changes in general, using the maximum parsimony principle has been one of the most powerful tools in phylogenetics, and it has been used in a variety of ...... uncertainty in the mapping. Recently developed probabilistic methods can incorporate statistical uncertainty in the character mappings. In these methods, focus is on a probability distribution of mutational mappings instead of a single estimate of the mutational mapping....

  3. Activation of JNK signaling links IgI mutations to disruption of the cell polarity and epithelial organization in Drosophila imaginal discs

    Institute of Scientific and Technical Information of China (English)

    Ming-wei Zhu; Tian-chi Xin; Shun-yan Weng; Yin Gao; Ying-jie Zhang; Qi Li; Ming-fa Li

    2010-01-01

    Dear Editor, Identification of Drosophila melanogaster as a model organism for cancer research has facilitated the exploration of human tumor malignancy. In Drosophila, lossof-function mutations in the neoplastic tumor suppressor genes (nTSGs) lethal(2)giant larvae (lgl), discs large (dlg) or scribble (scrib) cause a malignant tumor-like phenotype characteristic of disrupted cell polarity and overgrowth in epithelial tissues such as imaginal discs [1].

  4. Activation of Plant Immune Responses by a Gain-of-Function Mutation in an Atypical Receptor-Like Kinase1[C][W][OA

    Science.gov (United States)

    Bi, Dongling; Cheng, Yu Ti; Li, Xin; Zhang, Yuelin

    2010-01-01

    Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive1 (snc1) contains a gain-of-function mutation in a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat Resistance (R) protein and it has been a useful tool for dissecting R-protein-mediated immunity. Here we report the identification and characterization of snc4-1D, a semidominant mutant with snc1-like phenotypes. snc4-1D constitutively expresses defense marker genes PR1, PR2, and PDF1.2, and displays enhanced pathogen resistance. Map-based cloning of SNC4 revealed that it encodes an atypical receptor-like kinase with two predicted extracellular glycerophosphoryl diester phosphodiesterase domains. The snc4-1D mutation changes an alanine to threonine in the predicted cytoplasmic kinase domain. Wild-type plants transformed with the mutant snc4-1D gene displayed similar phenotypes as snc4-1D, suggesting that the mutation is a gain-of-function mutation. Epistasis analysis showed that NON-RACE-SPECIFIC DISEASE RESISTANCE1 is required for the snc4-1D mutant phenotypes. In addition, the snc4-1D mutant phenotypes are partially suppressed by knocking out MAP KINASE SUBSTRATE1, a positive defense regulator associated with MAP KINASE4. Furthermore, both the morphology and constitutive pathogen resistance of snc4-1D are partially suppressed by blocking jasmonic acid synthesis, suggesting that jasmonic acid plays an important role in snc4-1D-mediated resistance. Identification of snc4-1D provides us a unique genetic system for analyzing the signal transduction pathways downstream of receptor-like kinases. PMID:20508139

  5. Non-thermal atmospheric pressure plasma preferentially induces apoptosis in p53-mutated cancer cells by activating ROS stress-response pathways.

    Directory of Open Access Journals (Sweden)

    Yonghao Ma

    Full Text Available Non-thermal atmospheric pressure plasma (NTAPP is an ionized gas at room temperature and has potential as a new apoptosis-promoting cancer therapy that acts by generating reactive oxygen species (ROS. However, it is imperative to determine its selectivity and standardize the components and composition of NTAPP. Here, we designed an NTAPP-generating apparatus combined with a He gas feeding system and demonstrated its high selectivity toward p53-mutated cancer cells. We first determined the proper conditions for NTAPP exposure to selectively induce apoptosis in cancer cells. The apoptotic effect of NTAPP was greater for p53-mutated cancer cells; artificial p53 expression in p53-negative HT29 cells decreased the pro-apoptotic effect of NTAPP. We also examined extra- and intracellular ROS levels in NTAPP-treated cells to deduce the mechanism of NTAPP action. While NTAPP-mediated increases in extracellular nitric oxide (NO did not affect cell viability, intracellular ROS increased under NTAPP exposure and induced apoptotic cell death. This effect was dose-dependently reduced following treatment with ROS scavengers. NTAPP induced apoptosis even in doxorubicin-resistant cancer cell lines, demonstrating the feasibility of NTAPP as a potent cancer therapy. Collectively, these results strongly support the potential of NTAPP as a selective anticancer treatment, especially for p53-mutated cancer cells.

  6. Gestational mutations in radiation carcinogenesis

    Science.gov (United States)

    Meza, R.; Luebeck, G.; Moolgavkar, S.

    Mutations in critical genes during gestation could increase substantially the risk of cancer. We examine the consequences of such mutations using the Luebeck-Moolgavkar model for colorectal cancer and the Lea-Coulson modification of the Luria-Delbruck model for the accumulation of mutations during gestation. When gestational mutation rates are high, such mutations make a significant contribution to cancer risk even for adult tumors. Furthermore, gestational mutations ocurring at distinct times during emryonic developmemt lead to substantially different numbers of mutated cells at birth, with early mutations leading to a large number (jackpots) of mutated cells at birth and mutation occurring late leading to only a few mutated cells. Thus gestational mutations could confer considerable heterogeneity of the risk of cancer. If the fetus is exposed to an environmental mutagen, such as ionizing radiation, the gestational mutation rate would be expected to increase. We examine the consequences of such exposures during gestation on the subsequent development of cancer.

  7. Mutation and premating isolation.

    Science.gov (United States)

    Woodruff, R C; Thompson, J N

    2002-11-01

    While premating isolation might be traceable to different genetic mechanisms in different species, evidence supports the idea that as few as one or two genes may often be sufficient to initiate isolation. Thus, new mutation can theoretically play a key role in the process. But it has long been thought that a new isolation mutation would fail, because there would be no other individuals for the isolation-mutation-carrier to mate with. We now realize that premeiotic mutations are very common and will yield a cluster of progeny carrying the same new mutant allele. In this paper, we discuss the evidence for genetically simple premating isolation barriers and the role that clusters of an isolation mutation may play in initiating allopatric, and even sympatric, species divisions.

  8. Construction and characterization of mutations at codon 751 of the Escherichia coli gyrB gene that confer resistance to the antimicrobial peptide microcin B17 and alter the activity of DNA gyrase.

    Science.gov (United States)

    del Castillo, F J; del Castillo, I; Moreno, F

    2001-03-01

    Microcin B17 is a peptide antibiotic that inhibits DNA replication in Escherichia coli by targeting DNA gyrase. Previously, two independently isolated microcin B17-resistant mutants were shown to harbor the same gyrB point mutation that results in the replacement of tryptophan 751 by arginine in the GyrB polypeptide. We used site-directed mutagenesis to construct mutants in which tryptophan 751 was deleted or replaced by other amino acids. These mutants exhibit altered DNA gyrase activity and different levels of resistance to microcin B17.

  9. Analysis of RAS oncogene mutations in human lymphoid malignancies.

    OpenAIRE

    Neri, A.; Knowles, D M; Greco, A.; McCormick, F; Dalla-Favera, R

    1988-01-01

    We investigated the frequency of mutations activating RAS oncogenes in human lymphoid malignancies, including B- and T-cell-derived acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. By the polymerase chain reaction/oligonucleotide hybridization method, DNA from 178 cases was analyzed for activating mutations involving codons 12 and 61 of the HRAS, KRAS and NRAS genes and codon 13 of the NRAS gene. Mutations involving codons 12 or 13 of the NRAS gene were de...

  10. Mutations in Escherichia coli that relieve catabolite repression of tryptophanase synthesis. Tryptophanase promoter-like mutations.

    Science.gov (United States)

    Ward, D F; Yudkin, M D

    1976-01-01

    From a strain lacking adenyl cyclase and the catabolite-sensitive gene activator protein, two mutants were isolated that can synthesize tryptophanase. Each mutation is extremely closely linked to the tryptophanase structural gene. The mutations differ from one another in the rate of synthesis of tryptophanase that they permit in the genetic background in which they were isolated; they differ from one another and also from the wild type in the maximum rate of synthesis of tryptophanase that they permit in a genetic background with intact adenyl cyclase and catabolite-sensitive gene activator protein. Both mutations appear to lie in the tryptophanase promoter.

  11. Inherited cardiomyopathies caused by troponin mutations

    Institute of Scientific and Technical Information of China (English)

    Qun-Wei Lu; Xiao-Yan Wu; Sachio Morimoto

    2013-01-01

    Genetic investigations of cardiomyopathy in the recent two decades have revealed a large number of mutations in the genes encoding sarcomeric proteins as a cause of inherited hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), or restrictive cardiomyopathy (RCM). Most functional analyses of the effects of mutations on cardiac muscle contraction have revealed significant changes in the Ca2+-regulatory mechanism, in which cardiac troponin (cTn) plays important structural and functional roles as a key regulatory protein. Over a hundred mutations have been identified in all three subunits of cTn, i.e., cardiac troponins T, I, and C. Recent studies on cTn mutations have provided plenty of evidence that HCM- and RCM-linked mutations increase cardiac myofilament Ca2+ sensitivity, while DCM-linked mutations decrease it. This review focuses on the functional consequences of mutations found in cTn in terms of cardiac myofilament Ca2+ sensitivity, ATPase activity, force generation, and cardiac troponin I phosphorylation, to understand potential molecular and cellular pathogenic mechanisms of the three types of inherited cardiomyopathy.

  12. Predicting Resistance Mutations Using Protein Design Algorithms

    Energy Technology Data Exchange (ETDEWEB)

    Frey, K.; Georgiev, I; Donald, B; Anderson, A

    2010-01-01

    Drug resistance resulting from mutations to the target is an unfortunate common phenomenon that limits the lifetime of many of the most successful drugs. In contrast to the investigation of mutations after clinical exposure, it would be powerful to be able to incorporate strategies early in the development process to predict and overcome the effects of possible resistance mutations. Here we present a unique prospective application of an ensemble-based protein design algorithm, K*, to predict potential resistance mutations in dihydrofolate reductase from Staphylococcus aureus using positive design to maintain catalytic function and negative design to interfere with binding of a lead inhibitor. Enzyme inhibition assays show that three of the four highly-ranked predicted mutants are active yet display lower affinity (18-, 9-, and 13-fold) for the inhibitor. A crystal structure of the top-ranked mutant enzyme validates the predicted conformations of the mutated residues and the structural basis of the loss of potency. The use of protein design algorithms to predict resistance mutations could be incorporated in a lead design strategy against any target that is susceptible to mutational resistance.

  13. Mutational robustness of gene regulatory networks.

    Directory of Open Access Journals (Sweden)

    Aalt D J van Dijk

    Full Text Available Mutational robustness of gene regulatory networks refers to their ability to generate constant biological output upon mutations that change network structure. Such networks contain regulatory interactions (transcription factor-target gene interactions but often also protein-protein interactions between transcription factors. Using computational modeling, we study factors that influence robustness and we infer several network properties governing it. These include the type of mutation, i.e. whether a regulatory interaction or a protein-protein interaction is mutated, and in the case of mutation of a regulatory interaction, the sign of the interaction (activating vs. repressive. In addition, we analyze the effect of combinations of mutations and we compare networks containing monomeric with those containing dimeric transcription factors. Our results are consistent with available data on biological networks, for example based on evolutionary conservation of network features. As a novel and remarkable property, we predict that networks are more robust against mutations in monomer than in dimer transcription factors, a prediction for which analysis of conservation of DNA binding residues in monomeric vs. dimeric transcription factors provides indirect evidence.

  14. ATM Mutations in Cancer: Therapeutic Implications.

    Science.gov (United States)

    Choi, Michael; Kipps, Thomas; Kurzrock, Razelle

    2016-08-01

    Activation of checkpoint arrest and homologous DNA repair are necessary for maintenance of genomic integrity during DNA replication. Germ-line mutations of the ataxia telangiectasia mutated (ATM) gene result in the well-characterized ataxia telangiectasia syndrome, which manifests with an increased cancer predisposition, including a 20% to 30% lifetime risk of lymphoid, gastric, breast, central nervous system, skin, and other cancers. Somatic ATM mutations or deletions are commonly found in lymphoid malignancies, as well as a variety of solid tumors. Such mutations may result in chemotherapy resistance and adverse prognosis, but may also be exploited by existing or emerging targeted therapies that produce synthetic lethal states. Mol Cancer Ther; 15(8); 1781-91. ©2016 AACR. PMID:27413114

  15. Structural Effects of Oncogenic PI3K alpha Mutations

    Energy Technology Data Exchange (ETDEWEB)

    S Gabelli; C Huang; D Mandelker; O Schmidt-Kittler; B Vogelstein; L Amzel

    2011-12-31

    Physiological activation of PI3K{alpha} is brought about by the release of the inhibition by p85 when the nSH2 binds the phosphorylated tyrosine of activated receptors or their substrates. Oncogenic mutations of PI3K{alpha} result in a constitutively activated enzyme that triggers downstream pathways that increase tumor aggressiveness and survival. Structural information suggests that some mutations also activate the enzyme by releasing p85 inhibition. Other mutations work by different mechanisms. For example, the most common mutation, His1047Arg, causes a conformational change that increases membrane association resulting in greater accessibility to the substrate, an integral membrane component. These effects are examples of the subtle structural changes that result in increased activity. The structures of these and other mutants are providing the basis for the design of isozyme-specific, mutation-specific inhibitors for individualized cancer therapies.

  16. Mutations in Lettuce Improvement

    OpenAIRE

    2012-01-01

    Lettuce is a major vegetable in western countries. Mutations generated genetic variations and played an important role in the domestication of the crop. Many traits derived from natural and induced mutations, such as dwarfing, early flowering, male sterility, and chlorophyll deficiency, are useful in physiological and genetic studies. Mutants were also used to develop new lettuce products including miniature and herbicide-tolerant cultivars. Mutant analysis was critical in lettuce genomic stu...

  17. Mutation breeding in peas

    International Nuclear Information System (INIS)

    The pea as an ancient crop plant still today has wide uses and is an import source of food protein. It is also an important object for genetic studies and as such has been widely used in mutation induction experiments. However, in comparison with cereals this ancient crop plant (like several other grain legumes) has gained relatively little from advances in breeding. The review focuses on the prospects of genetic improvement of pea by induced mutations, discusses principles and gives methodological information. (author)

  18. Mutation Breeding in Sugarcane

    International Nuclear Information System (INIS)

    The present position of sugar industry particularly cane sugar production in the world has been discussed. The role of African Countries which can contribute more than the present 11% to world cane sugar production is presented. The breeding methods employed in cane growing court-tries indicate the biparental crossing and selection in F1 has been the major method used to develop varieties. Due to cytogenetical peculiarities, thousands of seedlings are grown to select the desirable genotype. Mutations or sports has been a source of variation for selection in nature. Induced mutations have only enhanced the mutation rate and has enabled the plant breeders to get better variation for selection. Though many mutagens have been used gamma rays have been most effective. Induced mutations for nonflowering, spineless leaf-sheath, higher sugar content, yield md resistance to diseases like smut and downy mildew have been reported. The methods of making mutated tissues express itself have been indicated. Mutation breeding holds out promise in sugarcane in that the basic variety or genotype can be kept intact and a few characters changed as desired by the plant breeder provided proper selection methods are employed. (author)

  19. Subquivers of mutation-acyclic quivers are mutation-acyclic

    CERN Document Server

    Warkentin, Matthias

    2011-01-01

    Quiver mutation plays a crucial role in the definition of cluster algebras by Fomin and Zelevinsky. It induces an equivalence relation on the set of all quivers without loops and two-cycles. A quiver is called mutation-acyclic if it is mutation-equivalent to an acyclic quiver. The aim of this note is to show that full subquivers of mutation-acyclic quivers are mutation-acyclic.

  20. 溶解氧突变指数对活性污泥重金属中毒的预警%Early Warning of Heavy Metal Poisoning of Activated Sludge Indicated with Dissolved Oxygen Mutation Index

    Institute of Scientific and Technical Information of China (English)

    陈亚松; 杜郁; 杨广文; 赵文玉; 陈振国

    2014-01-01

    以溶解氧(DO)突变为活性污泥中毒的预警指标,研究Cu2+、Cr6+、Pb2+、As5+4种重金属对活性污泥的毒性,并对预警活性污泥重金属中毒的测试参数进行优化。结果表明在相同的反应条件和重金属浓度(15 mg/L)下,4种重金属抑制活性污泥活性导致DO突变量分别为0.87、0.6、0.4和0.75 mg/L,其顺序为Cu2+> As5+> Cr6+> Pb2+,活性污泥对重金属铜中毒更加敏感。当As5+和Cr6+浓度由3 mg/L提高至15 mg/L时,活性污泥的DO突变量分别由0.32和0.27 mg/L上升至0.75和0.53 mg/L,表明随进水中As5+、Cr6+浓度增加,对活性污泥抑制性越强,存在剂量效应关系,因此溶解氧突变可作为活性污泥重金属离子中毒的判别指标,快速预警污水处理厂进水重金属引发活性污泥中毒与否,为污水处理厂稳定运行提供技术支持。%The early warning of activated sludge poisoned by heavy metals as Cu2+,Cr6+,Pb2+and As5+ was studied with dissolved oxygen mutation index. The parameters of early warning of activated sludge poisoning were optimized. With a certain condition and common dosage of heavy metals (15 mg/L),the dissolved oxygen mutation by these heavy metals are 0. 87,0. 6,0. 4 and 0. 75 mg/L,which indicates the biological inhibition order is Cu2+ >As5+ >Cr6+ >Pb2+. When concentrations of As5+and Cr6+ increase from 3 mg/L to 15 mg/L,the dissolved oxygen mutations increase from 0. 32,0. 27 mg/L up to 0. 75 and 0. 53 mg/L,respectively. With a rising dosage of heavy metals,the activated sludge ’s poison measured by dissolved oxygen mutation index is enhanced. Thus,the dissolved oxygen mutation index can be used as early warning of the activated sludge poisoned by heavy metals in wastewater treatment plant.

  1. Gamma-ray-induced dominant mutations that cause skeletal abnormalities in mice. II. Description of proved mutations

    Energy Technology Data Exchange (ETDEWEB)

    Selby, P.B.; Selby, P.R.

    1978-01-01

    In a mutation-rate experiment described earlier, 31 dominant skeletal mutations were confirmed by breeding tests. Skeletel abnormalities were detected in the skeletons of some of the sons of irradiated males, and for 31 of these sons the study of skeletons in subsequent generations showed that they transmitted abnormalities. The detailed descriptions of these mutations, together with descriptions of 6 presumed mutations found in a later paper, provide the basis for determining which mutations cause effects that would, if they occurred in humans, cause a serious handicap. Such a determination is necessary before these data can be used to estimate genetic hazard to humans. Furthermore, these descriptions of syndromes caused by individual dominant mutations should be useful to clinicians interested in skeletal defects. The statistical analysis of the frequency of each abnormality in the mutant line versus an approximation of the frequency of the malformation in the absence of new mutations is essential to be sure that a mutation is indeed the cause of each abnormality. These analyses, together with analyses of the correlation of abnormalities caused by individual mutations, clearly demonstrate that dominant mutations exhibit low penetrance for many of their effects. A few of the mutations also cause the death of some heterozygotes. No externally visible effects have been detected in heterozygotes for most of these mutations. Externally visible effects found in some of the heterozygotes for a few of the mutations include hydrocephalus, circling behavior, increased nervous activity, ray coat color, webbing of digits, and small size. Two coat-color mutations were found that caused no detected skeletal abnormalities. The data suggest that a few of the mutations may be reciprocal translocations. In most of the mutant lines tested cytologically, however, there was no indication of chromosomal aberrations.

  2. KRAS Mutations in Canine and Feline Pancreatic Acinar Cell Carcinoma.

    Science.gov (United States)

    Crozier, C; Wood, G A; Foster, R A; Stasi, S; Liu, J H W; Bartlett, J M S; Coomber, B L; Sabine, V S

    2016-07-01

    Companion animals may serve as valuable models for studying human cancers. Although KRAS is the most commonly mutated gene in human ductal pancreatic cancers (57%), with mutations frequently occurring at codons 12, 13 and 61, human pancreatic acinar cell carcinomas (ACCs) lack activating KRAS mutations. In the present study, 32 pancreatic ACC samples obtained from 14 dogs and 18 cats, including seven metastases, were analyzed for six common activating KRAS mutations located in codons 12 (n = 5) and 13 (n = 1) using Sequenom MassARRAY. No KRAS mutations were found, suggesting that, similar to human pancreatic ACC, KRAS mutations do not play a critical role in feline or canine pancreatic ACC. Due to the similarity of the clinical disease in dogs and cats to that of man, this study confirms that companion animals offer potential as a suitable model for investigating this rare subtype of pancreatic carcinoma. PMID:27290644

  3. Frequent GNAS mutations in low-grade appendiceal mucinous neoplasms

    OpenAIRE

    Nishikawa, G; Sekine, S; Ogawa, R; Matsubara, A.; Mori, T; Taniguchi, H; Kushima, R; Hiraoka, N.; Tsuta, K; Tsuda, H.; Kanai, Y.

    2013-01-01

    Background: The molecular basis for the development of appendiceal mucinous tumours, which can be a cause of pseudomyxoma peritonei, remains largely unknown. Methods: Thirty-five appendiceal mucinous neoplasms were analysed for GNAS and KRAS mutations. A functional analysis of mutant GNAS was performed using a colorectal cancer cell line. Results: A mutational analysis identified activating GNAS mutations in 16 of 32 low-grade appendiceal mucinous neoplasms (LAMNs) but in none of three mucino...

  4. Mutations in the P1 promoter region of Micromonospora echinospora.

    OpenAIRE

    Lin, L S; Rothstein, D. M.

    1992-01-01

    We demonstrated previously that the promoters P1a, P1b, and P1c are very closely spaced and are coordinately turned on during stationary phase in Micromonospora echinospora. To determine the nucleotides important for promoter recognition, we characterized mutations that were defective in transcription from the P1b start site, using Streptomyces lividans as the host. Two mutations upstream of the start site resulted in a drastic loss of promoter activity, while two mutations downstream of the ...

  5. Complementation analysis of eleven tryptophanase mutations in Escherichia coli.

    Science.gov (United States)

    White, M K; Yudkin, M D

    1979-10-01

    Nine independent mutants deficient in tryptophanase activity were isolated. Each mutation was transferred to a specialized transducing phage that carries the tryptophanase region of the Escherichia coli chromosome. The nine phages thus produced, and a tenth carrying a previously characterized tryptophanase mutation, were used to lysogenize a bacterial strain harbouring a mutation in the tryptophanase structural gene and also a suppressor of polarity. In no case was complementation observed; we conclude that there is no closely linked positive regulatory gene for tryptophanase.

  6. Mutational analysis of the encephalomyocarditis virus primary cleavage.

    OpenAIRE

    Hahn, H.; Palmenberg, A C

    1996-01-01

    Sixteen substitution mutations of the conserved DvExNPGP sequence, implicated in cardiovirus and aphthovirus primary polyprotein cleavage, were created in encephalomyocarditis virus cDNA, expressed, and characterized for processing activity. Nearly all the mutations severely decreased the efficiency of the primary cleavage reaction during cell-free synthesis of viral precursors, indicating a stringent requirement for the natural sequence in this processing event. When representative mutations...

  7. Recurrent NRAS mutations in pulmonary Langerhans cell histiocytosis.

    Science.gov (United States)

    Mourah, Samia; How-Kit, Alexandre; Meignin, Véronique; Gossot, Dominique; Lorillon, Gwenaël; Bugnet, Emmanuelle; Mauger, Florence; Lebbe, Celeste; Chevret, Sylvie; Tost, Jörg; Tazi, Abdellatif

    2016-06-01

    The mitogen-activated protein kinase (MAPK) pathway is constantly activated in Langerhans cell histiocytosis (LCH). Mutations of the downstream kinases BRAF and MAP2K1 mediate this activation in a subset of LCH lesions. In this study, we attempted to identify other mutations which may explain the MAPK activation in nonmutated BRAF and MAP2K1 LCH lesions.We analysed 26 pulmonary and 37 nonpulmonary LCH lesions for the presence of BRAF, MAP2K1, NRAS and KRAS mutations. Grossly normal lung tissue from 10 smoker patients was used as control. Patient spontaneous outcomes were concurrently assessed.BRAF(V600E) mutations were observed in 50% and 38% of the pulmonary and nonpulmonary LCH lesions, respectively. 40% of pulmonary LCH lesions harboured NRAS(Q61K) (/R) mutations, whereas no NRAS mutations were identified in nonpulmonary LCH biopsies or in lung tissue control. In seven out of 11 NRAS(Q61K) (/R)-mutated pulmonary LCH lesions, BRAF(V600) (E) mutations were also present. Separately genotyping each CD1a-positive area from the same pulmonary LCH lesion demonstrated that these concurrent BRAF and NRAS mutations were carried by different cell clones. NRAS(Q61K) (/R) mutations activated both the MAPK and AKT (protein kinase B) pathways. In the univariate analysis, the presence of concurrent BRAF(V600E) and NRAS(Q61K) (/R) mutations was significantly associated with patient outcome.These findings highlight the importance of NRAS genotyping of pulmonary LCH lesions because the use of BRAF inhibitors in this context may lead to paradoxical disease progression. These patients might benefit from MAPK kinase inhibitor-based treatments. PMID:27076591

  8. Mutations in lettuce improvement.

    Science.gov (United States)

    Mou, Beiquan

    2011-01-01

    Lettuce is a major vegetable in western countries. Mutations generated genetic variations and played an important role in the domestication of the crop. Many traits derived from natural and induced mutations, such as dwarfing, early flowering, male sterility, and chlorophyll deficiency, are useful in physiological and genetic studies. Mutants were also used to develop new lettuce products including miniature and herbicide-tolerant cultivars. Mutant analysis was critical in lettuce genomic studies including identification and cloning of disease-resistance genes. Mutagenesis combined with genomic technology may provide powerful tools for the discovery of novel gene alleles. In addition to radiation and chemical mutagens, unconventional approaches such as tissue or protoplast culture, transposable elements, and space flights have been utilized to generate mutants in lettuce. Since mutation breeding is considered nontransgenic, it is more acceptable to consumers and will be explored more in the future for lettuce improvement. PMID:22287955

  9. Silting mutation in triangulated categories

    CERN Document Server

    Aihara, Takuma

    2010-01-01

    In representation theory of algebras the notion of `mutation' often plays important roles, and two cases are well known, i.e. `cluster tilting mutation' and `exceptional mutation'. In this paper we focus on `tilting mutation', which has a disadvantage that it is often impossible, i.e. some of summands of a tilting object can not be replaced to get a new tilting object. The aim of this paper is to take away this disadvantage by introducing `silting mutation' for silting objects as a generalization of `tilting mutation'. We shall develope a basic theory of silting mutation. In particular, we introduce a partial order on the set of silting objects and establish the relationship with `silting mutation' by generalizing the theory of Riedmann-Schofield and Happel-Unger. We show that iterated silting mutation act transitively on the set of silting objects for local, hereditary or canonical algebras. Finally we give a bijection between silting subcategories and certain t-structures.

  10. MUTATIONS IN CALMODULIN GENES

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to an isolated polynucleotide encoding at least a part of calmodulin and an isolated polypeptide comprising at least a part of a calmodulin protein, wherein the polynucleotide and the polypeptide comprise at least one mutation associated with a cardiac disorder. The ...... the binding of calmodulin to ryanodine receptor 2 and use of such compound in a treatment of an individual having a cardiac disorder. The invention further provides a kit that can be used to detect specific mutations in calmodulin encoding genes....

  11. Are There Mutator Polymerases?

    Directory of Open Access Journals (Sweden)

    Miguel Garcia-Diaz

    2003-01-01

    Full Text Available DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.

  12. Artificial Bee Colony with Different Mutation Schemes: A comparative study

    Directory of Open Access Journals (Sweden)

    Iyad Abu Doush

    2014-03-01

    Full Text Available Artificial Bee Colony (ABC is a swarm-based metaheuristic for continuous optimization. Recent work hybridized this algorithm with other metaheuristics in order to improve performance. The work in this paper, experimentally evaluates the use of different mutation operators with the ABC algorithm. The introduced operator is activated according to a determined probability called mutation rate (MR. The results on standard benchmark function suggest that the use of this operator improves performance in terms of convergence speed and quality of final obtained solution. It shows that Power and Polynomial mutations give best results. The fastest convergence was for the mutation rate value (MR=0.2.

  13. A common Greenlandic Inuit BRCA1 RING domain founder mutation

    DEFF Research Database (Denmark)

    Hansen, T.v.O.; Ejlertsen, B.; Albrechtsen, Anders;

    2009-01-01

    of the families had members with ovarian cancer, suggesting that the RING domain may be an ovarian cancer hotspot. By SNP array analysis, we find that all 13 families share a 4.5 Mb genomic fragment containing the BRCA1 gene, showing that the mutation originates from a founder. Finally, analysis of 1152 Inuit......Germ-line mutations in the tumour suppressor proteins BRCA1 and BRCA2 predispose to breast and ovarian cancer. We examined 32 breast and/or ovarian cancer patients from Greenland for mutations in BRCA1 and BRCA2. Whereas no mutations were identified in 19 families, 13 families exhibited a BRCA1...... exon 3 nucleotide 234 T > G mutation, which has not previously been reported in the breast cancer information core (BIC) database. The mutation changes a conserved cysteine 39 to a glycine in the Zn(2+) site II of the RING domain, which is essential for BRCA1 ubiquitin ligase activity. Eight...

  14. How do oncoprotein mutations rewire protein-protein interaction networks?

    Science.gov (United States)

    Bowler, Emily H; Wang, Zhenghe; Ewing, Rob M

    2015-01-01

    The acquisition of mutations that activate oncogenes or inactivate tumor suppressors is a primary feature of most cancers. Mutations that directly alter protein sequence and structure drive the development of tumors through aberrant expression and modification of proteins, in many cases directly impacting components of signal transduction pathways and cellular architecture. Cancer-associated mutations may have direct or indirect effects on proteins and their interactions and while the effects of mutations on signaling pathways have been widely studied, how mutations alter underlying protein-protein interaction networks is much less well understood. Systematic mapping of oncoprotein protein interactions using proteomics techniques as well as computational network analyses is revealing how oncoprotein mutations perturb protein-protein interaction networks and drive the cancer phenotype. PMID:26325016

  15. Conversion from the "oncogene addiction" to "drug addiction" by intensive inhibition of the EGFR and MET in lung cancer with activating EGFR mutation.

    Science.gov (United States)

    Suda, Kenichi; Tomizawa, Kenji; Osada, Hirotaka; Maehara, Yoshihiko; Yatabe, Yasushi; Sekido, Yoshitaka; Mitsudomi, Tetsuya

    2012-06-01

    Emergence of acquired resistance is virtually inevitable in patients with a mutation in the epidermal growth factor receptor gene (EGFR) treated with EGFR tyrosine kinase inhibitors (TKIs). Several novel TKIs that may prevent or overcome the resistance mechanisms are now under clinical development. However, it is unknown how tumor cells will respond to intensive treatment using these novel TKIs. We previously established HCC827EPR cells, which are T790M positive, through combined treatment with erlotinib and a MET-TKI from erlotinib-hypersensitive HCC827 cells. In this study, we treated HCC827EPR cells sequentially with an irreversible EGFR-TKI, CL-387,785, to establish resistant cells (HCC827CLR), and we analyzed the mechanisms responsible for resistance. In HCC827CLR cells, PTEN expression was downregulated and Akt phosphorylation persisted in the presence of CL-387,785. Akt inhibition restored CL-387,785 sensitivity. In addition, withdrawal of CL-387,785 reduced cell viability in HCC827CLR cells, indicating that these cells were "addicted" to CL-387,785. HCC827CLR cells overexpressed the EGFR, and inhibition of the EGFR or MEK-ERK was needed to maintain cell proliferation. Increased senescence was observed in HCC827CLR cells in the drug-free condition. Through long-term culture of HCC827CLR cells without CL-387,785, we established HCC827-CL-387,785-independent cells, which exhibited decreased EGFR expression and a mesenchymal phenotype. In conclusion, PTEN downregulation is a newly identified mechanism underlying the acquired resistance to irreversible EGFR-TKIs after acquisition of T790M against erlotinib. This series of experiments highlights the flexibility of cancer cells that have adapted to environmental stresses induced by intensive treatment with TKIs. PMID:22133747

  16. An androgen receptor mutation in the MDA-MB-453 cell line model of molecular apocrine breast cancer compromises receptor activity.

    Science.gov (United States)

    Moore, Nicole L; Buchanan, Grant; Harris, Jonathan M; Selth, Luke A; Bianco-Miotto, Tina; Hanson, Adrienne R; Birrell, Stephen N; Butler, Lisa M; Hickey, Theresa E; Tilley, Wayne D

    2012-08-01

    Recent evidence indicates that the estrogen receptor-α-negative, androgen receptor (AR)-positive molecular apocrine subtype of breast cancer is driven by AR signaling. The MDA-MB-453 cell line is the prototypical model of this breast cancer subtype; its proliferation is stimulated by androgens such as 5α-dihydrotestosterone (DHT) but inhibited by the progestin medroxyprogesterone acetate (MPA) via AR-mediated mechanisms. We report here that the AR gene in MDA-MB-453 cells contains a G-T transversion in exon 7, resulting in a receptor variant with a glutamine to histidine substitution at amino acid 865 (Q865H) in the ligand binding domain. Compared with wild-type AR, the Q865H variant exhibited reduced sensitivity to DHT and MPA in transactivation assays in MDA-MB-453 and PC-3 cells but did not respond to non-androgenic ligands or receptor antagonists. Ligand binding, molecular modeling, mammalian two-hybrid and immunoblot assays revealed effects of the Q865H mutation on ligand dissociation, AR intramolecular interactions, and receptor stability. Microarray expression profiling demonstrated that DHT and MPA regulate distinct transcriptional programs in MDA-MB-453 cells. Gene Set Enrichment Analysis revealed that DHT- but not MPA-regulated genes were associated with estrogen-responsive transcriptomes from MCF-7 cells and the Wnt signaling pathway. These findings suggest that the divergent proliferative responses of MDA-MB-453 cells to DHT and MPA result from the different genetic programs elicited by these two ligands through the AR-Q865H variant. This work highlights the necessity to characterize additional models of molecular apocrine breast cancer to determine the precise role of AR signaling in this breast cancer subtype. PMID:22719059

  17. Mutations in antiquitin in individuals with pyridoxine-dependent seizures.

    NARCIS (Netherlands)

    Mills, P.B.; Struys, E.A.; Jakobs, C.; Plecko, B.; Baxter, P.; Baumgartner, M.; Willemsen, M.A.A.P.; Omran, H.; Tacke, U.; Uhlenberg, B.; Weschke, B.; Clayton, P.T.

    2006-01-01

    We show here that children with pyridoxine-dependent seizures (PDS) have mutations in the ALDH7A1 gene, which encodes antiquitin; these mutations abolish the activity of antiquitin as a delta1-piperideine-6-carboxylate (P6C)-alpha-aminoadipic semialdehyde (alpha-AASA) dehydrogenase. The accumulating

  18. Kin Selection - Mutation Balance

    DEFF Research Database (Denmark)

    Dyken, J. David Van; Linksvayer, Timothy Arnold; Wade, Michael J.

    2011-01-01

    selection-mutation balance, which provides an evolutionary null hypothesis for the statics and dynamics of cheating. When social interactions have linear fitness effects and Hamilton´s rule is satisfied, selection is never strong enough to eliminate recurrent cheater mutants from a population, but cheater...

  19. Neomorphic Mutations in PIK3R1 Confer Sensitivity to MAPK Inhibitors due to Activation of ERK and JNK Pathways | Office of Cancer Genomics

    Science.gov (United States)

    In a recent publication in Cancer Cell, CTD2 investigators discovered that a known cancer-associated gain-of-function alteration in phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) results in novel protein activity that confers sensitivity to mitogen-activated protein kinase (MAPK) inhibitors. The PIK3R1 gene encodes the p85α regulatory subunit of PIK3. Under normal conditions, p85α suppresses PIK3 mediated activation of downstream pathways that promote cell growth and survival.

  20. Positive Control Mutations in the MyoD Basic Region Fail to Show Cooperative DNA Binding and Transcriptional Activation in vitro

    Science.gov (United States)

    Bengal, Eyal; Flores, Osvaldo; Rangarajan, Pundi N.; Chen, Amy; Weintraub, Harold; Verma, Inder M.

    1994-06-01

    An in vitro transcription system from HeLa cells has been established in which MyoD and E47 proteins activate transcription both as homodimers and heterodimers. However, heterodimers activate transcription more efficiently than homodimers, and function synergistically from multiple binding sites. Positive control mutants in the basic region of MyoD that have previously been shown to be defective in initiating the myogenic program, can bind DNA but have lost their ability to function as transcriptional activators in vitro. Additionally, positive control mutants, unlike wild-type MyoD, fail to bind cooperatively to DNA. We propose that binding of MyoD complexes to high affinity MyoD binding sites induces conformational changes that facilitate cooperative binding to multiple sites and promote transcriptional activation.

  1. Evolutionary Stability Against Multiple Mutations

    CERN Document Server

    Ghatak, Anirban; Shaiju, A J

    2012-01-01

    It is known (see e.g. Weibull (1995)) that ESS is not robust against multiple mutations. In this article, we introduce robustness against multiple mutations and study some equivalent formulations and consequences.

  2. Mutation breeding newsletter. No. 45

    International Nuclear Information System (INIS)

    This issue of the Mutation Breeding newsletter contains 39 articles dealing with radiation induced mutations and chemical mutagenesis techniques in plant breeding programs with the aims of improving crop productivity and disease resistance as well as exploring genetic variabilities

  3. Mutation breeding newsletter. No. 33

    International Nuclear Information System (INIS)

    This issue of the newsletter reports a number of research news and research abstracts on application of radiation induced mutation techniques to increase mutagenesis and mutation frequency in plant breeding projects

  4. BRAF mutation in hairy cell leukemia

    Directory of Open Access Journals (Sweden)

    Ahmad Ahmadzadeh

    2014-09-01

    Full Text Available BRAF is a serine/threonine kinase with a regulatory role in the mitogen-activated protein kinase (MAPK signaling pathway. A mutation in the RAF gene, especially in BRAF protein, leads to an increased stimulation of this cascade, causing uncontrolled cell division and development of malignancy. Several mutations have been observed in the gene coding for this protein in a variety of human malignancies, including hairy cell leukemia (HCL. BRAF V600E is the most common mutation reported in exon15 of BRAF, which is observed in almost all cases of classic HCL, but it is negative in other B-cell malignancies, including the HCL variant. Therefore it can be used as a marker to differentiate between these B-cell disorders. We also discuss the interaction between miRNAs and signaling pathways, including MAPK, in HCL. When this mutation is present, the use of BRAF protein inhibitors may represent an effective treatment. In this review we have evaluated the role of the mutation of the BRAF gene in the pathogenesis and progression of HCL.

  5. Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro.

    Directory of Open Access Journals (Sweden)

    Yan Li

    Full Text Available RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH and ratchet domain. How the structural domains (arch, WH and ratchet domain coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.

  6. Understanding TERT Promoter Mutations: A Common Path to Immortality.

    Science.gov (United States)

    Bell, Robert J A; Rube, H Tomas; Xavier-Magalhães, Ana; Costa, Bruno M; Mancini, Andrew; Song, Jun S; Costello, Joseph F

    2016-04-01

    Telomerase (TERT) activation is a fundamental step in tumorigenesis. By maintaining telomere length, telomerase relieves a main barrier on cellular lifespan, enabling limitless proliferation driven by oncogenes. The recently discovered, highly recurrent mutations in the promoter ofTERTare found in over 50 cancer types, and are the most common mutation in many cancers. Transcriptional activation ofTERT, via promoter mutation or other mechanisms, is the rate-limiting step in production of active telomerase. AlthoughTERTis expressed in stem cells, it is naturally silenced upon differentiation. Thus, the presence ofTERTpromoter mutations may shed light on whether a particular tumor arose from a stem cell or more differentiated cell type. It is becoming clear thatTERTmutations occur early during cellular transformation, and activate theTERTpromoter by recruiting transcription factors that do not normally regulateTERTgene expression. This review highlights the fundamental and widespread role ofTERTpromoter mutations in tumorigenesis, including recent progress on their mechanism of transcriptional activation. These somatic promoter mutations, along with germline variation in theTERTlocus also appear to have significant value as biomarkers of patient outcome. Understanding the precise molecular mechanism ofTERTactivation by promoter mutation and germline variation may inspire novel cancer cell-specific targeted therapies for a large number of cancer patients.Mol Cancer Res; 14(4); 315-23. ©2016 AACR. PMID:26941407

  7. Novel Mutations in the PC Gene in Patients with Type B Pyruvate Carboxylase Deficiency

    DEFF Research Database (Denmark)

    Ostergaard, Elsebet; Duno, Morten; Møller, Lisbeth Birk;

    2013-01-01

    We have investigated seven patients with the type B form of pyruvate carboxylase (PC) deficiency. Mutation analysis revealed eight mutations, all novel. In a patient with exon skipping on cDNA analysis, we identified a homozygous mutation located in a potential branch point sequence, the first...... possible branch point mutation in PC. Two patients were homozygous for missense mutations (with normal protein amounts on western blot analysis), and two patients were homozygous for nonsense mutations. In addition, a duplication of one base pair was found in a patient who also harboured a splice site...... mutation. Another splice site mutation led to the activation of a cryptic splice site, shown by cDNA analysis.All patients reported until now with at least one missense mutation have had the milder type A form of PC deficiency. We thus report for the first time two patients with homozygous missense...

  8. Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation.

    Science.gov (United States)

    Lochhead, Pamela A; Clark, Jonathan; Wang, Lan-Zhen; Gilmour, Lesley; Squires, Matthew; Gilley, Rebecca; Foxton, Caroline; Newell, David R; Wedge, Stephen R; Cook, Simon J

    2016-01-01

    ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS(G12C/G13D) or BRAF(V600E). Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.

  9. Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation

    Science.gov (United States)

    Lochhead, Pamela A.; Clark, Jonathan; Wang, Lan-Zhen; Gilmour, Lesley; Squires, Matthew; Gilley, Rebecca; Foxton, Caroline; Newell, David R.; Wedge, Stephen R.; Cook, Simon J.

    2016-01-01

    Abstract ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRASG12C/G13D or BRAFV600E. Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target. PMID:26959608

  10. Rapid identification of HEXA mutations in Tay-Sachs patients.

    Science.gov (United States)

    Giraud, Carole; Dussau, Jeanne; Azouguene, Emilie; Feillet, François; Puech, Jean-Philippe; Caillaud, Catherine

    2010-02-19

    Tay-Sachs disease (TSD) is a recessively inherited neurodegenerative disorder due to mutations in the HEXA gene resulting in a beta-hexosaminidase A (Hex A) deficiency. The purpose of this study was to characterize the molecular abnormalities in patients with infantile or later-onset forms of the disease. The complete sequencing of the 14 exons and flanking regions of the HEXA gene was performed with a unique technical condition in 10 unrelated TSD patients. Eleven mutations were identified, including five splice mutations, one insertion, two deletions and three single-base substitutions. Four mutations were novel: two splice mutations (IVS8+5G>A, IVS2+4delAGTA), one missense mutation in exon 6 (c.621T>G (p.D207E)) and one small deletion (c.1211-1212delTG) in exon 11 resulting in a premature stop codon at residue 429. The c.621T>G missense mutation was found in a patient presenting an infantile form. Its putative role in the pathogenesis of TSD is suspected as residue 207 is highly conserved in human, mouse and rat. Moreover, structural modelling predicted changes likely to affect substrate binding and catalytic activity of the enzyme. The time-saving procedure reported here could be useful for the characterization of Tay-Sachs-causing mutations, in particular in non-Ashkenazi patients mainly exhibiting rare mutations. PMID:20100466

  11. Mutation breeding in pepper

    International Nuclear Information System (INIS)

    Pepper (Capsicum sp.) is an important vegetable and spice crop widely grown in tropical as well as in temperate regions. Until recently the improvement programmes were based mainly on using natural sources of germ plasma, crossbreeding and exploiting the heterosis of F1 hybrids. However, interest in using induced mutations is growing. A great number of agronomically useful mutants as well as mutants valuable for genetic, cytological and physiological studies have been induced and described. In this review information is presented about suitable mutagen treatment procedures with radiation as well as chemicals, M1 effects, handling the treated material in M1, M2 and subsequent generations, and mutant screening procedures. This is supplemented by a description of reported useful mutants and released cultivars. Finally, general advice is given on when and how to incorporate mutation induction in Capsicum improvement programmes. (author)

  12. Mutation of Auslander generators

    CERN Document Server

    Lada, Magdalini

    2009-01-01

    Let $\\Lambda$ be an artin algebra with representation dimension equal to three and $M$ an Auslander generator of $\\Lambda$. We show how, under certain assumptions, we can mutate $M$ to get a new Auslander generator whose endomorphism ring is derived equivalent to the endomorphism ring of $M$. We apply our results to selfinjective algebras with radical cube zero of infinite representation type, where we construct an infinite set of Auslander generators.

  13. The prevalence of PIK3CA mutations in gastric and colon cancer

    NARCIS (Netherlands)

    Velho, S; Oliveira, C; Ferreira, A; Ferreira, AC; Suriano, G; Schwartz, S; Duval, A; Carneiro, F; Machado, JC; Hamelin, R; Seruca, R

    2005-01-01

    A wide variety of tumours show PIK3CA mutations leading to increased phosphatidylinositol-3 kinase (PI3K) activity. We have determined the frequency of PIK3CA mutations in exons 9 and 20 that has previously been reported as mutational hotspot regions in distinct tumour models. One hundred and fifty

  14. Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

    Science.gov (United States)

    Boortz, Kayla A; Syring, Kristen E; Pound, Lynley D; Wang, Yingda; Oeser, James K; O'Brien, Richard M

    2016-01-01

    Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans. PMID:27611587

  15. Mucopolysaccharidosis IVA mutations in Chinese patients: 16 novel mutations.

    Science.gov (United States)

    Wang, Zheng; Zhang, Weimin; Wang, Yun; Meng, Yan; Su, Liang; Shi, Huiping; Huang, Shangzhi

    2010-08-01

    Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disease caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS) and transmitted as an autosomal recessive trait. This is the first systematic mutation screen in Chinese MPS IVA patients. Mutation detections in 24 unrelated Chinese MPS IVA patients were performed by PCR and direct sequencing of exons or the mRNA of GALNS. A total of 42 mutant alleles were identified, belonging to 27 different mutations. Out of the 27 mutations, 16 were novel, including 2 splicing mutations (c.567-1G>T and c.634-1G>A), 2 nonsense mutations (p.W325X and p.Q422X) and 12 missense mutations (p.T88I, p.H142R, p.P163H, p.G168L, p.H236D, p.N289S, p.T312A, p.G316V, p.A324E, p.L366P, p.Q422K and p.F452L). p.G340D was found to be a common mutation in the Chinese MPS IVA patients, accounting for 16.7% of the total number of mutant alleles. The results show that the mutations in Chinese MPS IVA patients are also family specific but have a different mutation spectrum as compared to those of other populations.

  16. Rice breeding with induced mutations

    International Nuclear Information System (INIS)

    The Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture decided in 1964 to organize a co-ordinated research programme on the use of induced mutations in rice breeding. The programme was organized within the framework of activities of the International Rice Commission. This is a report of the Third Co-ordination Meeting of the participants, which was held in Taipei, 5-9 June 1967. As the projects, which together make up the co-ordinated programme, are at different stages of progress, the report contains a variety of papers including completed studies, field and progress reports, and highlights of the discussions with some additional recommendations prepared by the participants. Refs, figs and tabs

  17. The Phenylmethylthiazolylthiourea Nonnucleoside Reverse Transcriptase (RT) Inhibitor MSK-076 Selects for a Resistance Mutation in the Active Site of Human Immunodeficiency Virus Type 2 RT

    OpenAIRE

    Auwerx, Joeri; Stevens, Miguel; Van Rompay, An R.; Bird, Louise E; Ren, Jingshan; De Clercq, Erik; Öberg, Bo; Stammers, David K.; Karlsson, Anna; Balzarini, Jan

    2004-01-01

    The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 μM) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 μM. As was also found for HIV-1 RT, MSK-076 inhibited HIV-2 RT in a noncompetitive manner with respect to dGT...

  18. Analysis of the matrix metalloproteinase family reveals that MMP8 is often mutated in melanoma

    OpenAIRE

    Palavalli, Lavanya H.; Prickett, Todd D.; Wunderlich, John R.; Wei, Xiaomu; Burrell, Allison S.; Porter-Gill, Patricia; Davis, Sean; Wang, Chenwei; Cronin, Julia C.; Agrawal, Neena S.; Lin, Jimmy C.; Westbroek, Wendy; Hoogstraten-Miller, Shelley; Molinolo, Alfredo A; Fetsch, Patricia

    2009-01-01

    A mutational analysis of the matrix metalloproteinase (MMP) gene family in human melanoma identified somatic mutations in 23% of melanomas. Five mutations in one of the most commonly mutated genes, MMP8, reduced MMP enzyme activity. Expression of wild-type but not mutant MMP8 in human melanoma cells inhibited growth on soft agar in vitro and tumor formation in vivo, suggesting that wild-type MMP-8 has the ability to inhibit melanoma progression.

  19. Mutation of Residue Arginine18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in Chlamydomonas reinhardtii

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    It has been known that arginine is used as the basic amino acid in the ?subunit of cytochrome b559 (Cyt b559) except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem (PS) Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSII complexes. In the present study,two arginine18 (R18) mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid (E) and glycine (G). The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type (WT) levels and that, after irradiation at high light intensity, oxygen evolution for the PSII of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSII (Fv/Fm) of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the ?subunit of Cyt b559 and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt b559 maintain the structure of the PSII complex and its activity,although it is not directly bound to the heme group.

  20. Multiple consequences of a single amino acid pathogenic RTK mutation: the A391E mutation in FGFR3.

    Directory of Open Access Journals (Sweden)

    Fenghao Chen

    Full Text Available The A391E mutation in fibroblast growth factor receptor 3 (FGFR3 is the genetic cause for Crouzon syndrome with Acanthosis Nigricans. Here we investigate the effect of this mutation on FGFR3 activation in HEK 293 T cells over a wide range of fibroblast growth factor 1 concentrations using a physical-chemical approach that deconvolutes the effects of the mutation on dimerization, ligand binding, and efficiency of phosphorylation. It is believed that the mutation increases FGFR3 dimerization, and our results verify this. However, our results also demonstrate that the increase in dimerization is not the sole effect of the mutation, as the mutation also facilitates the phosphorylation of critical tyrosines in the activation loop of FGFR3. The activation of mutant FGFR3 is substantially increased due to a combination of these two effects. The low expression of the mutant, however, attenuates its signaling and may explain the mild phenotype in Crouzon syndrome with Acanthosis Nigricans. The results presented here provide new knowledge about the physical basis behind growth disorders and highlight the fact that a single RTK mutation may affect multiple steps in RTK activation.

  1. KITLG Mutations Cause Familial Progressive Hyper- and Hypopigmentation

    DEFF Research Database (Denmark)

    Amyere, Mustapha; Vogt, Thomas; Hoo, Joe;

    2011-01-01

    by familial café-au-lait spots and skin fold freckling, caused by mutations in SPRED1. We performed a genome-wide linkage analysis in seven families with FPHH, and identified linkage on 12q21.12-q22, which overlaps with the DUH2 locus. We investigated whether KITLG in the locus is mutated in FPHH. We...... discovered three different mutations in four families. A reported FPH substitution was observed in two FPHH families, and two, to our knowledge, previously unreported substitutions, p.Val33Ala and p.Thr34Pro, cosegregated with FPHH in two separate families. All three mutations were located in a conserved β......-strand in KITLG, suggesting its important role in the activation of the KITLG receptor c-Kit. In aggregate, mutations in a single gene cause various pigmentation disorders: FPH, FPHH, and likely DUH2. Therefore, KITLG is an important modulator of skin pigmentation.Journal of Investigative Dermatology advance...

  2. Evaluation of the antibiotic activity and genetic mutation of microorganisms in the effluent treated with the electron-beam from waste-water treatment plant

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dong Hun; Nam, Ji Hyun; Shin, Ji Hye; Yun, Seo Yeon; Cho, Young Cheol; Oh, Kyoung hee [Chungbuk National University, Cheongju (Korea, Republic of)

    2011-04-15

    In this study, the residual concentrations and activities of antibiotics after UV or gamma-ray treatments were estimated, and the effect of irradiation of UV, gamma-ray, or electron beam was estimated on the survivability and less mutagenic effect on bacteria. The changes of bacterial communities and radiation resistant population in the effluent treated with UV and electron-beam were analyzed. The gamma-ray irradiation was more effective than UV in degradation of antibiotics. The extent of mutagenicity of electron-beam irradiation was less than those of UV or gamma-ray irradiations. The application of election-beam to the wastewater treatment system showed the high efficiency of destroying and removal effects on bacterial cells. The selective increase in population of radiation resistant bacteria was not observed. These results indicate that the application of ionizing radiation to the processes of wastewater treatment system will be suitable than UV irradiation because of its degradability of variable antibiotics, high removal rate of harmful bacteria, less mutagenicity of bacteria, and low selective effect on radiation resistant bacteria

  3. Evaluation of the antibiotic activity and genetic mutation of microorganisms in the effluent treated with the electron-beam from waste-water treatment plant

    International Nuclear Information System (INIS)

    In this study, the residual concentrations and activities of antibiotics after UV or gamma-ray treatments were estimated, and the effect of irradiation of UV, gamma-ray, or electron beam was estimated on the survivability and less mutagenic effect on bacteria. The changes of bacterial communities and radiation resistant population in the effluent treated with UV and electron-beam were analyzed. The gamma-ray irradiation was more effective than UV in degradation of antibiotics. The extent of mutagenicity of electron-beam irradiation was less than those of UV or gamma-ray irradiations. The application of election-beam to the wastewater treatment system showed the high efficiency of destroying and removal effects on bacterial cells. The selective increase in population of radiation resistant bacteria was not observed. These results indicate that the application of ionizing radiation to the processes of wastewater treatment system will be suitable than UV irradiation because of its degradability of variable antibiotics, high removal rate of harmful bacteria, less mutagenicity of bacteria, and low selective effect on radiation resistant bacteria

  4. Mutation breeding newsletter and reviews. No. 1

    International Nuclear Information System (INIS)

    Since the first issue of Mutation Breeding Newsletter (MBNL) was born in May 1972, and her sister Mutation Breeding Review (MBR) ten years later, there were 46 issues of MBNL and 13 MBR published, both MBNL and MBR, being the only specialised publications on mutation breeding worldwide. Our contributors and readers have enthusiastically supported them. During the past half century, mutation induction has matured from a focal research area to sophisticated technologies in modern plant improvement. Doubtlessly, these two publications played unique and important roles in fostering the development and application of mutation techniques in plant research, germplasm innovation, and new variety development. We are indebted to our predecessors at Plant Breeding and Genetics Section, and especially to Dr Alexander Micke, to have born and raised these publications and fostered their spread. In 1998, a new newsletter, Plant Breeding and Genetic Newsletter (PBGN), became a regular bulletin, covering all activities in our Section. Therefore, a major function of the MBNL was largely replaced by PBGN. On the other hand, the FAO/IAEA Mutant Variety Database (http://www-mvd.iaea.org/MVD/default.htm) has also taken over some functions of these two publications. Because of this and other reasons, we are facing a dwindling number of suitable submissions from outside for these two periodicals, and MBNL and MBR have become irregular publications since 2001. Even though, we, as well as many of you, contributors and readers, still believe that these two publications have reason to exist, but to keep them alive, significant evolution is inevitable. During the recent decade, mutation techniques are no longer used only as a tool for crop improvement of traditional traits, e.g. yield, resistance to disease and pests, but more frequently for diversified uses of crop end-products, enhancing quality and nutritional values and tolerances to abiotic stresses. Thanks to the massive progress in

  5. HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib.

    Science.gov (United States)

    Yang, Guang; Buhrlage, Sara J; Tan, Li; Liu, Xia; Chen, Jie; Xu, Lian; Tsakmaklis, Nicholas; Chen, Jiaji G; Patterson, Christopher J; Brown, Jennifer R; Castillo, Jorge J; Zhang, Wei; Zhang, Xiaofeng; Liu, Shuai; Cohen, Philip; Hunter, Zachary R; Gray, Nathanael; Treon, Steven P

    2016-06-23

    Activating mutations in MYD88 are present in ∼95% of patients with Waldenström macroglobulinemia (WM), as well as other B-cell malignancies including activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). In WM, mutated MYD88 triggers activation of Bruton tyrosine kinase (BTK). Ibrutinib, a pleiotropic kinase inhibitor that targets BTK, is highly active in patients with mutated MYD88. We observed that mutated MYD88 WM and ABC DLBCL cell lines, as well as primary WM cells show enhanced hematopoietic cell kinase (HCK) transcription and activation, and that HCK is activated by interleukin 6 (IL-6). Over-expression of mutated MYD88 triggers HCK and IL-6 transcription, whereas knockdown of HCK reduced survival and attenuated BTK, phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in mutated MYD88 WM and/or ABC DLBCL cells. Ibrutinib and the more potent HCK inhibitor A419259, blocked HCK activation and induced apoptosis in mutated MYD88 WM and ABC DLBCL cells. Docking and pull-down studies confirmed that HCK was a target of ibrutinib. Ibrutinib and A419259 also blocked adenosine triphosphate binding to HCK, whereas transduction of mutated MYD88 expressing WM cells with a mutated HCK gatekeeper greatly increased the half maximal effective concentration for ibrutinib and A419259. The findings support that HCK expression and activation is triggered by mutated MYD88, supports the growth and survival of mutated MYD88 WM and ABC DLBCL cells, and is a direct target of ibrutinib. HCK represents a novel target for therapeutic development in MYD88-mutated WM and ABC DLBCL, and possibly other diseases driven by mutated MYD88. PMID:27143257

  6. Canavan disease: Mutations among Jewish and non-Jewish patients

    Energy Technology Data Exchange (ETDEWEB)

    Kaul, R.; Gao, G.P.; Aloya, M.; Balamurugan, K.; Petrosky, A.; Michals, K.; Matalon, R. (Miami Children' s Hospital, FL (United States))

    1994-07-01

    Canavan disease is an autosomal recessive leukodystrophy caused by the deficiency of aspartoacylase (ASPA). Sixty-four probands were analyzed for mutations in the ASPA gene. Three point-mutations-693C[yields]A, 854[yields]C, and 914C[yields]A-were identified in the coding sequence. The 693C[yields]A and 914C[yields]A base changes, resulting in nonsense tyr231[yields]ter and missense ala305[yields]glu mutations, respectively, lead to complete loss of ASPA activity in in vitro expression studies. The 854A[yields]C transversion converted glu to ala in codon 285. The glu285[yields]ala mutant ASPA has 2.5% of the activity expressed by the wild-type enzyme. A fourth mutation, 433 -2(A[yields]G) transition, was identified at the splice-acceptor site in intron 2. The splice-site mutation would lead to skipping of exon 3, accompanied by a frameshift, and thus would produce aberrant ASPA. Of the 128 unrelated Canavan chromosomes analyzed, 88 were from probands of Ashkenazi Jewish descent. The glu285[yields]ala mutation was predominant (82.9%) in this population, followed by the tyr231[yields]ter (14.8%) and 433 -2(A[yields]G) (1.1%) mutations. The three mutations account for 98.8% of the Canavan chromosomes of Ashkenazi Jewish origin. The ala305-[yields]glu mutation was found exclusively in non-Jewish probands of European descent and constituted 60% of the 40 mutant chromosomes. Predominant occurrence of certain mutations among Ashkenazi Jewish and non-Jewish patients with Canavan disease would suggest a founding-father effect in propagation of these mutant chromosomes. 25 refs., 8 figs., 4 tabs.

  7. Muller's ratchet with compensatory mutations

    CERN Document Server

    Pfaffelhuber, Peter; Wakolbinger, Anton

    2011-01-01

    We consider an infinite dimensional system of stochastic differential equations which describes the evolution of type frequencies in a large population. Random reproduction is modeled by a Wright-Fisher noise whose inverse diffusion coefficient $N$ corresponds to the total population size. The type of an individual is the number $k$ of deleterious mutations it carries. We assume that fitness of individuals carrying $k$ mutations is decreased by $\\alpha k$ for some $\\alpha >0$. Along the individual lines of descent, (new) mutations accumulate at rate $\\lambda$ per generation, and each of these mutations has a small probability $\\gamma$ per generation to disappear. While the case $\\gamma =0 $ is known as (the Fleming-Viot version of) {\\em Muller's ratchet}, the case $\\gamma > 0$ is referred to as that of {\\em compensatory mutations} in the biological literature. In the former case ($\\gamma=0$), an ever increasing number of mutations is accumulated over time, while in the latter ($\\gamma > 0$) this is prevented ...

  8. Mutation Breeding Newsletter. No. 39

    International Nuclear Information System (INIS)

    This newsletter contains brief articles on the use of radiation to induce mutations in plants; radiation-induced mutants in Chrysanthemum; disrupting the association between oil and protein content in soybean seeds; mutation studies on bougainvillea; a new pepper cultivar; and the use of mutation induction to improve the quality of yam beans. A short review of the seminar on the use of mutation and related biotechnology for crop improvement in the Middle East and Mediterranean regions, and a description of a Co-ordinated Research Programme on the application of DNA-based marker mutations for the improvement of cereals and other sexually reproduced crop species are also included. Two tables are given: these are based on the ''FAO/IAEA Mutant Varieties Database'' and show the number of mutated varieties and the number of officially released mutant varieties in particular crops/species. Refs and tabs

  9. Filaggrin mutations and the skin.

    Science.gov (United States)

    De, Dipankar; Handa, Sanjeev

    2012-01-01

    Filaggrin is very important in the terminal differentiation of the skin and the formation of cornified envelope in the stratum corneum. Several mutations in the filaggrin gene have been identified in the last decade, mostly from the European countries. Loss of function mutations in the filaggrin gene results in reduced production of filaggrin, depending on the type and site of mutation. Such mutations in the filaggrin gene have been shown to be the most significant genetic risk factor for development of atopic dermatitis and undoubtedly has a role in the pathogenesis of ichthyosis vulgaris. Though there is theoretical possibility of association with hand eczema and allergic contact dermatitis; in clinical studies, the strength of these associations was not significantly strong. In this review, we have discussed the structure and function of filaggrin, basic genetics, type of mutations in filaggrin gene, and association of such mutations with different dermatoses.

  10. Filaggrin mutations and the skin

    Directory of Open Access Journals (Sweden)

    Dipankar De

    2012-01-01

    Full Text Available Filaggrin is very important in the terminal differentiation of the skin and the formation of cornified envelope in the stratum corneum. Several mutations in the filaggrin gene have been identified in the last decade, mostly from the European countries. Loss of function mutations in the filaggrin gene results in reduced production of filaggrin, depending on the type and site of mutation. Such mutations in the filaggrin gene have been shown to be the most significant genetic risk factor for development of atopic dermatitis and undoubtedly has a role in the pathogenesis of ichthyosis vulgaris. Though there is theoretical possibility of association with hand eczema and allergic contact dermatitis; in clinical studies, the strength of these associations was not significantly strong. In this review, we have discussed the structure and function of filaggrin, basic genetics, type of mutations in filaggrin gene, and association of such mutations with different dermatoses.

  11. Mutations and epimutations in the origin of cancer

    Energy Technology Data Exchange (ETDEWEB)

    Peltomaeki, Paeivi, E-mail: Paivi.Peltomaki@Helsinki.Fi

    2012-02-15

    Cancer is traditionally viewed as a disease of abnormal cell proliferation controlled by a series of mutations. Mutations typically affect oncogenes or tumor suppressor genes thereby conferring growth advantage. Genomic instability facilitates mutation accumulation. Recent findings demonstrate that activation of oncogenes and inactivation of tumor suppressor genes, as well as genomic instability, can be achieved by epigenetic mechanisms as well. Unlike genetic mutations, epimutations do not change the base sequence of DNA and are potentially reversible. Similar to genetic mutations, epimutations are associated with specific patterns of gene expression that are heritable through cell divisions. Knudson's hypothesis postulates that inactivation of tumor suppressor genes requires two hits, with the first hit occurring either in somatic cells (sporadic cancer) or in the germline (hereditary cancer) and the second one always being somatic. Studies on hereditary and sporadic forms of colorectal carcinoma have made it evident that, apart from genetic mutations, epimutations may serve as either hit or both. Furthermore, recent next-generation sequencing studies show that epigenetic genes, such as those encoding histone modifying enzymes and subunits for chromatin remodeling systems, are themselves frequent targets of somatic mutations in cancer and can act like tumor suppressor genes or oncogenes. This review discusses genetic vs. epigenetic origin of cancer, including cancer susceptibility, in light of recent discoveries. Situations in which mutations and epimutations occur to serve analogous purposes are highlighted.

  12. A common Greenlandic Inuit BRCA1 RING domain founder mutation

    DEFF Research Database (Denmark)

    Hansen, Thomas; Ejlertsen, Bent; Albrechtsen, Anders;

    2009-01-01

    Germ-line mutations in the tumour suppressor proteins BRCA1 and BRCA2 predispose to breast and ovarian cancer. We examined 32 breast and/or ovarian cancer patients from Greenland for mutations in BRCA1 and BRCA2. Whereas no mutations were identified in 19 families, 13 families exhibited a BRCA1 e...... possibility to reduce mortality in gene carriers, may warrant screening of the Greenlandic Inuit population. Provided screening is efficient, about 5% of breast- and 13% of ovarian cancers, respectively, may be prevented.......Germ-line mutations in the tumour suppressor proteins BRCA1 and BRCA2 predispose to breast and ovarian cancer. We examined 32 breast and/or ovarian cancer patients from Greenland for mutations in BRCA1 and BRCA2. Whereas no mutations were identified in 19 families, 13 families exhibited a BRCA1...... exon 3 nucleotide 234 T > G mutation, which has not previously been reported in the breast cancer information core (BIC) database. The mutation changes a conserved cysteine 39 to a glycine in the Zn(2+) site II of the RING domain, which is essential for BRCA1 ubiquitin ligase activity. Eight of the...

  13. Calreticulin Mutations in Myeloproliferative Neoplasms

    OpenAIRE

    Noa Lavi

    2014-01-01

    With the discovery of the JAK2V617F mutation in patients with Philadelphia chromosome-negative (Ph−) myeloproliferative neoplasms (MPNs) in 2005, major advances have been made in the diagnosis of MPNs, in understanding of their pathogenesis involving the JAK/STAT pathway, and finally in the development of novel therapies targeting this pathway. Nevertheless, it remains unknown which mutations exist in approximately one-third of patients with non-mutated JAK2 or MPL essential thrombocythemia (...

  14. Mutations induced in plant breeding

    Energy Technology Data Exchange (ETDEWEB)

    Barriga B, P. (Universidad Austral de Chile, Valdivia. Inst. de Produccion y Sanidad Vegetal)

    1984-10-01

    The most significant aspects of the use of ionizing radiations in plant breeding are reviewed. Aspects such as basic principles of mutation, expression and selection in obtention of mutants, methods for using induced mutations and sucess achieved with this methodology in plant breeding are reviewed. Results obtained in a program of induced mutation on wheat for high content of protein and lysine at the Universidad Austral de Chile are presented.

  15. Mutation breeding in mangosteen

    International Nuclear Information System (INIS)

    Mangosteen the queen of the tropical fruits is apomitic and only a cultivar is reported and it reproduces asexually. Conventional breeding is not possible and the other methods to create variabilities are through genetic engineering and mutation breeding. The former technique is still in the infantry stage in mangosteen research while the latter has been an established tool in breeding to improve cultivars. In this mutation breeding seeds of mangosteen were irradiated using gamma rays and the LD 50 for mangosteen was determined and noted to be very low at 10 Gy. After sowing in the seedbed, the seedlings were transplanted in polybags and observed in the nursery bed for about one year before planted in the field under old oil palm trees in Station MARDI, Kluang. After evaluation and screening, about 120 mutant mangosteen plants were selected and planted in Kluang. The plants were observed and some growth data taken. There were some mutant plants that have good growth vigour and more vigorous that the control plants. The trial are now in the fourth year and the plants are still in the juvenile stage. (Author)

  16. Mutation breeding in chickpea

    International Nuclear Information System (INIS)

    Chickpea is an important food legume in Turkey. Turkey is one of the most important gene centers in the world for legumes. Realizing the potential of induced mutations, a mutation breeding programme was initiated at the Nuclear Agriculture Section of the Saraykoy Nuclear Research and Training Center in 1994. The purpose of the study was to obtain high yielding chickpea mutants with large seeds, good cooking quality and high protein content. Beside this some characters such as higher adaptation ability, tolerant to cold and drought, increased machinery harvest type, higher yield, resistant to diseases especially to antracnose and pest were investigated too. Parent varieties were ILC-482, AK-7114 and AKCIN-91 had been used in these experiments. The irradiation doses were 0 (control), 50, 100, 150, 200, 250, 300, 350 and 400 Gy for field experiments, respectively. As a result of these experiments, two promising mutant lines were chosen and given to the Seed Registration and Certification Center for official registration These two promising mutants were tested at five different locations of Turkey, in 2004 and 2005 years. After 2 years of registration experiments one of outstanding mutants was officially released as mutant chickpea variety under the name TAEK-SAGEL, in 2006. Some basic characteristics of this mutant are; earliness (95-100 day), high yield capacity (180-220 kg/da), high seed protein (22-25 %), first pot height (20-25 cm), 100 seeds weight (42-48 g), cooking time (35-40 min) and resistance to Ascochyta blight.

  17. Tissue-specific mutation accumulation in human adult stem cells during life

    Science.gov (United States)

    Blokzijl, Francis; de Ligt, Joep; Jager, Myrthe; Sasselli, Valentina; Roerink, Sophie; Sasaki, Nobuo; Huch, Meritxell; Boymans, Sander; Kuijk, Ewart; Prins, Pjotr; Nijman, Isaac J.; Martincorena, Inigo; Mokry, Michal; Wiegerinck, Caroline L.; Middendorp, Sabine; Sato, Toshiro; Schwank, Gerald; Nieuwenhuis, Edward E. S.; Verstegen, Monique M. A.; van der Laan, Luc J. W.; de Jonge, Jeroen; Ijzermans, Jan N. M.; Vries, Robert G.; van de Wetering, Marc; Stratton, Michael R.; Clevers, Hans; Cuppen, Edwin; van Boxtel, Ruben

    2016-10-01

    The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.

  18. Driver Gene Mutations in Stools of Colorectal Carcinoma Patients Detected by Targeted Next-Generation Sequencing.

    Science.gov (United States)

    Armengol, Gemma; Sarhadi, Virinder K; Ghanbari, Reza; Doghaei-Moghaddam, Masoud; Ansari, Reza; Sotoudeh, Masoud; Puolakkainen, Pauli; Kokkola, Arto; Malekzadeh, Reza; Knuutila, Sakari

    2016-07-01

    Detection of driver gene mutations in stool DNA represents a promising noninvasive approach for screening colorectal cancer (CRC). Amplicon-based next-generation sequencing (NGS) is a good option to study mutations in many cancer genes simultaneously and from a low amount of DNA. Our aim was to assess the feasibility of identifying mutations in 22 cancer driver genes with Ion Torrent technology in stool DNA from a series of 65 CRC patients. The assay was successful in 80% of stool DNA samples. NGS results showed 83 mutations in cancer driver genes, 29 hotspot and 54 novel mutations. One to five genes were mutated in 75% of cases. TP53, KRAS, FBXW7, and SMAD4 were the top mutated genes, consistent with previous studies. Of samples with mutations, 54% presented concomitant mutations in different genes. Phosphatidylinositol 3-kinase/mitogen-activated protein kinase pathway genes were mutated in 70% of samples, with 58% having alterations in KRAS, NRAS, or BRAF. Because mutations in these genes can compromise the efficacy of epidermal growth factor receptor blockade in CRC patients, identifying mutations that confer resistance to some targeted treatments may be useful to guide therapeutic decisions. In conclusion, the data presented herein show that NGS procedures on stool DNA represent a promising tool to detect genetic mutations that could be used in the future for diagnosis, monitoring, or treating CRC. PMID:27155048

  19. Mutation breeding newsletter. No. 43

    International Nuclear Information System (INIS)

    This issue of the Newsletter includes articles dealing with radiation induced mutation based plant breeding research findings aimed at improving productivity, disease resistance and tolerance of stress conditions

  20. Guanine nucleotide dissociation inhibitor activity of the triple GoLoco motif protein G18: alanine-to-aspartate mutation restores function to an inactive second GoLoco motif.

    Science.gov (United States)

    Kimple, Randall J; Willard, Francis S; Hains, Melinda D; Jones, Miller B; Nweke, Gift K; Siderovski, David P

    2004-03-15

    GoLoco ('Galpha(i/o)-Loco' interaction) motif proteins have recently been identified as novel GDIs (guanine nucleotide dissociation inhibitors) for heterotrimeric G-protein alpha subunits. G18 is a member of the mammalian GoLoco-motif gene family and was uncovered by analyses of human and mouse genomes for anonymous open-reading frames. The encoded G18 polypeptide is predicted to contain three 19-amino-acid GoLoco motifs, which have been shown in other proteins to bind Galpha subunits and inhibit spontaneous nucleotide release. However, the G18 protein has thus far not been characterized biochemically. Here, we have cloned and expressed the G18 protein and assessed its ability to act as a GDI. G18 is capable of simultaneously binding more than one Galpha(i1) subunit. In binding assays with the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate, G18 exhibits GDI activity, slowing the exchange of GDP for GTP by Galpha(i1). Only the first and third GoLoco motifs within G18 are capable of interacting with Galpha subunits, and these bind with low micromolar affinity only to Galpha(i1) in the GDP-bound form, and not to Galpha(o), Galpha(q), Galpha(s) or Galpha12. Mutation of Ala-121 to aspartate in the inactive second GoLoco motif of G18, to restore the signature acidic-glutamine-arginine tripeptide that forms critical contacts with Galpha and its bound nucleotide [Kimple, Kimple, Betts, Sondek and Siderovski (2002) Nature (London) 416, 878-881], results in gain-of-function with respect to Galpha binding and GDI activity. PMID:14656218

  1. Neighborhood properties are important determinants of temperature sensitive mutations.

    Directory of Open Access Journals (Sweden)

    Svetlana Lockwood

    Full Text Available Temperature-sensitive (TS mutants are powerful tools to study gene function in vivo. These mutants exhibit wild-type activity at permissive temperatures and reduced activity at restrictive temperatures. Although random mutagenesis can be used to generate TS mutants, the procedure is laborious and unfeasible in multicellular organisms. Further, the underlying molecular mechanisms of the TS phenotype are poorly understood. To elucidate TS mechanisms, we used a machine learning method-logistic regression-to investigate a large number of sequence and structure features. We developed and tested 133 features, describing properties of either the mutation site or the mutation site neighborhood. We defined three types of neighborhood using sequence distance, Euclidean distance, and topological distance. We discovered that neighborhood features outperformed mutation site features in predicting TS mutations. The most predictive features suggest that TS mutations tend to occur at buried and rigid residues, and are located at conserved protein domains. The environment of a buried residue often determines the overall structural stability of a protein, thus may lead to reversible activity change upon temperature switch. We developed TS prediction models based on logistic regression and the Lasso regularized procedure. Through a ten-fold cross-validation, we obtained the area under the curve of 0.91 for the model using both sequence and structure features. Testing on independent datasets suggested that the model predicted TS mutations with a 50% precision. In summary, our study elucidated the molecular basis of TS mutants and suggested the importance of neighborhood properties in determining TS mutations. We further developed models to predict TS mutations derived from single amino acid substitutions. In this way, TS mutants can be efficiently obtained through experimentally introducing the predicted mutations.

  2. Hepatitis C virus induces a mutator phenotype: enhanced mutations of immunoglobulin and protooncogenes.

    Science.gov (United States)

    Machida, Keigo; Cheng, Kevin T-N; Sung, Vicky M-H; Shimodaira, Shigetaka; Lindsay, Karen L; Levine, Alexandra M; Lai, Ming-Yang; Lai, Michael M C

    2004-03-23

    Hepatitis C virus (HCV) is a nonretroviral oncogenic RNA virus, which is frequently associated with hepatocellular carcinoma (HCC) and B cell lymphoma. We demonstrated here that acute and chronic HCV infection caused a 5- to 10-fold increase in mutation frequency in Ig heavy chain, BCL-6, p53, and beta-catenin genes of in vitro HCV-infected B cell lines and HCV-associated peripheral blood mononuclear cells, lymphomas, and HCCs. The nucleotide-substitution pattern of p53 and beta-catenin was different from that of Ig heavy chain in HCV-infected cells, suggesting two different mechanisms of mutation. In addition, the mutated protooncogenes were amplified in HCV-associated lymphomas and HCCs, but not in lymphomas of nonviral origin or HBV-associated HCC. HCV induced error-prone DNA polymerase zeta, polymerase iota, and activation-induced cytidine deaminase, which together, contributed to the enhancement of mutation frequency, as demonstrated by the RNA interference experiments. These results indicate that HCV induces a mutator phenotype and may transform cells by a hit-and-run mechanism. This finding provides a mechanism of oncogenesis for an RNA virus.

  3. Canavan disease: mutations among Jewish and non-jewish patients.

    OpenAIRE

    Kaul, R; Gao, G. P.; Aloya, M.; K. Balamurugan; Petrosky, A.; Michals, K.; Matalon, R.

    1994-01-01

    Canavan disease is an autosomal recessive leukodystrophy caused by the deficiency of aspartoacylase (ASPA). Sixty-four probands were analyzed for mutations in the ASPA gene. Three point mutations--693C-->A, 854A-->C, and 914C-->A--were identified in the coding sequence. The 693C-->A and 914C-->A base changes, resulting in nonsense tyr231-->ter and missense ala305-->glu mutations, respectively, lead to complete loss of ASPA activity in in vitro expression studies. The 854A-->C transversion con...

  4. Cancer. TERT promoter mutations and telomerase reactivation in urothelial cancer.

    Science.gov (United States)

    Borah, Sumit; Xi, Linghe; Zaug, Arthur J; Powell, Natasha M; Dancik, Garrett M; Cohen, Scott B; Costello, James C; Theodorescu, Dan; Cech, Thomas R

    2015-02-27

    Reactivation of telomerase, the chromosome end-replicating enzyme, drives human cell immortality and cancer. Point mutations in the telomerase reverse transcriptase (TERT) gene promoter occur at high frequency in multiple cancers, including urothelial cancer (UC), but their effect on telomerase function has been unclear. In a study of 23 human UC cell lines, we show that these promoter mutations correlate with higher levels of TERT messenger RNA (mRNA), TERT protein, telomerase enzymatic activity, and telomere length. Although previous studies found no relation between TERT promoter mutations and UC patient outcome, we find that elevated TERT mRNA expression strongly correlates with reduced disease-specific survival in two independent UC patient cohorts (n = 35; n = 87). These results suggest that high telomerase activity may be a better marker of aggressive UC tumors than TERT promoter mutations alone. PMID:25722414

  5. Driver Mutations in Uveal Melanoma

    Science.gov (United States)

    Decatur, Christina L.; Ong, Erin; Garg, Nisha; Anbunathan, Hima; Bowcock, Anne M.; Field, Matthew G.; Harbour, J. William

    2016-01-01

    IMPORTANCE Frequent mutations have been described in the following 5 genes in uveal melanoma (UM): BAP1, EIF1AX, GNA11, GNAQ, and SF3B1. Understanding the prognostic significance of these mutations could facilitate their use in precision medicine. OBJECTIVE To determine the associations between driver mutations, gene expression profile (GEP) classification, clinicopathologic features, and patient outcomes in UM. DESIGN, SETTING, AND PARTICIPANTS Retrospective study of patients with UM treated by enucleation by a single ocular oncologist between November 1, 1998, and July 31, 2014. MAIN OUTCOMES AND MEASURES Clinicopathologic features, patient outcomes, GEP classification (class 1 or class 2), and mutation status were recorded. RESULTS The study cohort comprised 81 participants. Their mean age was 61.5 years, and 37% (30 of 81) were female. The GEP classification was class 1 in 35 of 81 (43%), class 2 in 42 of 81 (52%), and unknown in 4 of 81 (5%). BAP1 mutations were identified in 29 of 64 (45%), GNAQ mutations in 36 of 81 (44%), GNA11 mutations in 36 of 81 (44%), SF3B1 mutations in 19 of 81 (24%), and EIF1AX mutations in 14 of 81 (17%). Sixteen of the mutations in BAP1 and 6 of the mutations in EIF1AX were previously unreported in UM. GNAQ and GNA11 mutations were mutually exclusive. BAP1, SF3B1, and EIF1AX mutations were almost mutually exclusive with each other. Using multiple regression analysis, BAP1 mutations were associated with class 2 GEP and older patient. EIF1AX mutations were associated with class 1 GEP and the absence of ciliary body involvement. SF3B1 mutations were associated with younger patient age. GNAQ mutations were associated with the absence of ciliary body involvement and greater largest basal diameter. GNA11 mutations were not associated with any of the analyzed features. Using Cox proportional hazards modeling, class 2 GEP was the prognostic factor most strongly associated with metastasis (relative risk, 9.4; 95% CI, 3.1–28.5) and

  6. Mutation breeding in soybean

    International Nuclear Information System (INIS)

    In Indonesia, soybean is one of the important crop after rice. It is generally cultivated in the lowlands and rarely in the highlands. Seeds of soybean variety ORBA were treated with various doses of fast neutrons, gamma rays, EMS and NaN3 with the aims of studying the mutagen effects in M-1 and M-2 generations and also to select mutants adapted to highland conditions. D-50 doses for gamma rays, fast neutrons and EMS were around 23 krad, 2,300 rad, 0.3%, respectively. Much higher chlorophyll mutation frequency was observed in EMS treatment of 0.3%. Seven mutants were shorter and four early mutants matured from 4 to 20 days earlier than the control plants. Two early mutants were quite adaptable in both the low and highlands and produced better yields than the parental material. (author)

  7. Founder Mutations in Xeroderma Pigmentosum

    OpenAIRE

    Tamura, Deborah; DiGiovanna, John J.; Kraemer, Kenneth H.

    2010-01-01

    In this issue, Soufir et al. report a founder mutation in the XPC DNA repair gene in 74% of families with xeroderma pigmentosum (XP) in the Maghreb region (Algeria, Morocco, and Tunisia) of northern Africa. These patients have a high frequency of skin cancer. The presence of this founder mutation provides an opportunity for genetic counseling and early diagnosis of XP.

  8. Mucopolysaccharidosis type VII: Characterization of mutations and molecular heterogeneity

    OpenAIRE

    Tomatsu, Shunji; Fukuda, Seiji; Sukegawa, Kazuko; Ikedo, Yuko; Yamada, Shinji; Yamada, Yukiji; Sasaki, Toshiya; Okamoto, Hiroyuki; Kuwahara, Takashi; Yamaguchi, Seiji; Kiman, Tadashi; Shintaku, Haruo; Isshiki, Gen; Orii, Tadao

    1991-01-01

    We identified two different exonic point mutations causing β-glucuronidase (βGl) deficiency in two Japanese patients with mucopolysaccharidosis type VII (MPSVII). Enzyme assay of lysates of the lymphocytes and cultured fibroblasts showed little residual activity. The βGl-specific mRNA levels were normal, as determined by northern blot analysis. Mutated cDNA clones, including the entire coding sequence, were isolated using the polymerase chain reaction (PCR) products derived from βGl-deficient...

  9. Artificial Bee Colony with Different Mutation Schemes: A comparative study

    OpenAIRE

    Iyad Abu Doush; Hasan, Basima Hani F.; Mohammed Azmi Al-Betar; Eslam Al Maghayreh; Faisal Alkhateeb; Mohammad Hamdan

    2014-01-01

    Artificial Bee Colony (ABC) is a swarm-based metaheuristic for continuous optimization. Recent work hybridized this algorithm with other metaheuristics in order to improve performance. The work in this paper, experimentally evaluates the use of different mutation operators with the ABC algorithm. The introduced operator is activated according to a determined probability called mutation rate (MR). The results on standard benchmark function suggest that the use of this operator improves p...

  10. Une autre mondialisation : les mutations du blanchiment contemporain

    OpenAIRE

    Bernard Castelli

    2005-01-01

    In this article, we notice three major mutations which caracterize today the forms of concealing and integrating the illegal funds : the emergence of offshore finance centers, the formation of financial underground networks and the presence of skilled launderers represent the main trends of this illégal financial activity. It is very important to take into account these last mutations for understanding better and perhaps controling the recent development of the money laundering. The author co...

  11. Myeloproliferative neoplasms: From JAK2 mutations discovery to JAK2 inhibitor therapies

    OpenAIRE

    Passamonti, Francesco; Maffioli, Margherita; Caramazza, Domenica; Cazzola, Mario

    2011-01-01

    Most BCR-ABL1-negative myeloproliferative neoplasms (MPN) carry an activating JAK2 mutation. Approximately 96% of patients with polycythemia vera (PV) harbors the V617F mutation in JAK2 exon 14, whereas the minority of JAK2 (V617F)-negative subjects shows several mutations in exon 12. Other mutation events as MPL, TET2, LNK, EZH2 have been described in chronic phase, while NF1, IDH1, IDH2, ASX1, CBL and Ikaros in blast phase of MPN. The specific pathogenic implication of these mutations is un...

  12. Studies of human mutation rates, December 1, 1985--November 30, 1986

    International Nuclear Information System (INIS)

    This program seeks to quantify native human mutation rates and to determine how man's activities may affect these rates. The program is divided into six tasks, i.e. The American Indian mutation rate, monitoring populations for frequency of mutation by electrophoresis of blood proteins, application of molecular biological approaches to the detection and study of mutational events in human populations, development of two-dimensional electrophoresis for identification of mutant proteins, co-operative program with the Radiation Effects Research Foundation in Hiroshima and Nagasaki, Japan, and statistical problems associated with the estimation of mutation rates. Progress of each of the above tasks is related in detail. (DT)

  13. Two Case Reports of Rare BRAF Mutations in Exon 11 and Exon 15 with Discussion of Potential Treatment Options

    Directory of Open Access Journals (Sweden)

    Georg Richtig

    2016-09-01

    Full Text Available BRAF mutations occur in up to 50% of melanomas. Mutations in the BRAF gene directly influence the patient’s treatment because several inhibitors are available that only target BRAFV600 mutations. Herein, we describe two cases of patients with metastatic melanomas, each carrying a ‘nonstandard’ mutation in the BRAF gene: BRAFK601E and BRAFG466E, respectively. The first patient was treated with a MEK inhibitor and the second one with ipilimumab. However, not all BRAF mutations result in increased BRAF kinase activity, and clinical data for ‘nonstandard’ mutations, such as those described in our case report, are sparse. Therefore, treatment with MEK inhibitors can be helpful in cases where BRAF mutations result in increased activity, whereas immune checkpoint inhibitors might be used in cases where the mutations lead to activity levels below those of the wild type.

  14. Mutation-selection dynamics and error threshold in an evolutionary model for Turing Machines

    OpenAIRE

    Musso, Fabio; Feverati, Giovanni

    2011-01-01

    We investigate the mutation-selection dynamics for an evolutionary computation model based on Turing Machines that we introduced in a previous article. The use of Turing Machines allows for very simple mechanisms of code growth and code activation/inactivation through point mutations. To any value of the point mutation probability corresponds a maximum amount of active code that can be maintained by selection and the Turing machines that reach it are said to be at the error threshold. Simulat...

  15. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Krauthammer, Michael; Kong, Yong; Ha, Byung Hak; Evans, Perry; Bacchiocchi, Antonella; McCusker, James P.; Cheng, Elaine; Davis, Matthew J.; Goh, Gerald; Choi, Murim; Ariyan, Stephan; Narayan, Deepak; Dutton-Regester, Ken; Capatana, Ana; Holman, Edna C.; Bosenberg, Marcus; Sznol, Mario; Kluger, Harriet M.; Brash, Douglas E.; Stern, David F.; Materin, Miguel A.; Lo, Roger S.; Mane, Shrikant; Ma, Shuangge; Kidd, Kenneth K.; Hayward, Nicholas K.; Lifton, Richard P.; Schlessinger, Joseph; Boggon, Titus J.; Halaban, Ruth (Yale-MED); (UCLA); (Queens)

    2012-10-11

    We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1{sup P29S}) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1{sup P29S} showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

  16. Mutation in domain II of IAA1 confers diverse auxin-related phenotypes and represses auxin-activated expression of Aux/IAA genes in steroid regulator-inducible system.

    Science.gov (United States)

    Park, Jin-Young; Kim, Hye-Joung; Kim, Jungmook

    2002-12-01

    Most of Aux/IAA genes are rapidly induced by auxin. The Aux/IAA proteins are short-lived nuclear proteins sharing the four conserved domains. Domain II is critical for rapid degradation of Aux/IAA proteins. Among these gene family members, IAA1 is one of the earliest auxin-inducible genes. We used a steroid hormone-inducible system to reveal putative roles and downstream signaling of IAA1 in auxin response. Arabidopsis transgenic plants were generated expressing fusion protein of IAA1 (IAA1-GR) or IAA1 with a mutation in domain II (iaa1-GR) and the glucocorticoid hormone-binding domain (GR). IAA1-GR transgenic plants did not exhibit any discernable phenotypic differences by DEX treatment that allows nuclear translocation of the fusion protein. In contrast, diverse auxin-related physiological processes including gravitropism and phototropism were impaired by DEX treatment in roots, hypocotyls, stems, and leaves in iaa1-GR transgenic plants. Auxin induction of seven Aux/IAA mRNAs including IAA1 itself was repressed by DEX treatment, suggesting that IAA1 functions in the nucleus by mediating auxin response and might act as a negative feedback regulator for the expression of Aux/IAA genes including IAA1 itself. Auxin induction of Aux/IAA genes in the presence of cycloheximide can be repressed by DEX treatment, showing that the repression of transcription of the Aux/IAAs by the iaa1 mutant protein is primary. Wild-type IAA1-GR could not suppress auxin induction of IAA1 and IAA2. These results indicate that inhibition of auxin-activated transcription of Aux/IAA genes by the iaa1 mutant protein might be responsible for alteration of various auxin responses.

  17. The Gly482Ser Missense Mutation of the Peroxisome Proliferator-Activated Receptor γ Coactivator-1α (PGC-1α Gene Associates with Reduced Insulin Sensitivity in Normal and Glucose-Intolerant Obese Subjects

    Directory of Open Access Journals (Sweden)

    Marzia Fanelli

    2005-01-01

    Full Text Available Among the putative candidate genes for insulin resistance, the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α is a transcriptional coactivator of PPARγ and α, regulating a wide range of processes involved in energy production and utilization, such as thermogenesis, liver gluconeogenesis, glucose uptake in muscle. In population studies a Gly482Ser substitution in PGC-1α has been reported to be associated with increased risk of type diabetes 2 and insulin resistance. In the present study we have analysed the association between the Gly482Ser missense mutation of the PGC-1α gene and insulin sensitivity and glucose tolerance in a population of obese non-diabetic subjects. The Gly482Ser SNPs was detected by PCR-RFLP in a cohort of 358 Caucasian obese subjects (223 with normal glucose tolerance (NGT and 125 with impaired glucose tolerance (IGT. We observed a significant association (p < 0.007 between carriers of the Gly482Ser variant of the PGC-1α gene and insulin resistance measured by HOMAIR. Multivariate analysis confirmed that the Gly482Ser SNP was a significant (p < 0.02 determinant of decreased insulin sensitivity, independently from other well-known modulators of insulin action. In conclusion, we have found significant association between the Gly482Ser variant of the PGC-1α gene and reduced insulin sensitivity in obese subjects. This association resulted independent from all other known modulators of insulin resistance, and suggests a primary role for the PGC-1α gene on the genetic susceptibility to insulin resistance in obesity.

  18. Molecular characterization of two galactosemia mutations: correlation of mutations with highly conserved domains in galactose-1-phosphate uridyl transferase.

    OpenAIRE

    Reichardt, J K; Packman, S; Woo, S L

    1991-01-01

    Galactosemia is an autosomal recessive disorder of human galactose metabolism caused by deficiency of the enzyme galactose-1-phosphate uridyl transferase (GALT). The molecular basis of this disorder is at present not well understood. We report here two missense mutations which result in low or undetectable enzymatic activity. First, we identified at nucleotide 591 a transition which substitutes glutamine 188 by arginine. The mutated glutamine is not only highly conserved in evolution (conserv...

  19. Mutational meltdown in laboratory yeast populations

    NARCIS (Netherlands)

    Zeyl, C.; Mizesko, M.; Visser, de J.A.G.M.

    2001-01-01

    In small or repeatedly bottlenecked populations, mutations are expected to accumulate by genetic drift, causing fitness declines. In mutational meltdown models, such fitness declines further reduce population size, thus accelerating additional mutation accumulation and leading to extinction. Because

  20. BRAF V600 mutations and pathological features in Japanese melanoma patients.

    Science.gov (United States)

    Yamazaki, Naoya; Tanaka, Ryota; Tsutsumida, Arata; Namikawa, Kenjiro; Eguchi, Hironobu; Omata, Wataru; Oashi, Kohei; Ogawa, Toru; Hayashi, Amiko; Nakamura, Noriyuki; Tsuta, Koji

    2015-02-01

    Ultraviolet radiation is a risk factor for BRAF V600 mutations frequently found in melanomas that cause constitutive BRAF activation. Primary sites of melanoma and the frequency of BRAF mutations might differ between races. Melanoma is rare in Japan (1500-2000 cases/year compared with 132 000/year worldwide) and the frequency and distribution of BRAF V600 mutations are unknown. We aimed to investigate the frequency of BRAF V600 mutations in a cohort of Japanese patients with melanoma and determine the relationship between mutations and clinical/pathologic features. DNA was extracted from 80 formalin-fixed, paraffin-embedded tumours from individuals diagnosed with melanoma. BRAF V600 mutations were detected using the Cobas 4800 System with z480 Analyzer and Cobas 4800 BRAF V600 Mutation Test reagents. BRAF V600 mutations were detected in 41.8% of tested tumours, with an invalid rate of 1.3%. The mutation rate was more than 60% in patients aged less than 60 years and more than 36% in patients with stage III/IV disease. No sex difference in the mutation rate was observed. BRAF V600 mutations were detected in 18.8% of acral lentiginous melanomas (ALMs), 64.7% of superficial spreading melanomas, 50.0% of lentigo maligna melanomas and 20.0% of nodular melanomas. Although the mutation rate was low in ALMs, 36.4% were mutation positive at stage III/IV compared with 9.5% at stage I/II. This study confirmed associations among BRAF V600 mutations, pathological features and subtypes of melanoma. BRAF V600 mutations were more frequent in late-stage ALMs than in early-stage ALMs. Superficial spreading melanomas had similar mutation rates at all stages. These insights suggest improved treatment predictions for stage III/IV melanoma patients. PMID:25051202

  1. Mutational analysis of a ras catalytic domain

    DEFF Research Database (Denmark)

    Willumsen, B M; Papageorge, A G; Kung, H F;

    1986-01-01

    We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal...... transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic...

  2. Evolution of evolvability via adaptation of mutation rates.

    Science.gov (United States)

    Bedau, Mark A; Packard, Norman H

    2003-05-01

    We examine a simple form of the evolution of evolvability-the evolution of mutation rates-in a simple model system. The system is composed of many agents moving, reproducing, and dying in a two-dimensional resource-limited world. We first examine various macroscopic quantities (three types of genetic diversity, a measure of population fitness, and a measure of evolutionary activity) as a function of fixed mutation rates. The results suggest that (i) mutation rate is a control parameter that governs a transition between two qualitatively different phases of evolution, an ordered phase characterized by punctuated equilibria of diversity, and a disordered phase of characterized by noisy fluctuations around an equilibrium diversity, and (ii) the ability of evolution to create adaptive structure is maximized when the mutation rate is just below the transition between these two phases of evolution. We hypothesize that this transition occurs when the demands for evolutionary memory and evolutionary novelty are typically balanced. We next allow the mutation rate itself to evolve, and we observe that evolving mutation rates adapt to values at this transition. Furthermore, the mutation rates adapt up (or down) as the evolutionary demands for novelty (or memory) increase, thus supporting the balance hypothesis. PMID:12689727

  3. Mutation rates and the evolution of germline structure.

    Science.gov (United States)

    Scally, Aylwyn

    2016-07-19

    Genome sequencing studies of de novo mutations in humans have revealed surprising incongruities in our understanding of human germline mutation. In particular, the mutation rate observed in modern humans is substantially lower than that estimated from calibration against the fossil record, and the paternal age effect in mutations transmitted to offspring is much weaker than expected from our long-standing model of spermatogenesis. I consider possible explanations for these discrepancies, including evolutionary changes in life-history parameters such as generation time and the age of puberty, a possible contribution from undetected post-zygotic mutations early in embryo development, and changes in cellular mutation processes at different stages of the germline. I suggest a revised model of stem-cell state transitions during spermatogenesis, in which 'dark' gonial stem cells play a more active role than hitherto envisaged, with a long cycle time undetected in experimental observations. More generally, I argue that the mutation rate and its evolution depend intimately on the structure of the germline in humans and other primates.This article is part of the themed issue 'Dating species divergences using rocks and clocks'. PMID:27325834

  4. Distinct Viral and Mutational Spectrum of Endemic Burkitt Lymphoma.

    Directory of Open Access Journals (Sweden)

    Francesco Abate

    2015-10-01

    Full Text Available Endemic Burkitt lymphoma (eBL is primarily found in children in equatorial regions and represents the first historical example of a virus-associated human malignancy. Although Epstein-Barr virus (EBV infection and MYC translocations are hallmarks of the disease, it is unclear whether other factors may contribute to its development. We performed RNA-Seq on 20 eBL cases from Uganda and showed that the mutational and viral landscape of eBL is more complex than previously reported. First, we found the presence of other herpesviridae family members in 8 cases (40%, in particular human herpesvirus 5 and human herpesvirus 8 and confirmed their presence by immunohistochemistry in the adjacent non-neoplastic tissue. Second, we identified a distinct latency program in EBV involving lytic genes in association with TCF3 activity. Third, by comparing the eBL mutational landscape with published data on sporadic Burkitt lymphoma (sBL, we detected lower frequencies of mutations in MYC, ID3, TCF3 and TP53, and a higher frequency of mutation in ARID1A in eBL samples. Recurrent mutations in two genes not previously associated with eBL were identified in 20% of tumors: RHOA and cyclin F (CCNF. We also observed that polyviral samples showed lower numbers of somatic mutations in common altered genes in comparison to sBL specimens, suggesting dual mechanisms of transformation, mutation versus virus driven in sBL and eBL respectively.

  5. P16 UV mutations in human skin epithelial tumors.

    Science.gov (United States)

    Soufir, N; Molès, J P; Vilmer, C; Moch, C; Verola, O; Rivet, J; Tesniere, A; Dubertret, L; Basset-Seguin, N

    1999-09-23

    The p16 gene expresses two alternative transcripts (p16alpha and p16beta) involved in tumor suppression via the retinoblastoma (Rb) or p53 pathways. Disruption of these pathways can occur through inactivation of p16 or p53, or activating mutations of cyclin dependant kinase 4 gene (Cdk4). We searched for p16, Cdk4 and p53 gene mutations in 20 squamous cell carcinomas (SSCs), 1 actinic keratosis (AK), and 28 basal cell carcinomas (BCCs), using PCR-SSCP. A deletion and methylation analysis of p16 was also performed. Six different mutations (12%) were detected in exon 2 of p16 (common to p16alpha and p16beta), in five out of 21 squamous lesions (24%) (one AK and four SCCs) and one out of 28 BCCs (3.5%). These included four (66%) ultraviolet (UV)-type mutations (two tandems CC : GG to TT : AA transitions and two C : G to T : A transitions at dipyrimidic site) and two transversions. P53 mutations were present in 18 samples (37%), mostly of UV type. Of these, only two (one BCC and one AK) harboured simultaneously mutations of p16, but with no consequence on p16beta transcript. Our data demonstrate for the first time the presence of p16 UV induced mutations in non melanoma skin cancer, particularly in the most aggressive SCC type, and support that p16 and p53 are involved in two independent pathways in skin carcinogenesis.

  6. Mutations in PIK3CA are infrequent in neuroblastoma

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    Mazanek Pavel

    2006-07-01

    Full Text Available Abstract Background Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessed PIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as these have not been previously reported. Methods Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines were sequenced for PIK3CA activating mutations within the C2, helical and kinase domain "hot spots" where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations to assess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. Results We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%. Neither mutation (R524M and E982D has been studied to date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively. Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model

  7. Minisequencing mitochondrial DNA pathogenic mutations

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    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  8. Functional characterization of human cancer-derived TRKB mutations.

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    Thomas R Geiger

    Full Text Available Cancer originates from cells that have acquired mutations in genes critical for controlling cell proliferation, survival and differentiation. Often, tumors continue to depend on these so-called driver mutations, providing the rationale for targeted anticancer therapies. To date, large-scale sequencing analyses have revealed hundreds of mutations in human tumors. However, without their functional validation it remains unclear which mutations correspond to driver, or rather bystander, mutations and, therefore, whether the mutated gene represents a target for therapeutic intervention. In human colorectal tumors, the neurotrophic receptor TRKB has been found mutated on two different sites in its kinase domain (TRKB(T695I and TRKB(D751N. Another site, in the extracellular part of TRKB, is mutated in a human lung adenocarcinoma cell line (TRKB(L138F. Lastly, our own analysis has identified one additional TRKB point mutation proximal to the kinase domain (TRKB(P507L in a human melanoma cell line. The functional consequences of all these point mutations, however, have so far remained elusive. Previously, we have shown that TRKB is a potent suppressor of anoikis and that TRKB-expressing cells form highly invasive and metastatic tumors in nude mice. To assess the functional consequences of these four TRKB mutations, we determined their potential to suppress anoikis and to form tumors in nude mice. Unexpectedly, both colon cancer-derived mutants, TRKB(T695I and TRKB(D751N, displayed reduced activity compared to that of wild-type TRKB. Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK. The two mutants derived from human tumor cell lines (TRKB(L138F and TRKB(P507L were functionally indistinguishable from wild-type TRKB in both in-vitro and in-vivo assays. In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

  9. HNPCC: Six new pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Epplen Joerg T

    2004-06-01

    Full Text Available Abstract Background Hereditary non-polyposis colorectal cancer (HNPCC is an autosomal dominant disease with a high risk for colorectal and endometrial cancer caused by germline mutations in DNA mismatch-repair genes (MMR. HNPCC accounts for approximately 2 to 5% of all colorectal cancers. Here we present 6 novel mutations in the DNA mismatch-repair genes MLH1, MSH2 and MSH6. Methods Patients with clinical diagnosis of HNPCC were counselled. Tumor specimen were analysed for microsatellite instability and immunohistochemistry for MLH1, MSH2 and MSH6 protein was performed. If one of these proteins was not detectable in the tumor mutation analysis of the corresponding gene was carried out. Results We identified 6 frameshift mutations (2 in MLH1, 3 in MSH2, 1 in MSH6 resulting in a premature stop: two mutations in MLH1 (c.2198_2199insAACA [p.N733fsX745], c.2076_2077delTG [p.G693fsX702], three mutations in MSH2 (c.810_811delGT [p.C271fsX282], c.763_766delAGTGinsTT [p.F255fsX282], c.873_876delGACT [p.L292fsX298] and one mutation in MSH6 (c.1421_1422dupTG [p.C475fsX480]. All six tumors tested for microsatellite instability showed high levels of microsatellite instability (MSI-H. Conclusions HNPCC in families with MSH6 germline mutations may show an age of onset that is comparable to this of patients with MLH1 and MSH2 mutations.

  10. Radiation mutation breeding

    International Nuclear Information System (INIS)

    In order to develop an advanced technical knowledge for the selection of better mutants, some of the crops were irradiated and the mutation rate, the survival rate and the method for selction of a mutant were studied. Furthermore, this study aimed to obtain basic data applicable to the development of genetic resources by evaluation and analysis the specific character for selection of the superior mutant and its plant breeding. 1. selection of the mutant with a superior resistance against environment in the principal crops 1) New varieties of mutant rices such as Wonpyeongbyeo, Wongwangbyeo, Winmibyeo, and heogseon chalbeyeo (sticky forma) were registered in the national variety list and made an application to crop variety protection right. They are under review now. 2) We also keep on studying on the number of a grain of 8 lines of excellent mutant rice for the purpose of improvement of breeding . 3) We selected 3 lines which have a resistance to pod and stem blight in large soybean, 31 lines with small grain size and higher yield, 112 lines of soybean of cooking, 7 lines of low lipoxygenase content, and 12 lines with decreased phytic acid content by 20 % compared to the previous level. 2. Selection of advanced Mugunwha (Rose of Sharon) mutant 1) Bagseul, a new variety of mutant, was developed and 30 plantlets of it are being proliferated. 2) Fifty-three lines of a mutant having a various morphologies were selected

  11. Radiation mutation breeding

    Energy Technology Data Exchange (ETDEWEB)

    Song, Hi Sup; Kim, Jae Sung; Kim, Jin Kyu; Shin, In Chul; Lim, Young Taek

    1998-04-01

    In order to develop an advanced technical knowledge for the selection of better mutants, some of the crops were irradiated and the mutation rate, the survival rate and the method for selction of a mutant were studied. Furthermore, this study aimed to obtain basic data applicable to the development of genetic resources by evaluation and analysis the specific character for selection of the superior mutant and its plant breeding. 1. selection of the mutant with a superior resistance against environment in the principal crops 1) New varieties of mutant rices such as Wonpyeongbyeo, Wongwangbyeo, Winmibyeo, and heogseon chalbeyeo (sticky forma) were registered in the national variety list and made an application to crop variety protection right. They are under review now. 2) We also keep on studying on the number of a grain of 8 lines of excellent mutant rice for the purpose of improvement of breeding . 3) We selected 3 lines which have a resistance to pod and stem blight in large soybean, 31 lines with small grain size and higher yield, 112 lines of soybean of cooking, 7 lines of low lipoxygenase content, and 12 lines with decreased phytic acid content by 20 % compared to the previous level. 2. Selection of advanced Mugunwha (Rose of Sharon) mutant 1) Bagseul, a new variety of mutant, was developed and 30 plantlets of it are being proliferated. 2) Fifty-three lines of a mutant having a various morphologies were selected.

  12. Mutation breeding by ion implantation

    Science.gov (United States)

    Yu, Zengliang; Deng, Jianguo; He, Jianjun; Huo, Yuping; Wu, Yuejin; Wang, Xuedong; Lui, Guifu

    1991-07-01

    Ion implantation as a new mutagenic method has been used in the rice breeding program since 1986, and for mutation breeding of other crops later. It has been shown, in principle and in practice, that this method has many outstanding advantages: lower damage rate; higher mutation rate and wider mutational spectrum. Many new lines of rice with higher yield rate; broader disease resistance; shorter growing period but higher quality have been bred from ion beam induced mutants. Some of these lines have been utilized for the intersubspecies hybridization. Several new lines of cotton, wheat and other crops are now in breeding. Some biophysical effects of ion implantation for crop seeds have been studied.

  13. Induction of mutations in wheat

    International Nuclear Information System (INIS)

    Studies on toxicity of various mutagenic factors including gamma radiation, X-rays and fast neutrons and their optimal doses for valuble mutation breeding of bread winter, spring and durum wheat have been carried out. Data on sublethal doses of mutagens and chromosome aberration frequency for different doses of mutagens have been collected. It is observed that the greater general frequency of visible mutations by a mutagen does not ensure the greater relative yield of valuable mutations. In the course of the investigations, about 90 valuable mutant forms and mutant hybrids are selected for further breeding studies. (M.G.B.)

  14. Mutations induced by ultraviolet light

    Energy Technology Data Exchange (ETDEWEB)

    Pfeifer, Gerd P. [Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States)]. E-mail: gpfeifer@coh.org; You, Young-Hyun [Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States); Besaratinia, Ahmad [Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States)

    2005-04-01

    The different ultraviolet (UV) wavelength components, UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm), have distinct mutagenic properties. A hallmark of UVC and UVB mutagenesis is the high frequency of transition mutations at dipyrimidine sequences containing cytosine. In human skin cancers, about 35% of all mutations in the p53 gene are transitions at dipyrimidines within the sequence 5'-TCG and 5'-CCG, and these are localized at several mutational hotspots. Since 5'-CG sequences are methylated along the p53 coding sequence in human cells, these mutations may be derived from sunlight-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. Cyclobutane pyrimidine dimers (CPDs) form preferentially at dipyrimidines containing 5-methylcytosine when cells are irradiated with UVB or sunlight. In order to define the contribution of 5-methylcytosine to sunlight-induced mutations, the lacI and cII transgenes in mouse fibroblasts were used as mutational targets. After 254 nm UVC irradiation, only 6-9% of the base substitutions were at dipyrimidines containing 5-methylcytosine. However, 24-32% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of these mutations were transitions. Thus, CPDs forming preferentially at dipyrimidines with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells. Using mouse cell lines harboring photoproduct-specific photolyases and mutational reporter genes, we showed that CPDs (rather than 6-4 photoproducts or other lesions) are responsible for the great majority of UVB-induced mutations. An important component of UVB mutagenesis is the deamination of cytosine and 5-methylcytosine within CPDs. The mutational specificity of long-wave UVA (340-400 nm) is distinct from that of the shorter wavelength UV and is characterized mainly by G to T transversions presumably arising through mechanisms

  15. Lung Adenocarcinoma with Pulmonary Miliary Metastases and Complex Somatic Heterozygous EGFR Mutation

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    Alexandre Schaller

    2014-11-01

    Full Text Available The pretreatment detection of an activating mutation of EGFR is now routinely performed in metastatic nonsquamous non-small cell lung cancer (NSCLC. The therapeutic impact of such a detection is major, as patients with advanced NSCLC exhibiting a mutation of exon 19 or 21 will benefit from EGFR-tyrosine kinase inhibitors (TKI. The presence of an EGFR resistance mutation, such as T790M in EGFR-TKI-naïve patients, is seldom looked for and is related either to a germinal mutation or to somatically mutated subclones. It has a negative predictive impact. We present the case of a patient with a lung papillary adenocarcinoma and miliary intrapulmonary metastases whose tumor displays a somatic complex heterozygous EGFR mutation, combining L858R (exon 21 and a primary resistance mutation T790M (exon 20, both detected by direct sequencing.

  16. Breast cancer in BRCA mutation carriers: medical treatment.

    Science.gov (United States)

    Milani, Andrea; Geuna, Elena; Zucchini, Giorgia; Aversa, Caterina; Martinello, Rossella; Montemurro, Filippo

    2016-10-01

    About 10% of breast cancers are associated with the inheritance of autosomal dominant breast cancer susceptibility alleles BRCA1 and BRCA2. Until recently, the medical management of BRCA mutation-associated breast cancer has not differed from that of the sporadic breast cancer counterpart. However, there is mounting evidence that this molecular alteration confers sensitivity or resistance to systemic therapies that can be exploited in terms of medical management. For example, studies support the use of platinum salts chemotherapy in BRCA mutated cancers. Moreover, a number of targeted therapies are showing activity in BRCA mutation carriers. Above all, BRCA defective tumor cells are particularly sensitive to Poly(ADP-ribose) polymerase (PARP) inhibitors. This review will summarize the state of the art of the medical treatment of breast cancer in BRCA mutation carriers, with a particular focus on chemotherapies and targeted therapies. PMID:26799758

  17. Mutations in SLC26A1 Cause Nephrolithiasis.

    Science.gov (United States)

    Gee, Heon Yung; Jun, Ikhyun; Braun, Daniela A; Lawson, Jennifer A; Halbritter, Jan; Shril, Shirlee; Nelson, Caleb P; Tan, Weizhen; Stein, Deborah; Wassner, Ari J; Ferguson, Michael A; Gucev, Zoran; Sayer, John A; Milosevic, Danko; Baum, Michelle; Tasic, Velibor; Lee, Min Goo; Hildebrandt, Friedhelm

    2016-06-01

    Nephrolithiasis, a condition in which urinary supersaturation leads to stone formation in the urinary system, affects about 5%-10% of individuals worldwide at some point in their lifetime and results in significant medical costs and morbidity. To date, mutations in more than 30 genes have been described as being associated with nephrolithiasis, and these mutations explain about 15% of kidney stone cases, suggesting that additional nephrolithiasis-associated genes remain to be discovered. To identify additional genes whose mutations are linked to nephrolithiasis, we performed targeted next-generation sequencing of 18 hypothesized candidate genes in 348 unrelated individuals with kidney stones. We detected biallelic mutations in SLC26A1 (solute carrier family 26 member 1) in two unrelated individuals with calcium oxalate kidney stones. We show by immunofluorescence, immunoblotting, and glycosylation analysis that the variant protein mimicking p.Thr185Met has defects in protein folding or trafficking. In addition, by measuring anion exchange activity of SLC26A1, we demonstrate that all the identified mutations in SLC26A1 result in decreased transporter activity. Our data identify SLC26A1 mutations as causing a recessive Mendelian form of nephrolithiasis. PMID:27210743

  18. Whole-genome sequencing reveals oncogenic mutations in mycosis fungoides.

    Science.gov (United States)

    McGirt, Laura Y; Jia, Peilin; Baerenwald, Devin A; Duszynski, Robert J; Dahlman, Kimberly B; Zic, John A; Zwerner, Jeffrey P; Hucks, Donald; Dave, Utpal; Zhao, Zhongming; Eischen, Christine M

    2015-07-23

    The pathogenesis of mycosis fungoides (MF), the most common cutaneous T-cell lymphoma (CTCL), is unknown. Although genetic alterations have been identified, none are considered consistently causative in MF. To identify potential drivers of MF, we performed whole-genome sequencing of MF tumors and matched normal skin. Targeted ultra-deep sequencing of MF samples and exome sequencing of CTCL cell lines were also performed. Multiple mutations were identified that affected the same pathways, including epigenetic, cell-fate regulation, and cytokine signaling, in MF tumors and CTCL cell lines. Specifically, interleukin-2 signaling pathway mutations, including activating Janus kinase 3 (JAK3) mutations, were detected. Treatment with a JAK3 inhibitor significantly reduced CTCL cell survival. Additionally, the mutation data identified 2 other potential contributing factors to MF, ultraviolet light, and a polymorphism in the tumor suppressor p53 (TP53). Therefore, genetic alterations in specific pathways in MF were identified that may be viable, effective new targets for treatment.

  19. Ferredoxin Gene Mutation in Iranian Trichomonas Vaginalis Isolates

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    Soudabeh Heidari

    2013-09-01

    Full Text Available Background: Trichomonas vaginalis causes trichomoniasis and metronidazole is its chosen drug for treatment. Ferredoxin has role in electron transport and carbohydrate metabolism and the conversion of an inactive form of metronidazole (CO to its active form (CPR. Ferredoxin gene mutations reduce gene expression and increase its resistance to metronidazole. In this study, the frequency of ferredoxin gene mutations in clinical isolates of T.vaginalis in Tehran has been studied.Methods: Forty six clinical T. vaginalis isolates of vaginal secretions and urine sediment were collected from Tehran Province since 2011 till 2012. DNA was extracted and ferredoxin gene was amplified by PCR technique. The ferredoxin gene PCR products were sequenced to determine gene mutations.Results: In four isolates (8.69% point mutation at nucleotide position -239 (the translation start codon of the ferredoxin gene were detected in which adenosine were converted to thymine.Conclusion: Mutation at nucleotide -239 ferredoxin gene reduces translational regulatory protein’s binding affinity which concludes reduction of ferredoxin expression. For this reduction, decrease in activity and decrease in metronidazole drug delivery into the cells occur. Mutations in these four isolates may lead to resistance of them to metronidazole.

  20. Mutations in SYNGAP1 Cause Intellectual Disability, Autism, and a Specific Form of Epilepsy by Inducing Haploinsufficiency

    DEFF Research Database (Denmark)

    Berryer, Martin H; Hamdan, Fadi F; Klitten, Laura L;

    2013-01-01

    De novo mutations in SYNGAP1, which codes for a RAS/RAP GTP-activating protein, cause nonsyndromic intellectual disability (NSID). All disease-causing point mutations identified until now in SYNGAP1 are truncating, raising the possibility of an association between this type of mutations and NSID...

  1. Markov models for accumulating mutations

    CERN Document Server

    Beerenwinkel, Niko

    2007-01-01

    We introduce and analyze a waiting time model for the accumulation of genetic changes. The continuous time conjunctive Bayesian network is defined by a partially ordered set of mutations and by the rate of fixation of each mutation. The partial order encodes constraints on the order in which mutations can fixate in the population, shedding light on the mutational pathways underlying the evolutionary process. We study a censored version of the model and derive equations for an EM algorithm to perform maximum likelihood estimation of the model parameters. We also show how to select the maximum likelihood poset. The model is applied to genetic data from different cancers and from drug resistant HIV samples, indicating implications for diagnosis and treatment.

  2. Mutation breeding newsletter. No. 12

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  3. Plant improvement by induced mutations

    International Nuclear Information System (INIS)

    Genetic variability is required for the plant breeder to select better traits. Mutation induction by radiation and other mutagens is a means of altering genes and creating genetic variability for the breeder. A list is given of the number of mutant varieties of vegetables, fruits and ornamental flowers. Data given in the tables show that using induced mutations, 227 improved varieties of agricultural crops have been developed and released to farmers in 35 different countries. The IAEA has been involved in fostering mutation breeding since its foundation through training and direct research support. The Joint FAO/IAEA Division has published the Mutation Breeding Newsletter for plant breeders all over the world to keep abreast with developments in this field

  4. Dynamical Mutation of Dark Energy

    CERN Document Server

    Abramo, L R; Liberato, L; Rosenfeld, R

    2007-01-01

    We discuss the intriguing possibility that dark energy may change its equation of state in situations where large dark energy fluctuations are present. We show indications of this dynamical mutation in some generic models of dark energy.

  5. Mutation breeding newsletter. No. 4

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents reports and rea search abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  6. Mutation breeding newsletter. No. 3

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents reports and rea search abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  7. Mutation breeding newsletter. No. 11

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  8. Mutation breeding newsletter. No. 23

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  9. Mutation breeding newsletter. No. 30

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  10. Mutation breeding newsletter. No. 29

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents reports and rea search abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  11. Mutation breeding newsletter. No. 26

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents reports and research abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  12. Mutation breeding newsletter. No. 2

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents reports and rea search abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  13. Mutation breeding newsletter. No. 8

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  14. Mutation breeding newsletter. No. 16

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  15. Mutation breeding newsletter. No. 32

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  16. Mutation breeding newsletter. No. 15

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  17. Mutation Breeding Newsletter. No. 37

    International Nuclear Information System (INIS)

    This newsletter contains a brief account of FAO/IAEA meetings held in 1990 on plant breeding involving the use of induced mutations. It also features a list of commercially available plant cultivars produced by such techniques. Refs and tabs

  18. Mutation breeding newsletter. No. 7

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents reports and rea search abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  19. Mutation breeding newsletter. No. 5

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents reports and rea search abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  20. Mutation breeding newsletter. No. 28

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents reports and research abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  1. Mutation breeding newsletter. No. 17

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  2. Mutation breeding newsletter. No. 24

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents reports and research abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  3. Mutation breeding newsletter. No. 10

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  4. Mutation breeding newsletter. No. 9

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  5. Mutation breeding newsletter. No. 14

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  6. Mutation breeding newsletter. No. 25

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents reports and research abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  7. Mutation breeding newsletter. No. 27

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents reports and research abstracts on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  8. Mutation breeding newsletter. No. 22

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  9. Mutation breeding newsletter. No. 13

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  10. Mutation breeding newsletter. No. 18

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  11. Mutation breeding newsletter. No. 31

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  12. Mutation breeding newsletter. No. 36

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents abstracts and short communications of research results on radiation and chemical induced mutation breeding projects. Positive traits such as disease resistance and increased productivity are highlighted

  13. Mutation breeding newsletter. No. 19

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents new reports on mutation breeding programs using radiation or chemical mutagenesis to improve productivity, introduce disease resistance or induce morphological changes in crop plants

  14. Mutation breeding newsletter. No. 34

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents abstracts and short communications of research results on radiation and chemical induced mutation breeding projects. Positive traits such as disease resistance and increased productivity are highlighted

  15. Mutation breeding newsletter. No. 44

    International Nuclear Information System (INIS)

    This issue of the Newsletter presents research reports on the role of radiation induced mutation and chemical mutagens in improving productivity, disease resistance; cold and salinity tolerance of various crops and ornamental plants

  16. Parkin Somatic Mutations Link Melanoma and Parkinson's Disease.

    Science.gov (United States)

    Levin, Lotan; Srour, Shani; Gartner, Jared; Kapitansky, Oxana; Qutob, Nouar; Dror, Shani; Golan, Tamar; Dayan, Roy; Brener, Ronen; Ziv, Tamar; Khaled, Mehdi; Schueler-Furman, Ora; Samuels, Yardena; Levy, Carmit

    2016-06-20

    Epidemiological studies suggest a direct link between melanoma and Parkinson's disease (PD); however, the underlying molecular basis is unknown. Since mutations in Parkin are the major driver of early-onset PD and Parkin was recently reported to play a role in cancer development, we hypothesized that Parkin links melanoma and PD. By analyzing whole exome/genome sequencing of Parkin from 246 melanoma patients, we identified five non-synonymous mutations, three synonymous mutations, and one splice region variant in Parkin in 3.6% of the samples. In vitro analysis showed that wild-type Parkin plays a tumor suppressive role in melanoma development resulting in cell-cycle arrest, reduction of metabolic activity, and apoptosis. Using a mass spectrometry-based analysis, we identified potential Parkin substrates in melanoma and generated a functional protein association network. The activity of mutated Parkin was assessed by protein structure modeling and examination of Parkin E3 ligase activity. The Parkin-E28K mutation impairs Parkin ubiquitination activity and abolishes its tumor suppressive effect. Taken together, our analysis of genomic sequence and in vitro data indicate that Parkin is a potential link between melanoma and Parkinson's disease. Our findings suggest new approaches for early diagnosis and treatment against both diseases. PMID:27297116

  17. Parkin Somatic Mutations Link Melanoma and Parkinson's Disease.

    Science.gov (United States)

    Levin, Lotan; Srour, Shani; Gartner, Jared; Kapitansky, Oxana; Qutob, Nouar; Dror, Shani; Golan, Tamar; Dayan, Roy; Brener, Ronen; Ziv, Tamar; Khaled, Mehdi; Schueler-Furman, Ora; Samuels, Yardena; Levy, Carmit

    2016-06-20

    Epidemiological studies suggest a direct link between melanoma and Parkinson's disease (PD); however, the underlying molecular basis is unknown. Since mutations in Parkin are the major driver of early-onset PD and Parkin was recently reported to play a role in cancer development, we hypothesized that Parkin links melanoma and PD. By analyzing whole exome/genome sequencing of Parkin from 246 melanoma patients, we identified five non-synonymous mutations, three synonymous mutations, and one splice region variant in Parkin in 3.6% of the samples. In vitro analysis showed that wild-type Parkin plays a tumor suppressive role in melanoma development resulting in cell-cycle arrest, reduction of metabolic activity, and apoptosis. Using a mass spectrometry-based analysis, we identified potential Parkin substrates in melanoma and generated a functional protein association network. The activity of mutated Parkin was assessed by protein structure modeling and examination of Parkin E3 ligase activity. The Parkin-E28K mutation impairs Parkin ubiquitination activity and abolishes its tumor suppressive effect. Taken together, our analysis of genomic sequence and in vitro data indicate that Parkin is a potential link between melanoma and Parkinson's disease. Our findings suggest new approaches for early diagnosis and treatment against both diseases.

  18. Mutations in G protein beta subunits promote transformation and kinase inhibitor resistance

    OpenAIRE

    Yoda, Akinori; Adelmant, Guillaume; Tamburini, Jerome; Chapuy, Bjoern; Shindoh, Nobuaki; Yoda, Yuka; Weigert, Oliver; Kopp, Nadja; Wu, Shuo-chieh; Kim, Sunhee S.; Liu, Huiyun; Tivey, Trevor; Christie, Amanda L.; Elpek, Kutlu G; Card, Joseph

    2014-01-01

    Activating mutations of G protein alpha subunits (Gα) occur in 4–5% of all human cancers 1 but oncogenic alterations in beta subunits (Gβ) have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors, and disrupt Gα-Gβγ interactions. Different mutations in Gβ protein...

  19. PPARγ mutations, lipodystrophy and diabetes.

    Science.gov (United States)

    Astapova, Olga; Leff, Todd

    2014-11-01

    The focus of this review is the lipodystrophy syndrome caused by mutation in the PPARγ nuclear receptor - partial familial lipodystrophy FPLD3. To provide a broader context for how these mutations act to generate the clinical features of partial lipodystrophy we will review the basic biology of PPARγ and also survey the set PPARγ genetic variants that do not cause lipodystrophy, but are nonetheless associated with clinically related syndromes, specifically type 2 diabetes.

  20. Activating BRAF Mutations Detected in Mixed Hürthle Cell Carcinoma and Multifocal Papillary Carcinoma of the Thyroid Gland: Report of an Unusual Case and Review of the Literature.

    Science.gov (United States)

    Sinno, Sara; Choucair, Mahmoud; Nasrallah, Mona; Wadi, Lara; Jabbour, Mark N; Nassif, Samer

    2016-09-01

    Despite the increase in the incidence of thyroid carcinomas, the occurrence of collision tumors in the thyroid remains a rare event. We present the case of a 69-year-old female who presented to the emergency department with a chief complaint of painful neck swelling. Imaging revealed a large right hemithyroid mass and a left hemithyroid nodule. Fine needle aspiration of the lesions and subsequent total thyroidectomy revealed a Hürthle cell carcinoma in the right lobe and bilateral multicentric papillary carcinoma foci, including 2 foci with a classical pattern and 1 encapsulated follicular variant in the isthmus. BRAF gene mutation analysis revealed V600E gene mutation in the classical variants of papillary carcinoma and in the Hürthle cell carcinoma. The focus of follicular variant of papillary carcinoma in the isthmus and a sample from normal thyroid tissue did not harbor BRAF mutations. This case is remarkable in being an unusual report of a follicular Hürthle cell carcinoma harboring the BRAF V600E mutation and occurring in collision with multifocal papillary carcinoma. Documentation of such cases is important as it helps better understand the pathogenesis, clinical behavior, and radiologic findings of such rare lesions and to determine the optimal treatment modalities. PMID:27006301

  1. Activating BRAF Mutations Detected in Mixed Hürthle Cell Carcinoma and Multifocal Papillary Carcinoma of the Thyroid Gland: Report of an Unusual Case and Review of the Literature.

    Science.gov (United States)

    Sinno, Sara; Choucair, Mahmoud; Nasrallah, Mona; Wadi, Lara; Jabbour, Mark N; Nassif, Samer

    2016-09-01

    Despite the increase in the incidence of thyroid carcinomas, the occurrence of collision tumors in the thyroid remains a rare event. We present the case of a 69-year-old female who presented to the emergency department with a chief complaint of painful neck swelling. Imaging revealed a large right hemithyroid mass and a left hemithyroid nodule. Fine needle aspiration of the lesions and subsequent total thyroidectomy revealed a Hürthle cell carcinoma in the right lobe and bilateral multicentric papillary carcinoma foci, including 2 foci with a classical pattern and 1 encapsulated follicular variant in the isthmus. BRAF gene mutation analysis revealed V600E gene mutation in the classical variants of papillary carcinoma and in the Hürthle cell carcinoma. The focus of follicular variant of papillary carcinoma in the isthmus and a sample from normal thyroid tissue did not harbor BRAF mutations. This case is remarkable in being an unusual report of a follicular Hürthle cell carcinoma harboring the BRAF V600E mutation and occurring in collision with multifocal papillary carcinoma. Documentation of such cases is important as it helps better understand the pathogenesis, clinical behavior, and radiologic findings of such rare lesions and to determine the optimal treatment modalities.

  2. Quantitative and Sensitive Detection of GNAS Mutations Causing McCune-Albright Syndrome with Next Generation Sequencing

    OpenAIRE

    Satoshi Narumi; Kumihiro Matsuo; Tomohiro Ishii; Yusuke Tanahashi; Tomonobu Hasegawa

    2013-01-01

    Somatic activating GNAS mutations cause McCune-Albright syndrome (MAS). Owing to low mutation abundance, mutant-specific enrichment procedures, such as the peptide nucleic acid (PNA) method, are required to detect mutations in peripheral blood. Next generation sequencing (NGS) can analyze millions of PCR amplicons independently, thus it is expected to detect low-abundance GNAS mutations quantitatively. In the present study, we aimed to develop an NGS-based method to detect low-abundance somat...

  3. Whole Genome Sequencing of Mycobacterium tuberculosis Reveals Slow Growth and Low Mutation Rates during Latent Infections in Humans

    OpenAIRE

    Roberto Colangeli; Vic L Arcus; Cursons, Ray T.; Ali Ruthe; Noel Karalus; Kathy Coley; Manning, Shannon D.; Soyeon Kim; Emily Marchiano; David Alland

    2014-01-01

    Very little is known about the growth and mutation rates of Mycobacterium tuberculosis during latent infection in humans. However, studies in rhesus macaques have suggested that latent infections have mutation rates that are higher than that observed during active tuberculosis disease. Elevated mutation rates are presumed risk factors for the development of drug resistance. Therefore, the investigation of mutation rates during human latency is of high importance. We performed whole genome mut...

  4. Induced mutations in sorghum improvement

    International Nuclear Information System (INIS)

    A critical review of different aspects of mutagen sensitivity, considering the importance of such factors as genotypic constitution of the material, pre- and post-treatment modifications, type of mutagen and dose, techniques of handling the material and treatment procedures to maximize the induction of mutations together with the scope of induced mutations in sorghum improvement is presented. Hydrazine was found to be a more effective and efficient mutagen for inducing chlorophyll and viable mutations in sorghum than ethyl methanesulphonate, methyl methanesulphonate or γ-rays. Ethyl methane-sulphonate among the alkylating agents and nitroso methyl urea among nitroso compounds were the most potent mutagens. The efficient radiation dose was within the 20-35 kr range whereas 0.015M was the effective dosage for hydrazine and ethylmethane sulphonate. The combination treatments of various physical and chemical mutagens failed to yield significant increase in the recovery of mutations, while cysteine post-treatments of γ-irradiated and hydrazine-treated material reduced seedling injury, seed sterility and increased the recovery of viable mutations compared to single treatments. There is scope for induced mutations in solving some of the current problems of sorghum improvement such as, increasing the recombination potential of tropical X temperate crosses, improving the nutritional quality of grain and forage sorghums, diversification of male sterile cytoplasmic sources, better understanding of mechanism of apomixis and augmenting the levels of resistance to sorghum insects, pests and diseases. (author)

  5. A multiparametric computational algorithm for comprehensive assessment of genetic mutations in mucopolysaccharidosis type IIIA (Sanfilippo syndrome.

    Directory of Open Access Journals (Sweden)

    Krastyu G Ugrinov

    Full Text Available Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo syndrome is a Lysosomal Storage Disease caused by cellular deficiency of N-sulfoglucosamine sulfohydrolase (SGSH. Given the large heterogeneity of genetic mutations responsible for the disease, a comprehensive understanding of the mechanisms by which these mutations affect enzyme function is needed to guide effective therapies. We developed a multiparametric computational algorithm to assess how patient genetic mutations in SGSH affect overall enzyme biogenesis, stability, and function. 107 patient mutations for the SGSH gene were obtained from the Human Gene Mutation Database representing all of the clinical mutations documented for Sanfilippo syndrome. We assessed each mutation individually using ten distinct parameters to give a comprehensive predictive score of the stability and misfolding capacity of the SGSH enzyme resulting from each of these mutations. The predictive score generated by our multiparametric algorithm yielded a standardized quantitative assessment of the severity of a given SGSH genetic mutation toward overall enzyme activity. Application of our algorithm has identified SGSH mutations in which enzymatic malfunction of the gene product is specifically due to impairments in protein folding. These scores provide an assessment of the degree to which a particular mutation could be treated using approaches such as chaperone therapies. Our multiparametric protein biogenesis algorithm advances a key understanding in the overall biochemical mechanism underlying Sanfilippo syndrome. Importantly, the design of our multiparametric algorithm can be tailored to many other diseases of genetic heterogeneity for which protein misfolding phenotypes may constitute a major component of disease manifestation.

  6. GNAS gene mutation may be present only transiently during colorectal tumorigenesis

    Science.gov (United States)

    Zauber, Peter; Marotta, Stephen P; Sabbath-Solitare, Marlene

    2016-01-01

    Mutations of the gene GNAS have been shown to activate the adenylate cyclase gene and lead to constitutive cAMP signaling. Several preliminary reports have suggested a role for GNAS gene mutations during colorectal carcinogenesis, particularly mucinous carcinomas. The aim of this study was to clarify the incidence of GNAS mutations in adenomas (tubular, tubulovillous, and villous), carcinomas with residual adenoma, and carcinomas, and to relate these findings to mutations of the KRAS gene and to the mucinous status of the tumors. We used standard PCR techniques and direct gene sequencing to evaluate tumors for gene mutations. No GNAS mutations were identified in 25 tubular adenomas, but were present in 6.4% of tubulovillous adenomas and 11.2% of villous adenomas. A GNAS mutation was found in 9.7% of the benign portion of carcinoma with residual adenoma, but in none of 86 carcinomas. A similar trend was seen for KRAS mutation across the five groups of tumors. GNAS mutations may function as an important driver mutation during certain phases of colorectal carcinogenesis, but may then be lost once the biological advantage gained by the mutated gene is no longer necessary to sustain or advance tumor development. PMID:27186325

  7. Genome Destabilizing Mutator Alleles Drive Specific Mutational Trajectories in Saccharomyces cerevisiae

    Science.gov (United States)

    Stirling, Peter C.; Shen, Yaoqing; Corbett, Richard; Jones, Steven J. M.; Hieter, Philip

    2014-01-01

    In addition to environmental factors and intrinsic variations in base substitution rates, specific genome-destabilizing mutations can shape the mutational trajectory of genomes. How specific alleles influence the nature and position of accumulated mutations in a genomic context is largely unknown. Understanding the impact of genome-destabilizing alleles is particularly relevant to cancer genomes where biased mutational signatures are identifiable. We first created a more complete picture of cellular pathways that impact mutation rate using a primary screen to identify essential Saccharomyces cerevisiae gene mutations that cause mutator phenotypes. Drawing primarily on new alleles identified in this resource, we measure the impact of diverse mutator alleles on mutation patterns directly by whole-genome sequencing of 68 mutation-accumulation strains derived from wild-type and 11 parental mutator genotypes. The accumulated mutations differ across mutator strains, displaying base-substitution biases, allele-specific mutation hotspots, and break-associated mutation clustering. For example, in mutants of POLα and the Cdc13–Stn1–Ten1 complex, we find a distinct subtelomeric bias for mutations that we show is independent of the target sequence. Together our data suggest that specific genome-instability mutations are sufficient to drive discrete mutational signatures, some of which share properties with mutation patterns seen in tumors. Thus, in a population of cells, genome-instability mutations could influence clonal evolution by establishing discrete mutational trajectories for genomes. PMID:24336748

  8. 有源滤波器的模糊阈值变环宽滞环电流跟踪控制策略%A fuzzy threshold mutative bandwidth hysteresis current tracking control method for active power filter

    Institute of Scientific and Technical Information of China (English)

    刘心旸; 王杰; 王昕; 姚钢

    2011-01-01

    A fuzzy threshold mutative bandwidth hysteresis current tracking control method for the Active Power Filter (APF) current tracking module is presented. Based on the construction of APF device model and the analysis of the variation of hysteresis width, the purpose to achieve a limited frequency of switching is reached by using an internal fuzzy controller to dynamically adjust the loop width of hysteresis comparator device. Compared with the traditional hysteresis method, the proposed method has more excellent dynamic response and tracking precision, taking full advantage of conduction capability of the power switches, moreover,a reduction of the maximum switching frequency ensures that the device operates safely. Finally, the current tracking efficiency of the method above and the harmonic compensating capability to nonlinear loads are demonstrated by the simulation and experiment results in this paper.This work is supported by National Natural Science Foundation of China (No. 60674035).%针对有源电力滤波器(APF)的电流跟踪功能模块,提出了一种模糊阈值变环宽滞环电流跟踪控制算法.在构建APF装置模型并分析滞环宽度变化规律的基础上,通过使用内部模糊控制器用以动态地调节滞环比较器的环宽,从而达到了限定开关频率的目的.该方法相比于传统滞环比较方法具有更好的跟踪精度和动态性能,充分利用了开关器件的导通能力;同时由于降低了最大开关频率,从而保证了装置的安全运行.通过仿真与实际实验结果进一步验证了该算法的电流跟踪效果以及对于非线性装置产生的系统谐波的补偿能力.

  9. Adaptive mutations produce resistance to ciprofloxacin.

    OpenAIRE

    Riesenfeld, C; Everett, M.; Piddock, L J; Hall, B G

    1997-01-01

    Mutation to ciprofloxacin resistance continually occurred in nondividing Escherichia coli cells during a 7-day exposure to ciprofloxacin in agar, while no accumulation of rifampin resistance mutations was detected in those cells. We propose that the resistance mutations result from adaptive mutations, which preferentially produce phenotypes that promote growth in nondividing cells.

  10. Consensus for EGFR mutation testing in non-small cell lung cancer: results from a European workshop

    DEFF Research Database (Denmark)

    Pirker, Robert; Herth, Felix J F; Kerr, Keith M;

    2010-01-01

    Activating somatic mutations of the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have recently been characterized in a subset of patients with advanced non-small cell lung cancer (NSCLC). Patients harboring these mutations in their tumors show excellent response to EGFR...... tyrosine kinase inhibitors (EGFR-TKIs). The EGFR-TKI gefitinib has been approved in Europe for the treatment of adult patients with locally advanced or metastatic NSCLC with activating mutations of the EGFR TK. Because EGFR mutation testing is not yet well established across Europe, biomarker......-directed therapy only slowly emerges for the subset of NSCLC patients most likely to benefit: those with EGFR mutations....

  11. Carcinogen-specific mutations in preferred Ras-Raf pathway oncogenes directed by strand bias.

    Science.gov (United States)

    Keller, Ross R; Gestl, Shelley A; Lu, Amy Q; Hoke, Alicia; Feith, David J; Gunther, Edward J

    2016-08-01

    Carcinogen exposures inscribe mutation patterns on cancer genomes and sometimes bias the acquisition of driver mutations toward preferred oncogenes, potentially dictating sensitivity to targeted agents. Whether and how carcinogen-specific mutation patterns direct activation of preferred oncogenes remains poorly understood. Here, mouse models of breast cancer were exploited to uncover a mechanistic link between strand-biased mutagenesis and oncogene preference. When chemical carcinogens were employed during Wnt1-initiated mammary tumorigenesis, exposure to either 7,12-dimethylbenz(a)anthracene (DMBA) or N-ethyl-N-nitrosourea (ENU) dramatically accelerated tumor onset. Mammary tumors that followed DMBA exposure nearly always activated the Ras pathway via somatic Hras(CAA61CTA) mutations. Surprisingly, mammary tumors that followed ENU exposure typically lacked Hras mutations, and instead activated the Ras pathway downstream via Braf(GTG636GAG) mutations. Hras(CAA61CTA) mutations involve an A-to-T change on the sense strand, whereas Braf(GTG636GAG) mutations involve an inverse T-to-A change, suggesting that strand-biased mutagenesis may determine oncogene preference. To examine this possibility further, we turned to an alternative Wnt-driven tumor model in which carcinogen exposures augment a latent mammary tumor predisposition in Apc(min) mice. DMBA and ENU each accelerated mammary tumor onset in Apc(min) mice by introducing somatic, "second-hit" Apc mutations. Consistent with our strand bias model, DMBA and ENU generated strikingly distinct Apc mutation patterns, including stringently strand-inverse mutation signatures at A:T sites. Crucially, these contrasting signatures precisely match those proposed to confer bias toward Hras(CAA61CTA) versus Braf(GTG636GAG) mutations in the original tumor sets. Our findings highlight a novel mechanism whereby exposure history acts through strand-biased mutagenesis to specify activation of preferred oncogenes. PMID:27207659

  12. Alleged Detrimental Mutations in the SMPD1 Gene in Patients with Niemann-Pick Disease

    OpenAIRE

    Cosima Rhein; Christiane Mühle; Johannes Kornhuber; Martin Reichel

    2015-01-01

    Loss-of-function mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene are associated with decreased catalytic activity of acid sphingomyelinase (ASM) and are the cause of the autosomal recessive lysosomal storage disorder Niemann-Pick disease (NPD) types A and B. Currently, >100 missense mutations in SMPD1 are listed in the Human Gene Mutation Database. However, not every sequence variation in SMPD1 is detrimental and gives rise to NPD. We have analysed several alleged SMPD1 mis...

  13. Mutations in uvrD induce the SOS response in Escherichia coli.

    OpenAIRE

    Ossanna, N; Mount, D W

    1989-01-01

    We have isolated three new mutations in uvrD that increase expression of the Escherichia coli SOS response in the absence of DNA damage. Like other uvrD (DNA helicase II) mutants, these strains are sensitive to UV irradiation and have high spontaneous mutation frequencies. Complementation studies with uvrD+ showed that UV sensitivity and spontaneous mutator activity were recessive in these new mutants. The SOS-induction phenotype, however, was not completely complemented, which indicated that...

  14. Detection of PIK3CA Mutations in Breast Cancer Bone Metastases

    OpenAIRE

    Manijeh Daneshmand; Hanson, Jennifer E. L.; Mitra Nabavi; Hilton, John F.; Lisa Vandermeer; Femina Kanji; Dent, Susan F; Mark Clemons; Ian A. J. Lorimer

    2012-01-01

    Background. An important goal of personalized cancer therapy is to tailor specific therapies to the mutational profile of individual patients. However, whole genome sequencing studies have shown that the mutational profiles of cancers evolve over time and often differ between primary and metastatic sites. Activating point mutations in the PIK3CA gene are common in primary breast cancer tumors, but their presence in breast cancer bone metastases has not been assessed previously. Results. Fourt...

  15. Protection by dietary compounds against mutation in a transgenic rodent.

    Science.gov (United States)

    de Boer, J G

    2001-11-01

    One of the most relevant biomarkers of genotoxicity and, potentially, carcinogenesis is the occurrence of mutations. Data indicate that carcinogens are highly specific with regard to their target tissue in inducing both tumors and mutations. This specificity may reflect the dependence on tissue-specific metabolic activation, the organ-specific environment or both. Ideally, therefore, mutation should be determined in a real animal rather than in a cell culture system. The lacI transgenic rodent model provides such a system. We have used this model to investigate tissue, species and sex specificity of mutation induced by selected dietary carcinogens and to examine how some compounds may alter the induction of mutation. We have studied mutation using several chemicals, including the dietary heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the environmentally important aromatic hydrocarbon benzo[a]pyrene and the food contaminant aflatoxin B1. We have shown that the mutagenic potency of these chemicals can be modulated by other dietary compounds, including green tea and conjugated linoleic acid, and the dioxin 2,3,7,8-tetrachlorodibenzo[b,e][1,4]dioxin (TCDD). These results demonstrate that the lacI transgenic rodent is a useful model for the study of chemoprevention in vivo.

  16. Multigene mutational profiling of cholangiocarcinomas identifies actionable molecular subgroups

    Science.gov (United States)

    Mafficini, Andrea; Wood, Laura D.; Corbo, Vincenzo; Melisi, Davide; Malleo, Giuseppe; Vicentini, Caterina; Malpeli, Giorgio; Antonello, Davide; Sperandio, Nicola; Capelli, Paola; Tomezzoli, Anna; Iacono, Calogero; Lawlor, Rita T.; Bassi, Claudio; Hruban, Ralph H.; Guglielmi, Alfredo; Tortora, Giampaolo; de Braud, Filippo; Scarpa, Aldo

    2014-01-01

    One-hundred-fifty-three biliary cancers, including 70 intrahepatic cholangiocarcinomas (ICC), 57 extrahepatic cholangiocarcinomas (ECC) and 26 gallbladder carcinomas (GBC) were assessed for mutations in 56 genes using multigene next-generation sequencing. Expression of EGFR and mTOR pathway genes was investigated by immunohistochemistry. At least one mutated gene was observed in 118/153 (77%) cancers. The genes most frequently involved were KRAS (28%), TP53 (18%), ARID1A (12%), IDH1/2 (9%), PBRM1 (9%), BAP1 (7%), and PIK3CA (7%). IDH1/2 (p=0.0005) and BAP1 (p=0.0097) mutations were characteristic of ICC, while KRAS (p=0.0019) and TP53 (p=0.0019) were more frequent in ECC and GBC. Multivariate analysis identified tumour stage and TP53 mutations as independent predictors of survival. Alterations in chromatin remodeling genes (ARID1A, BAP1, PBRM1, SMARCB1) were seen in 31% of cases. Potentially actionable mutations were seen in 104/153 (68%) cancers: i) KRAS/NRAS/BRAF mutations were found in 34% of cancers; ii) mTOR pathway activation was documented by immunohistochemistry in 51% of cases and by mutations in mTOR pathway genes in 19% of cancers; iii) TGF-ß/Smad signaling was altered in 10.5% cancers; iv) mutations in tyrosine kinase receptors were found in 9% cases. Our study identified molecular subgroups of cholangiocarcinomas that can be explored for specific drug targeting in clinical trials. PMID:24867389

  17. A novel somatic MAPK1 mutation in primary ovarian mixed germ cell tumors.

    Science.gov (United States)

    Zou, Yang; Deng, Wei; Wang, Feng; Yu, Xiao-Hong; Liu, Fa-Ying; Yang, Bi-Cheng; Huang, Mei-Zhen; Guo, Jiu-Bai; Xie, Qiu-Hua; He, Ming; Huang, Ou-Ping

    2016-02-01

    A recent exome-sequencing study revealed prevalent mitogen-activated protein kinase 1 (MAPK1) p.E322K mutation in cervical carcinoma. It remains largely unknown whether ovarian carcinomas also harbor MAPK1 mutations. As paralogous gene mutations co‑occur frequently in human malignancies, we analyzed here a total of 263 ovarian carcinomas for the presence of MAPK1 and paralogous MAPK3 mutations by DNA sequencing. A previously unreported MAPK1 p.D321N somatic mutation was identified in 2 out of 18 (11.1%) ovarian mixed germ cell tumors, while no other MAPK1 or MAPK3 mutation was detected in our samples. Of note, OCC‑115, the MAPK1‑mutated sample with bilateral cancerous ovaries affected, harbored MAPK1 mutation in the right ovary while retained the left ovary intact, implicating that the genetic alterations underlying ovarian mixed germ cell tumor may be different, even in patients with similar genetic backgrounds and tumor microenvironments. The results of evolutionary conservation and protein structure modeling analysis implicated that MAPK1 p.D321N mutation may be pathogenic. Additionally, mutations in protein phosphatase 2 regulatory subunit α (PPP2R1A), ring finger protein 43 (RNF43), DNA directed polymerase ε (POLE1), ribonuclease type III (DICER1), CCCTC‑binding factor (CTCF), ribosomal protein L22 (RPL22), DNA methyltransferase 3α (DNMT3A), transformation/transcription domain‑associated protein (TRRAP), isocitrate dehydrogenase (IDH)1 and IDH2 were not detected in ovarian mixed germ cell tumors, implicating these genetic alterations may be not associated with MAPK1 mutation in the development of this malignancy. The present study identified a previously unreported MAPK1 mutation in ovarian mixed germ cell tumors for the first time, and this mutation may be actively involved in the tumorigenesis of this disease. PMID:26548627

  18. Identification of High-Impact cis-Regulatory Mutations Using Transcription Factor Specific Random Forest Models.

    Directory of Open Access Journals (Sweden)

    Dmitry Svetlichnyy

    2015-11-01

    Full Text Available Cancer genomes contain vast amounts of somatic mutations, many of which are passenger mutations not involved in oncogenesis. Whereas driver mutations in protein-coding genes can be distinguished from passenger mutations based on their recurrence, non-coding mutations are usually not recurrent at the same position. Therefore, it is still unclear how to identify cis-regulatory driver mutations, particularly when chromatin data from the same patient is not available, thus relying only on sequence and expression information. Here we use machine-learning methods to predict functional regulatory regions using sequence information alone, and compare the predicted activity of the mutated region with the reference sequence. This way we define the Predicted Regulatory Impact of a Mutation in an Enhancer (PRIME. We find that the recently identified driver mutation in the TAL1 enhancer has a high PRIME score, representing a "gain-of-target" for MYB, whereas the highly recurrent TERT promoter mutation has a surprisingly low PRIME score. We trained Random Forest models for 45 cancer-related transcription factors, and used these to score variations in the HeLa genome and somatic mutations across more than five hundred cancer genomes. Each model predicts only a small fraction of non-coding mutations with a potential impact on the function of the encompassing regulatory region. Nevertheless, as these few candidate driver mutations are often linked to gains in chromatin activity and gene expression, they may contribute to the oncogenic program by altering the expression levels of specific oncogenes and tumor suppressor genes.

  19. Common Β- Thalassaemia Mutations in

    Directory of Open Access Journals (Sweden)

    P Azarfam

    2005-01-01

    Full Text Available Introduction: β –Thalassaemia was first explained by Thomas Cooly as Cooly’s anaemia in 1925. The β- thalassaemias are hereditary autosomal disorders with decreased or absent β-globin chain synthesis. The most common genetic defects in β-thalassaemias are caused by point mutations, micro deletions or insertions within the β-globin gene. Material and Methods: In this research , 142 blood samples (64 from childrens hospital of Tabriz , 15 samples from Shahid Gazi hospital of Tabriz , 18 from Urumia and 45 samples from Aliasghar hospital of Ardebil were taken from thalassaemic patients (who were previously diagnosed .Then 117 non-familial samples were selected . The DNA of the lymphocytes of blood samples was extracted by boiling and Proteinase K- SDS procedure, and mutations were detected by ARMS-PCR methods. Results: From the results obtained, eleven most common mutations,most of which were Mediterranean mutations were detected as follows; IVS-I-110(G-A, IVS-I-1(G-A ،IVS-I-5(G-C ,Frameshift Codon 44 (-C,( codon5(-CT,IVS-1-6(T-C, IVS-I-25(-25bp del ,Frameshift 8.9 (+G ,IVS-II-1(G-A ,Codon 39(C-T, Codon 30(G-C the mutations of the samples were defined. The results showed that Frameshift 8.9 (+G, IVS-I-110 (G-A ,IVS-II-I(G-A, IVS-I-5(G-C, IVS-I-1(G-A , Frameshift Codon 44(-C , codon5(-CT , IVS-1-6(T-C , IVS-I-25(-25bp del with a frequency of 29.9%, 25.47%,17.83%, 7.00%, 6.36% , 6.63% , 3.8% , 2.5% , 0.63% represented the most common mutations in North - west Iran. No mutations in Codon 39(C-T and Codon 30(G-C were detected. Cunclusion: The frequency of the same mutations in patients from North - West of Iran seems to be different as compared to other regions like Turkey, Pakistan, Lebanon and Fars province of Iran. The pattern of mutations in this region is more or less the same as in the Mediterranean region, but different from South west Asia and East Asia.

  20. [Founder mutation in Lynch syndrome].

    Science.gov (United States)

    Cajal, Andrea R; Piñero, Tamara A; Verzura, Alicia; Santino, Juan Pablo; Solano, Angela R; Kalfayan, Pablo G; Ferro, Alejandra; Vaccaro, Carlos

    2016-01-01

    Lynch syndrome is the most frequent syndrome in hereditary colorectal cancer, a family-specific deleterious mutations in genes encoding DNA reparation proteins: MLH1 (mutL homolog 1), MSH2, MSH6 (mutS homolog 2 y 6, respectively), PMS2 (PMS1 homolog 2, mismatch repair system component) y MUTYH (mutY DNA glycosylase). The c.2252_2253delAA, p.Lys751Serfs*3 mutation in MLH1 gene segregates with a haplotype reported in the northern region of Italy and whose origin was attributed to a founder effect. This mutation co-segregates with typical characteristics of Lynch syndrome, including early age at onset and multiple primary tumors in the same individual, a high frequency of pancreatic cancer, high microsatellite instability and lack of PMS2 expression. This report describes a mutation in an Argentinian patient with endometrioid adenocarcinoma of uterus. Her first-degree relatives had a history of colon cancer diagnosed before 50 years, fulfilling the Amsterdam Criteria I and Lynch syndrome II. The high pathogenicity associated to this mutation makes necessary the study of all members from families with hereditary cancer, allowing pre-symptomatic genetic diagnosis, early assessment and the instauration of preventive treatments.

  1. [Founder mutation in Lynch syndrome].

    Science.gov (United States)

    Cajal, Andrea R; Piñero, Tamara A; Verzura, Alicia; Santino, Juan Pablo; Solano, Angela R; Kalfayan, Pablo G; Ferro, Alejandra; Vaccaro, Carlos

    2016-01-01

    Lynch syndrome is the most frequent syndrome in hereditary colorectal cancer, a family-specific deleterious mutations in genes encoding DNA reparation proteins: MLH1 (mutL homolog 1), MSH2, MSH6 (mutS homolog 2 y 6, respectively), PMS2 (PMS1 homolog 2, mismatch repair system component) y MUTYH (mutY DNA glycosylase). The c.2252_2253delAA, p.Lys751Serfs*3 mutation in MLH1 gene segregates with a haplotype reported in the northern region of Italy and whose origin was attributed to a founder effect. This mutation co-segregates with typical characteristics of Lynch syndrome, including early age at onset and multiple primary tumors in the same individual, a high frequency of pancreatic cancer, high microsatellite instability and lack of PMS2 expression. This report describes a mutation in an Argentinian patient with endometrioid adenocarcinoma of uterus. Her first-degree relatives had a history of colon cancer diagnosed before 50 years, fulfilling the Amsterdam Criteria I and Lynch syndrome II. The high pathogenicity associated to this mutation makes necessary the study of all members from families with hereditary cancer, allowing pre-symptomatic genetic diagnosis, early assessment and the instauration of preventive treatments. PMID:27295708

  2. Infantile onset spinocerebellar ataxia caused by compound heterozygosity for Twinkle mutations and modeling of Twinkle mutations causing recessive disease

    Science.gov (United States)

    Gulsuner, Suleyman; Stapleton, Gail A.; Walsh, Tom; Lee, Ming K.; Mandell, Jessica B.; Morales, Augusto; Klevit, Rachel E.; King, Mary-Claire; Rogers, R. Curtis

    2016-01-01

    Mutations in nuclear genes required for the replication and maintenance of mitochondrial DNA cause progressive multisystemic neuromuscular disorders with overlapping phenotypes. Biallelic mutations in C10orf2, encoding the Twinkle mitochondrial DNA helicase, lead to infantile-onset cerebellar ataxia (IOSCA), as well as milder and more severe phenotypes. We present a 13-year-old girl with ataxia, severe hearing loss, optic atrophy, peripheral neuropathy, and hypergonadotropic hypogonadism. Whole-exome sequencing revealed that the patient is compound heterozygous for previously unreported variants in the C10orf2 gene: a paternally inherited frameshift variant (c.333delT; p.L112Sfs*3) and a maternally inherited missense variant (c.904C>T; p.R302W). The identification of novel C10orf2 mutations extends the spectrum of mutations in the Twinkle helicase causing recessive disease, in particular the intermediate IOSCA phenotype. Structural modeling suggests that the p.R302W mutation and many other recessively inherited Twinkle mutations impact the position or interactions of the linker region, which is critical for the oligomeric ring structure and activity of the helicase. This study emphasizes the utility of whole-exome sequencing for the genetic diagnosis of a complex multisystemic disorder. PMID:27551684

  3. Infantile onset spinocerebellar ataxia caused by compound heterozygosity for Twinkle mutations and modeling of Twinkle mutations causing recessive disease.

    Science.gov (United States)

    Pierce, Sarah B; Gulsuner, Suleyman; Stapleton, Gail A; Walsh, Tom; Lee, Ming K; Mandell, Jessica B; Morales, Augusto; Klevit, Rachel E; King, Mary-Claire; Rogers, R Curtis

    2016-07-01

    Mutations in nuclear genes required for the replication and maintenance of mitochondrial DNA cause progressive multisystemic neuromuscular disorders with overlapping phenotypes. Biallelic mutations in C10orf2, encoding the Twinkle mitochondrial DNA helicase, lead to infantile-onset cerebellar ataxia (IOSCA), as well as milder and more severe phenotypes. We present a 13-year-old girl with ataxia, severe hearing loss, optic atrophy, peripheral neuropathy, and hypergonadotropic hypogonadism. Whole-exome sequencing revealed that the patient is compound heterozygous for previously unreported variants in the C10orf2 gene: a paternally inherited frameshift variant (c.333delT; p.L112Sfs*3) and a maternally inherited missense variant (c.904C>T; p.R302W). The identification of novel C10orf2 mutations extends the spectrum of mutations in the Twinkle helicase causing recessive disease, in particular the intermediate IOSCA phenotype. Structural modeling suggests that the p.R302W mutation and many other recessively inherited Twinkle mutations impact the position or interactions of the linker region, which is critical for the oligomeric ring structure and activity of the helicase. This study emphasizes the utility of whole-exome sequencing for the genetic diagnosis of a complex multisystemic disorder. PMID:27551684

  4. Intra-organ variation in age-related mutation accumulation in the mouse.

    Directory of Open Access Journals (Sweden)

    Rita A Busuttil

    Full Text Available Using a transgenic mouse model harboring chromosomally integrated lacZ mutational target genes, we previously demonstrated that mutations accumulate with age much more rapidly in the small intestine than in the brain. Here it is shown that in the small intestine point mutations preferentially accumulate in epithelial cells of the mucosa scraped off the underlying serosa. The mucosal cells are the differentiated villus cells that have undergone multiple cell divisions. A smaller age-related increase, also involving genome rearrangements, was observed in the serosa, which consists mainly of the remaining crypts and non-dividing smooth muscle cells. In the brain we observed an accumulation of only point mutations in no other areas than hypothalamus and hippocampus. To directly test for cell division as the determining factor in the generation of point mutations we compared mutation induction between mitotically active and quiescent embryonic fibroblasts from the same lacZ mice, treated with either UV (a point mutagen or hydrogen peroxide (a clastogen. The results indicate that while point mutations are highly replication-dependent, genome rearrangements are as easily induced in non-dividing cells as in mitotically active ones. This strongly suggests that the point mutations found to have accumulated in the mucosal part of the small intestine are the consequence of replication errors. The same is likely true for point mutations accumulating in hippocampus and hypothalamus of the brain since neurogenesis in these two areas continues throughout life. The observed intra-organ variation in mutation susceptibility as well as the variation in replication dependency of different types of mutations indicates the need to not only extend observations made on whole organs to their sub-structures but also take the type of mutations and mitotic activity of the cells into consideration. This should help elucidating the impact of genome instability and its

  5. The Role of gsp Mutations on the Development of Adrenocortical Tumors and Adrenal Hyperplasia

    Science.gov (United States)

    Villares Fragoso, Maria Candida Barisson; Wanichi, Ingrid Quevedo; Cavalcante, Isadora Pontes; Mariani, Beatriz Marinho de Paula

    2016-01-01

    Somatic GNAS point mutations, commonly known as gsp mutations, are involved in the pathogenesis of McCune–Albright syndrome (MAS) and have also been described in autonomous hormone-producing tumors, such as somatotropinoma, corticotrophoma, thyroid cancer, ovarian and testicular Leydig cell tumors, and primary macronodular adrenocortical hyperplasia (PMAH) (1–3). The involvement of gsp mutations in adrenal tumors was first described by Lyons et al. Since then, several studies have detected the presence of gsp mutations in adrenal tumors, but none of them could explain its presence along or the mechanism that leads to tumor formation and hormone hypersecretion. As a result, the molecular pathogenesis of the majority of sporadic adrenocortical tumors remains unclear (3). PMAH has also been reported with gsp somatic mutations in a few cases. Fragoso et al. identified two distinct gsp somatic mutations affecting arginine residues on codon 201 of GNAS in a few patients with PMAH who lacked any features or manifestations of MAS. Followed by this discovery, other studies have continued looking for gsp mutations based on strong prior evidence demonstrating that increased cAMP signaling is sufficient for cell proliferation and cortisol production (2, 4). With consideration for the previously reported findings, we conjecture that although somatic activating mutations in GNAS are a rare molecular event, these mutations could probably be sufficient to induce the development of macronodule hyperplasia and variable cortisol secretion. In this manuscript, we revised the presence of gsp mutations associated with adrenal cortical tumors and hyperplasia. PMID:27512387

  6. BRAF V600 mutations and pathological features in Japanese melanoma patients

    OpenAIRE

    Yamazaki, Naoya; Tanaka, Ryota; Tsutsumida, Arata; Namikawa, Kenjiro; Eguchi, Hironobu; Omata, Wataru; Oashi, Kohei; Ogawa, Toru; Hayashi, Amiko; Nakamura, Noriyuki; Tsuta, Koji

    2014-01-01

    Ultraviolet radiation is a risk factor for BRAF V600 mutations frequently found in melanomas that cause constitutive BRAF activation. Primary sites of melanoma and the frequency of BRAF mutations might differ between races. Melanoma is rare in Japan (1500–2000 cases/year compared with 132 000/year worldwide) and the frequency and distribution of BRAF V600 mutations are unknown. We aimed to investigate the frequency of BRAF V600 mutations in a cohort of Japanese patients with melanoma and dete...

  7. [Some behavioral features in Drosophila melanogaster lines carrying a flamenco gene mutation].

    Science.gov (United States)

    Subocheva, E A; Romanova, L G; Romanova, N I; Kim, A I

    2001-11-01

    Olfactory sensitivity and locomotor activity was assayed in Drosophila melanogaster strains carrying a mutation of the flamenco gene, which controls transposition of the mobile genetic element 4 (MGE4) retrotransposon the gypsy mobile element. A change in olfactory sensitivity was detected. The reaction to the odor of acetic acid was inverted in flies of the mutator strain (MS), which carried the flam mutation and active MGE4 copies and were characterized by genetic instability. Flies of the genetically unstable strains displayed a lower locomotor activity. The behavioral changes in MS flies can be explained by the pleiotropic effect of the flam mutation or by insertion mutations which arise in behavior genes as a result of genome destabilization by MGE4. PMID:11771305

  8. De novo mutations in ATP1A3 cause alternating hemiplegia of childhood

    DEFF Research Database (Denmark)

    Heinzen, Erin L; Swoboda, Kathryn J; Hitomi, Yuki;

    2012-01-01

    Alternating hemiplegia of childhood (AHC) is a rare, severe neurodevelopmental syndrome characterized by recurrent hemiplegic episodes and distinct neurological manifestations. AHC is usually a sporadic disorder and has unknown etiology. We used exome sequencing of seven patients with AHC...... and their unaffected parents to identify de novo nonsynonymous mutations in ATP1A3 in all seven individuals. In a subsequent sequence analysis of ATP1A3 in 98 other patients with AHC, we found that ATP1A3 mutations were likely to be responsible for at least 74% of the cases; we also identified one inherited mutation...... in a case of familial AHC. Notably, most AHC cases are caused by one of seven recurrent ATP1A3 mutations, one of which was observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset dystonia-parkinsonism, AHC-causing mutations in this gene caused consistent reductions in ATPase activity without...

  9. ANALYSIS OF C-HA-RAS GENE AMPLIFICATION AND MUTATION IN LARYNGEAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    刘世喜; 林代诚; 洪邦泰; 黄光琦

    1995-01-01

    In order to study the ahered molecular events during laryngeal carcinogenesis and elucidate the role of Ha-ras oncogene amplification and mutation, we have examined their profile by polymerase chain reaction (PCR) and selective oligonucleoride hybridization. We analyzed the mutational status of codon 12 of Ha-ras in 22 laryngeal carcinomas and 10 normal tissues, and found that 7 of 22 laryngeal carcinomas con-tained a Ha-ras mutation at codon 12. The frequency of mutation was 32%. None of the normal tissues re-vealed mutation. Moreover, no amplification was found in cancers when compared to the normal. Our findings indicated that the aefivmed Ha-ras gene existed in laryngeal carcinoma, and activation of the Ha-ras gene by mutation at codon 12 might play a key role in laryngeal carcinogenesis.

  10. Molecular Investigation of Leber’s Hereditary Optic Neuropathy Common Mutations in Suspected Patients

    Directory of Open Access Journals (Sweden)

    HR Soleimanpour

    2004-08-01

    Full Text Available LHON is a mitochondrial neurodegenerative disorder often manifesting itself in the second or third decade of life, and hence resulting in progressive central vision loss usually in a short period of 2-8 weeks within which different degrees of blindness may occur. Etiologically, more than twenty missense mutations have been reported for LHON, amongst which the three mutations of G11778A, G3460A and T14484C, affecting NADH dehydrogenase complex activity, are recognized as primary mutations. The three primary mutations account for 90% of LHON patients, emphasizing the importance of molecular investigation of these mutations for differential diagnosis of LHON. Using PCR-RFLP, this research resulted in the detection of two LHON families carrying the G11778A mutation in homoplasmy and described the clinical and molecular features of the disease in the patients.

  11. Heterozygous carriers of a Parkin or PINK1 mutation share a common functional endophenotype

    DEFF Research Database (Denmark)

    van Nuenen, BF; Siebner, Hartwig; Weiss, MM;

    2008-01-01

    inherited Parkinson disease alters the cortical control of sequential finger movements. METHODS: Nonmanifesting individuals carrying a single heterozygous Parkin (n = 13) or PINK1 (n = 9) mutation and 23 healthy controls without these mutations were studied with functional MRI (fMRI). During f......MRI, participants performed simple sequences of three thumb-to-finger opposition movements with their right dominant hand. Since heterozygous Parkin and PINK1 mutations cause a latent dopaminergic nigrostriatal dysfunction, we predicted a compensatory recruitment of those rostral premotor areas that are normally...... rostral dorsal premotor cortex in mutation carriers but not in controls. Task-related activation of these premotor areas was similar in carriers of a Parkin or PINK1 mutation. CONCLUSION: Mutations in different genes linked to recessively inherited Parkinson disease are associated with an additional...

  12. CBL mutations do not frequently occur in paediatric acute myeloid leukaemia

    NARCIS (Netherlands)

    Coenen, Eva A.; Driessen, Emma M. C.; Zwaan, C. Michel; Stary, Jan; Baruchel, Andre; de Haas, Valerie; de Bont, Eveline S. J. M.; Reinhardt, Dirk; Kaspers, Gertjan J. L.; Arentsen-Peters, Susan T. C. J. M.; Meyer, Claus; Marschalek, Rolf; Pieters, Rob; Stam, Ronald W.; van den Heuvel-Eibrink, Marry M.

    2012-01-01

    RAS-pathway mutations, causing a proliferative advantage, occur in acute myeloid leukaemia (AML) and MLL-rearranged leukaemia. Recently, mutations in the Casitas B lineage lymphoma (CBL) gene were reported to be involved in RAS-pathway activation in various myeloid malignancies, but their role in pa

  13. A cancer-predisposing "hot spot" mutation of the fumarase gene creates a dominant negative protein.

    Science.gov (United States)

    Lorenzato, Annalisa; Olivero, Martina; Perro, Mario; Brière, Jean Jacques; Rustin, Pierre; Di Renzo, Maria Flavia

    2008-02-15

    The Fumarase (Fumarate Hydratase, FH) is a tumor suppressor gene whose germline heterozygous mutations predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). The FH gene encodes an enzyme of the Krebs cycle, functioning as a homotetramer and catalyzing the hydration of fumarate to malate. Among the numerous FH mutations reported so far, the R190H missense mutation is the most frequent in HLRCC patients. Here we show the functional analyses of the R190H, in comparison to the better characterized E319Q mutation. We first expressed wild-type and mutated proteins in FH deficient human skin fibroblasts, using lentiviral vectors. The wild-type transgene was able to restore the FH enzymatic activity in cells, while the R190H- and E319Q-FH were not. More interestingly, when the same transgenes were expressed in normal, FH-proficient cells, only the R190H-FH reduced the endogenous FH enzymatic activity. By enforcing the expression of equal amount of wild-type and R190H-FH in the same cell, we showed that the mutated FH protein directly inhibited enzymatic activity by nearly abrogating the FH homotetramer formation. These data demonstrate the dominant negative effect of the R190H missense mutation in the FH gene and suggest that the FH tumor-suppressing activity might be impaired in cells carrying a heterozygous mutation. PMID:17960613

  14. Novel allelic mutations in murine Serca2 induce differential development of squamous cell tumors.

    Science.gov (United States)

    Toki, Hideaki; Minowa, Osamu; Inoue, Maki; Motegi, Hiromi; Karashima, Yuko; Ikeda, Ami; Kaneda, Hideki; Sakuraba, Yoshiyuki; Saiki, Yuriko; Wakana, Shigeharu; Suzuki, Hiroshi; Gondo, Yoichi; Shiroishi, Toshihiko; Noda, Tetsuo

    2016-08-01

    Dominant mutations in the Serca2 gene, which encodes sarco(endo)plasmic reticulum calcium-ATPase, predispose mice to gastrointestinal epithelial carcinoma [1-4] and humans to Darier disease (DD) [14-17]. In this study, we generated mice harboring N-ethyl-N-nitrosourea (ENU)-induced allelic mutations in Serca2: three missense mutations and one nonsense mutation. Mice harboring these Serca2 mutations developed tumors that were categorized as either early onset squamous cell tumors (SCT), with development similar to null-type knockout mice [2,4] (aggressive form; M682, M814), or late onset tumors (mild form; M1049, M1162). Molecular analysis showed no aberration in Serca2 mRNA or protein expression levels in normal esophageal cells of any of the four mutant heterozygotes. There was no loss of heterozygosity at the Serca2 locus in the squamous cell carcinomas in any of the four lines. The effect of each mutation on Ca(2+)-ATPase activity was predicted using atomic-structure models and accumulated mutated protein studies, suggesting that putative complete loss of Serca2 enzymatic activity may lead to early tumor onset, whereas mutations in which Serca2 retains residual enzymatic activity result in late onset. We propose that impaired Serca2 gene product activity has a long-term effect on squamous cell carcinogenesis from onset to the final carcinoma stage through an as-yet unrecognized but common regulatory pathway. PMID:27131742

  15. Limited diagnostic value of enzyme analysis in patients with mitochondrial tRNA mutations

    DEFF Research Database (Denmark)

    Wibrand, Flemming; Jeppesen, Tina Dysgaard; Frederiksen, Anja L;

    2010-01-01

    We evaluated the diagnostic value of respiratory chain (RC) enzyme analysis of muscle in adult patients with mitochondrial myopathy (MM). RC enzyme activity was measured in muscle biopsies from 39 patients who carry either the 3243A>G mutation, other tRNA point mutations, or single, large...

  16. The Relationship of the Factor V Leiden Mutation and Pregnancy Outcomes for Mother and Fetus

    Science.gov (United States)

    Dizon-Townson, Donna; Miller, Connie; Sibai, Baha; Spong, Catherine Y.; Thom, Elizabeth; Wendel, George; Wenstrom, Katharine; Samuels, Philip; Cotroneo, Margaret A.; Moawad, Atef; Sorokin, Yoram; Meis, Paul; Miodovnik, Menachem; O’Sullivan, Mary J.; Conway, Deborah; Wapner, Ronald J.; Gabbe, Steven G.

    2013-01-01

    Objective We sought to estimate the frequency of pregnancy-related thromboembolic events among carriers of the factor V Leiden (FVL) mutation without a personal history of thromboembolism, and to evaluate the impact of maternal and fetal FVL mutation carriage or other thrombophilias on the risk of adverse outcomes. Methods Women with a singleton pregnancy and no history of thromboembolism were recruited at 13 clinical centers before 14 weeks of gestation from April 2000 to August 2001. Each was tested for the FVL mutation, as was the resultant conceptus after delivery or after miscarriage, when available. The incidence of thromboembolism (primary outcome), and of other adverse outcomes, was compared between FVL mutation carriers and noncarriers. We also compared adverse outcomes in a secondary nested carrier-control analysis of FVL mutation and other coagulation abnormalities. In this secondary analysis, we defined carriers as women having one or more of the following traits: carrier for FVL mutation, protein C deficiency, protein S deficiency, antithrombin III deficiency, activated protein C resistance, or lupus anticoagulant-positive, heterozygous for prothrombin G20210A or homozygous for the 5,10 methylenetetrahydrofolate reductase mutations. Carriers of the FVL mutation alone (with or without activated protein C resistance) were compared with those having one or more other coagulation abnormalities and with controls with no coagulation abnormality. Results One hundred thirty-four FVL mutation carriers were identified among 4,885 gravidas (2.7%), with both FVL mutation status and pregnancy outcomes available. No thromboembolic events occurred among the FVL mutation carriers (0%, 95% confidence interval 0–2.7%). Three pulmonary emboli and one deep venous thrombosis occurred (0.08%, 95% confidence interval 0.02–0.21%), all occurring in FVL mutation noncarriers. In the nested carrier-control analysis (n = 339), no differences in adverse pregnancy outcomes were

  17. Frequent POLE1 p.S297F mutation in Chinese patients with ovarian endometrioid carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Yang; Liu, Fa-Ying; Liu, Huai; Wang, Feng [Key Laboratory of Women' s Reproductive Health of Jiangxi Province, Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi 330006 (China); Central Laboratory, Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi 330006 (China); Li, Wei [Key Laboratory of Women' s Reproductive Health of Jiangxi Province, Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi 330006 (China); Central Laboratory, Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi 330006 (China); Graduate School of Nanchang University, Nanchang, Jiangxi 330031 (China); Huang, Mei-Zhen [Graduate School of Nanchang University, Nanchang, Jiangxi 330031 (China); Jiangxi Provincial Cancer Institute, Jiangxi Provincial Cancer Hospital, Nanchang, Jiangxi 330029 (China); Huang, Yan; Yuan, Xiao-Qun [Key Laboratory of Women' s Reproductive Health of Jiangxi Province, Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi 330006 (China); Central Laboratory, Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi 330006 (China); Graduate School of Nanchang University, Nanchang, Jiangxi 330031 (China); Xu, Xiao-Yun [Graduate School of Nanchang University, Nanchang, Jiangxi 330031 (China); Jiangxi Provincial Cancer Institute, Jiangxi Provincial Cancer Hospital, Nanchang, Jiangxi 330029 (China); Huang, Ou-Ping, E-mail: huangouping@gmail.com [Jiangxi Provincial Cancer Institute, Jiangxi Provincial Cancer Hospital, Nanchang, Jiangxi 330029 (China); He, Ming, E-mail: jxhm56@hotmail.com [Department of Pharmacology and Molecular Therapeutics, Nanchang University School of Pharmaceutical Science, Nanchang 330006 (China)

    2014-03-15

    The catalytic subunit of DNA polymerase epsilon (POLE1) functions primarily in nuclear DNA replication and repair. Recently, POLE1 mutations were detected frequently in colorectal and endometrial carcinomas while with lower frequency in several other types of cancer, and the p.P286R and p.V411L mutations were the potential mutation hotspots in human cancers. Nevertheless, the mutation frequency of POLE1 in ovarian cancer still remains largely unknown. Here, we screened a total of 251 Chinese samples with distinct subtypes of ovarian carcinoma for the presence of POLE1 hotspot mutations by direct sequencing. A heterozygous somatic POLE1 mutation, p.S297F (c.890C>T), but not p.P286R and p.V411L hotspot mutations observed in other cancer types, was identified in 3 out of 37 (8.1%) patients with ovarian endometrioid carcinoma; this mutation was evolutionarily highly conserved from Homo sapiens to Schizosaccharomyces. Of note, the POLE1 mutation coexisted with mutation in the ovarian cancer-associated PPP2R1A (protein phosphatase 2, regulatory subunit A, α) gene in a 46-year-old patient, who was also diagnosed with ectopic endometriosis in the benign ovary. In addition, a 45-year-old POLE1-mutated ovarian endometrioid carcinoma patient was also diagnosed with uterine leiomyoma while the remaining 52-year-old POLE1-mutated patient showed no additional distinctive clinical manifestation. In contrast to high frequency of POLE1 mutations in ovarian endometrioid carcinoma, no POLE1 mutations were identified in patients with other subtypes of ovarian carcinoma. Our results showed for the first time that the POLE1 p.S297F mutation, but not p.P286R and p.V411L hotspot mutations observed in other cancer types, was frequent in Chinese ovarian endometrioid carcinoma, but absent in other subtypes of ovarian carcinoma. These results implicated that POLE1 p.S297F mutation might be actively involved in the pathogenesis of ovarian endometrioid carcinoma, but might not be actively

  18. Frequent POLE1 p.S297F mutation in Chinese patients with ovarian endometrioid carcinoma

    International Nuclear Information System (INIS)

    The catalytic subunit of DNA polymerase epsilon (POLE1) functions primarily in nuclear DNA replication and repair. Recently, POLE1 mutations were detected frequently in colorectal and endometrial carcinomas while with lower frequency in several other types of cancer, and the p.P286R and p.V411L mutations were the potential mutation hotspots in human cancers. Nevertheless, the mutation frequency of POLE1 in ovarian cancer still remains largely unknown. Here, we screened a total of 251 Chinese samples with distinct subtypes of ovarian carcinoma for the presence of POLE1 hotspot mutations by direct sequencing. A heterozygous somatic POLE1 mutation, p.S297F (c.890C>T), but not p.P286R and p.V411L hotspot mutations observed in other cancer types, was identified in 3 out of 37 (8.1%) patients with ovarian endometrioid carcinoma; this mutation was evolutionarily highly conserved from Homo sapiens to Schizosaccharomyces. Of note, the POLE1 mutation coexisted with mutation in the ovarian cancer-associated PPP2R1A (protein phosphatase 2, regulatory subunit A, α) gene in a 46-year-old patient, who was also diagnosed with ectopic endometriosis in the benign ovary. In addition, a 45-year-old POLE1-mutated ovarian endometrioid carcinoma patient was also diagnosed with uterine leiomyoma while the remaining 52-year-old POLE1-mutated patient showed no additional distinctive clinical manifestation. In contrast to high frequency of POLE1 mutations in ovarian endometrioid carcinoma, no POLE1 mutations were identified in patients with other subtypes of ovarian carcinoma. Our results showed for the first time that the POLE1 p.S297F mutation, but not p.P286R and p.V411L hotspot mutations observed in other cancer types, was frequent in Chinese ovarian endometrioid carcinoma, but absent in other subtypes of ovarian carcinoma. These results implicated that POLE1 p.S297F mutation might be actively involved in the pathogenesis of ovarian endometrioid carcinoma, but might not be actively

  19. P53 MUTATIONS IN HUMAN LUNG-TUMORS

    NARCIS (Netherlands)

    MILLER, CW; ASLO, A; KOK, K; YOKOTA, J; BUYS, CHCM; TERADA, M; KOEFFLER, HP; Simon, K.

    1992-01-01

    Mutation of one p53 allele and loss of the normal p53 allele [loss of heterozygosity (LOH)] occur in many tumors including lung cancers. These alterations apparently contribute to development of cancer by interfering with the tumor suppressor activity of p53. We directly sequenced amplified DNA in t

  20. Induced mutation of Dendrobium orchid

    International Nuclear Information System (INIS)

    Dendrobiiim orchids serve as the main orchid cut flower export of Malaysia. The wide range of colour and forms presently available in the market are obtained through hybridisation. Induced mutation breeding program was initiated on a commercial variety Dendrobium 'Sonia Kai' to explore the possibilities of obtaining new colour and forms. Matured seeds from self pollination were cultured and irradiated at 35 Gy at the protocorm-like bodies (PLBS) stage. Selection of induced mutations was done after the first flowering of the plants regenerated from the irradiated protocorms. Results showed changes in flower colour, shape and size. Most of these chances are expressed in different combinations in the petals, sepals and lip of the flowers. Thus, resulting. in a very wide spectrum of mutations. Some of these chances are not stable. To date, mutants that showed stable characteristics changes are grouped into 11 categories based on flower colour and form. These results show that the combination of its vitro technique and induced mutation can be applied in orchid breeding to produce new interesting and attractive variety for the market